Objective: This paper aims to investigate the effect of applying recombinant human brain natriuretic peptide in patients with heart failure combined with hypotension. Recombinant human brain natriuretic peptide is a s...Objective: This paper aims to investigate the effect of applying recombinant human brain natriuretic peptide in patients with heart failure combined with hypotension. Recombinant human brain natriuretic peptide is a synthetic polypeptide drug that is primarily used to treat acute heart failure. Its mechanism of action closely mimics that of human endogenous brain natriuretic peptide. By binding to receptors on cardiomyocytes, it exerts its pharmacological effects. Methods: For the study, 76 heart failure patients with hypotension were selected from our hospital between May 2022 and June 2023. These patients were divided into two groups: a control group and an observation group, each comprising 38 patients. The control group received dopamine treatment, while the observation group was treated with recombinant brain natriuretic peptide. The objective was to compare the effects of the treatments in both groups by analyzing cardiac function indices and levels of vasoactive substances to identify any significant differences in outcomes. Results: The overall response rate of the patients in the observation group and the control group was 94.74% and 73.68%, significantly higher as compared with the observation group (P 0.05). After the following treatment, BNP, ANNP and urine output in the observation group were significantly different compared with the control group, of the statistical significance (P Conclusion: For the treatment of heart failure patients with hypotension, the clinical application of recombinant human brain natriuretic peptide is the most ideal, and significantly improves the cardiac function of patients, which is worth popularizing.展开更多
Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare ...Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare efficacy based on in vitro level was evaluated by detecting the inhibition rate of elastase,the inhibition rate of collagenase,the protein content of type I collagen in human fibroblasts,the inhibition of reactive oxygen species(ROS)with human keratinocytes,and the effects of the recombinant humanized collagen on the expression of hyaluronic acid(HA),filaggrin(FLG)and transglutaminase 1(TGM1)in keratinocytes.The results showed that recombinant humanized collagen was able to maintain stability at temperatures below 70℃.With regard to its skincare efficacy,recombinant humanized collagen could inhibit elastase and collagenase activities and promote the increase of type I collagen content in human fibroblasts.It also showed good inhibition of ROS in keratinocytes in vitro and could increase the expression of HA,FLG,and TGM1 in keratinocytes.In short,the recombinant humanized collagen exhibited a favourable skin care effect in vitro level.This study proved that it has potential firming,anti-wrinkle,moisturizing,and repairing efficacy,and is a valuable cosmetic raw material.展开更多
BACKGROUND Dry eye syndrome(DES)after diabetic cataract surgery can seriously affect the patient’s quality of life.Therefore,effective alleviation of symptoms in patients with this disease has important clinical sign...BACKGROUND Dry eye syndrome(DES)after diabetic cataract surgery can seriously affect the patient’s quality of life.Therefore,effective alleviation of symptoms in patients with this disease has important clinical significance.AIM To explore the clinical effect of recombinant human epidermal growth factor(rhEGF)plus sodium hyaluronate(SH)eye drops on DES after cataract surgery in patients with diabetes.METHODS We retrospectively evaluated 82 patients with diabetes who experienced DES after cataract surgery at Tianjin Beichen Hospital,Affiliated Hospital of Nankai University between April 2021 and April 2023.They were classified into an observation group(42 cases,rhEGF+SH eye drops)and a control group(40 cases,SH eye drops alone),depending on the different treatment schemes.The therapeutic efficacy,dry eye symptom score,tear film breakup time(TFBUT),basic tear secretion score[assessed using Schirmer I test(SIt)],corneal fluorescein staining(FL)score,tear inflammatory markers,adverse reactions during treat-ment,and treatment satisfaction were compared between the two groups.RESULTS Therapeutic efficacy was higher in the observation group compared with the control group.Both groups showed improved TFBUT and dry eye,as well as improved SIt and FL scores after treatment,with a more pronounced improvement in the observation group.Although no marked differences in adverse reactions were observed between the two groups,treatment satisfaction was higher in the observation group.CONCLUSION rhEGF+SH eye drops rendered clinical benefits to patients by effectively ameliorating dry eye and visual impairment with favorable efficacy,fewer adverse reactions,and high safety levels.Thus,this treatment should be promoted in clinical practice.展开更多
Objective:To evaluate the therapeutic effect of recombinant bovine basic fibroblast growth factor(rbFGF)eye gel combined with tobramycin-dexamethasone(TOB-Dex)eye drops on dry eye syndrome(DES)after cataract surgery.M...Objective:To evaluate the therapeutic effect of recombinant bovine basic fibroblast growth factor(rbFGF)eye gel combined with tobramycin-dexamethasone(TOB-Dex)eye drops on dry eye syndrome(DES)after cataract surgery.Methods:86 patients with DES after cataract surgery,admitted from November 2021 to November 2023,were randomly divided into groups.The observation group included 43 patients treated with rbFGF eye gel combined with TOB-Dex eye drops.The reference group included 43 patients treated with TOB-Dex eye drops alone.Multiple indicators,including total effective rate and clinical symptom scores,were compared between the two groups.Results:The total effective rate in the observation group was higher than in the reference group(P<0.05).Before treatment,there were no differences in clinical symptom scores,serum factors,or disease severity scores between the two groups(P>0.05).Three weeks after treatment,the observation group had lower clinical symptom scores,serum factors,and disease severity scores compared to the reference group(P<0.05).The adverse reaction rate in the observation group was lower than in the reference group(P<0.05).Conclusion:rbFGF eye gel combined with TOB-Dex eye drops can improve the clinical efficacy for patients with DES after cataract surgery,alleviate disease symptoms,reduce inflammatory responses,and have fewer adverse reactions.展开更多
BACKGROUND Chronic heart failure(CHF)is a serious and prevalent condition characterized by impaired cardiac function and inflammation.Standard therapy for CHF has limitations,prompting the exploration of alternative t...BACKGROUND Chronic heart failure(CHF)is a serious and prevalent condition characterized by impaired cardiac function and inflammation.Standard therapy for CHF has limitations,prompting the exploration of alternative treatments.Recombinant human brain natriuretic peptide(BNP)has emerged as a potential therapy,with evidence suggesting that it can improve cardiac function and reduce inflammation in patients with CHF.However,further research is required to determine the efficacy and safety of lyophilized recombinant human BNP in CHF patients and its impact on microinflammatory status.This study aimed to investigate the effects of lyophilized recombinant human BNP therapy on CHF patients’cardiac function and microinflammatory status.AIM To investigate the effects of freeze-dried recombinant human BNP therapy on cardiac function and microinflammatory status in patients with CHF.METHODS In total,102 CHF patients admitted to our hospital from January 2021 to January 2022 were randomly assigned to control and observation groups(n=51 patients/group).The control patients were treated with standard HF therapy for 3 d,whereas the observational patients were injected with the recombinant human BNP for 3 d.Clinical efficacy,inflammatory factor levels,myocardial damage,cardiac function before and after the treatment,and adverse reactions during treatment were compared between the two groups.RESULTS The overall clinical efficacy was higher in the observation group than in the control group.Compared with baseline,serum hypersensitive C-reactive protein,N-terminal proBNP,and troponin I level,and physical,emotional,social,and economic scores were lower in both groups after treatment,with greater reductions in levels and scores noted in the observation group than in the control group.The overall incidence of adverse reactions in the observation group was not significantly different compared with that in the control group(P>0.05).CONCLUSION Freeze-dried recombinant human BNP therapy can improve heart function and enhance microinflammatory status,thereby improving overall quality of life without any obvious side effects.This therapy is safe and reliable.展开更多
Interferon-gamma(IFN-γ)plays a dual role in cancer;it is both a pro-and an antitumorigenic cytokine,depending on the type of cancer.The deregulation of the IFN-γcanonic pathway is associated with several disorders,i...Interferon-gamma(IFN-γ)plays a dual role in cancer;it is both a pro-and an antitumorigenic cytokine,depending on the type of cancer.The deregulation of the IFN-γcanonic pathway is associated with several disorders,including vulner-ability to viral infections,inflammation,and cancer progression.In particular,the interplay between lung adenocarcinoma(LUAD)and viral infections appears to exist in association with the deregulation of IFN-γsignaling.In this mini-review,we investigated the status of the IFN-γsignaling pathway and the expression level of its components in LUAD.Interestingly,a reduction in IFNGR1 expression seems to be associated with LUAD progression,affecting defenses against viruses such as severe acute respiratory syndrome coronavirus 2.In addition,alterations in the expression of IFNGR1 may inhibit the antiproliferative action of IFN-γsignaling in LUAD.展开更多
In this editorial,we comment on an article published in a recent issue of the World Journal of Clinical Cases.There is a pressing need for reliable tools for diagnosing tuberculosis(TB)of the gastrointestinal tract.De...In this editorial,we comment on an article published in a recent issue of the World Journal of Clinical Cases.There is a pressing need for reliable tools for diagnosing tuberculosis(TB)of the gastrointestinal tract.Despite advancements in the diagnosis and treatment,TB remains a global health challenge.Ali et al demon-strated that TB may mimic gastrointestinal conditions,such as gastric outlet obstruction,causing a delay in the diagnosis.Furthermore,the latter complication is frequently observed during infections,including Helicobacter pylori,and rarely is related to TB,as in the presented case.In line with this,we think that laboratory tests based on interferon-gamma release assays can be a helpful tool for diagnosing latent TB paced in the gastrointestinal tract.Innovative strategies and approaches for diagnosing latent/active extra pulmonary TB are crucial for establishing the diagnosis early and enhancing treatment strategies to mitigate the global burden of TB.展开更多
Lyophilized recombinant brain natriuretic peptide(BNP)is an exogenous peptide synthesized by artificial recombination technology,with a similar structure and similar physiological effects with the endogenous natriuret...Lyophilized recombinant brain natriuretic peptide(BNP)is an exogenous peptide synthesized by artificial recombination technology,with a similar structure and similar physiological effects with the endogenous natriuretic peptide secreted by the human body.It’s main mechanism of action is to increase cyclic guanosine monophosphate by binding with its corresponding receptor in the body,regulating,thus,the imbalance of the vascular system and cardiac hemodynamics,improving the heart’s pumping capacity,and inhibiting sympathetic excitability and myocardial remodeling.Moreover,it can promote mitochondrial metabolism and enhance the use of adenosine triphosphate in cardiomyocytes.In the present study,102 chronic heart failure(HF)patients were randomly assigned to a control and an observation group consisting of 51 patients each.Patients of the control group were treated with standard HF therapy for 3 d including oral metoprolol tartrate tablets,spironolactone,and olmesartanate while patients of the observation group were administered the recombinant human BNP injection for the same time-period,plus the standard HF therapy.The recombinant human BNP group(observation group)demonstrated better physical,emotional,social,and economic scores,as well as cardiac and inflammatory biomarkers such as serum hypersensitive C-reactive protein,N-terminal pro BNP and troponin I levels,compared to the control group.Moreover,cardiac function was also improved,as left ventricular ejection fraction and stroke volume were significantly higher in the observation group than in the control group.Interestingly,adverse reactions were not different between the 2 groups.However,these results are not generalizable and the need of large multicenter randomized controlled trials examining the safety and efficacy of recombinant human BNP in HF patients is of major importance.展开更多
AIM:To characterize changes of corneal nerve morphology and tear indices in patients with neurotrophic keratitis(NK)treated with recombinant human nerve growth factor(rhNGF).METHODS:In a prospective observational stud...AIM:To characterize changes of corneal nerve morphology and tear indices in patients with neurotrophic keratitis(NK)treated with recombinant human nerve growth factor(rhNGF).METHODS:In a prospective observational study,six patients(nine eyes)were locally treated with rhNGF.Visual acuity,corneal fluorescein staining score,the heights of the tear river,lipid layer thickness(LLT),tear ferning(TF)test,conjunctival impression cytology(CIC)examination,the densities of cornea subbasal nerve fibers were determined before and after treatment.RESULTS:Compared with baseline,there was a significant difference in corneal fluorescence staining scores(P<0.01);all patient corneal epithelial defects recovered completely within 8wk,but there was no significant improvement in the height of the tear river(P=0.202).LLT was significantly increased when compared with baseline(P=0.042);however,the function of conjunctival goblet cells and mucin content did not significantly improve using the TF test and CIC examination(P=0.557,P=0.539).After 8wk of treatment,the average corneal subbasal nerve fiber density increased significantly(P<0.01),as did the number of corneal nerve fiber branches(P=0.001).CONCLUSION:RhNGF can increase the density of corneal subbasal nerve fibers,promote the healing of persistent corneal epithelial defects and corneal ulcers in patients with NK,also improving tear function partially.展开更多
Lymphatic filariasis (LF) remains a public health concern as it can cause permanent morbidity and disability to those infected. While the global elimination of LF in these endemic areas is ongoing through mass drug ad...Lymphatic filariasis (LF) remains a public health concern as it can cause permanent morbidity and disability to those infected. While the global elimination of LF in these endemic areas is ongoing through mass drug administration, there is the need to develop diagnostic tools that would be utilized to track the progress of total global eradication as well as perform surveillance for the recurrence of lymphatic filariasis transmission. Currently, approved LF diagnosis tools are faced with lack of specificity, low sensitivity, and periodicity dependence. Recombinant filarial antigen-based assays can address these drawbacks and offer practical instruments for LF diagnosis and surveillance. This present study, evaluated rWb-SXP-1 and rWb-123 antigens as potential diagnostic biomarker tools for Wuchereria banchrofti in human sera using microspheres-based multiplex serological assay. Based on statistical analysis using XLSTAT 2019 (Addinsoft) on data generated from multiplex technology assay, generated ROC curves for both rWb-SXP-1 and rWb-123 demonstrated 87.1% sensitivity to Wuchereria banchrofti human sera with rWb-SXP-1 antigens having the highest specificity of 96%. Indication that rWb-SXP-1 and rWb-123 antigens are capable of detecting immunoglobulin G4 (IgG4) antibodies in human sera synthesized specifically against W. banchrofti infections. Therefore, rWb-SXP-1 and rWb-123 antigens can be utilized to detect W. banchrofti infections by antibody profiling with excellent diagnostic sensitivity and specificity using microsphere-based multiplex serological tests. This method can be particularly practical for screening a large number of sera samples and/or for quick, extensive field-testing due to the high-throughput and quick formats applied.展开更多
The destruction of recombinant bamboo depends on many factors,and the complex ambient temperature is an important factor affecting its basic mechanical properties.To investigate the failure mechanism and stress–strai...The destruction of recombinant bamboo depends on many factors,and the complex ambient temperature is an important factor affecting its basic mechanical properties.To investigate the failure mechanism and stress–strain relationship of recombinant bamboo at different temperatures,eighteen tensile specimens of recombinant bamboo were tested.The results showed that with increasing ambient temperature,the typical failure modes of recombinant bamboo were flush fracture,toothed failure,and serrated failure.The ultimate tensile strength,ultimate strain and elastic modulus of recombinant bamboo decreased with increasing temperature,and the ultimate tensile stress decreased from 154.07 to 96.55 MPa,a decrease of 37.33%,and the ultimate strain decreased from 0.011 to 0.008,a decrease of 26.57%.Based on the Ramberg-Osgood model and the pseudo‒elastic design method,a predictive model was established for the tensile stress–strain relationship of recombinant bamboo considering the temperature level.The model can accurately evaluate the tensile stress–strain relationship of recombinant bamboo under different temperature conditions.展开更多
A recombinant skin model is a model in which skin cells are grown in vitro on a bioactive scaffold and provided with adequate nutrition to promote cell proliferation and differentiation to produce a mock skin structur...A recombinant skin model is a model in which skin cells are grown in vitro on a bioactive scaffold and provided with adequate nutrition to promote cell proliferation and differentiation to produce a mock skin structure and biological features. The development of recombinant skin models allows for effective and scientifically sound in vitro evaluation tests. This review briefly summarizes the overview of recombinant skin models and the progress of their application in in vitro evaluation which focuses on three aspects: skin irritation, skin corrosivity, and anti-skin aging. Moreover, an outlook on the future development of recombinant skin models is also provided in this review.展开更多
Objective Bloom’s syndrome is an autosomal recessive disorder characterized by genomic instability and a predisposition to many cancers. Mutations of the BLM gene (encoding a BLM helicase) may form a structure of t...Objective Bloom’s syndrome is an autosomal recessive disorder characterized by genomic instability and a predisposition to many cancers. Mutations of the BLM gene (encoding a BLM helicase) may form a structure of the etiology of this disease. As a global pollutant, mercury poses a major threat to human health. The current study was conducted to elucidate the effects of Hg^2+ on the structure and activity of BLM642‐1290 recombinant helicase, and to further explore the molecular mechanisms of mercury toxicity to the DNA helicase. Methods The effects of Hg^2+ on biological activity and structure of BLM642‐1290 recombinant helicase were determined by fluorescence polarized, ultraviolet spectroscopic, and free‐phosphorus assay technologies, respectively. Results The helicase activity, the DNA‐binding activity, and the ATPase activity of BLM642‐1290 recombinant helicase were inhibited by Hg^2+ treatment. The LMCT (ligand‐to‐metal charge transition) peaks of the helicase were enhanced with the increase of the Hg^2+ level. The LMCT peaks of the same concentration of helicase gradually increased over time. Conclusions The biological activity of BLM642‐1290 recombinant helicase is inhibited by Hg^2+ treatment. The conformation of the helicase is significantly altered by Hg^2+ . There exist two binding sites between Hg^2+ and the helicase, which are located in the amino acid residues 1063‐1066 and 940‐944 of the helicase, respectively.展开更多
Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column...Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column chromatography steps with macroporous resin,DEAE cellulose DES2 and preparative RP-HPLC, respectively, and the optimal conditions were obtained.Purity of the product was determined by SDS-PAGE and analytical RP-HPLC. The molecular weight wasdetermined by mass spec-trometry. The structure of the product was analyzed by peptide map.ResultsThe product with purity of 95.4786% was obtained after three purification steps in the optimumconditions with a total yield of 39%. The molecular weight of the product was 6 913.32 ± 6.55 Da,coincident to the theoretical molecular weight of r-hirudin 3. The structure of the product wascoincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfullyemployed for purification of r-hirudin 3 from E. coli using batch-type approaches. The productobtained with high purity was confirmed to be r-hirudin 3.展开更多
The present study aimed at breeding new rice germplasms with similar genome but significantly differed in heat tolerance during the grain filling stage.A total of 791 BC1F8 backcross recombinant lines,derived from the...The present study aimed at breeding new rice germplasms with similar genome but significantly differed in heat tolerance during the grain filling stage.A total of 791 BC1F8 backcross recombinant lines,derived from the cross of Xieqingzao B /N22 //Xieqingzao B,were used as materials.Each rice line was separated evenly into two groups,and the heat tolerance of all rice lines were evaluated at natural high temperature in fields.The rice lines with heat tolerant index higher than 90% or lower than 40% were selected to compare the phenotypic characters and further identify heat tolerance at the early milky stage in a phytotron.Rice lines with similar phenotypic characters but significantly differed in heat tolerance at the milky stage were analyzed by 887 simple sequence repeat markers that were evenly distributed on the 12 rice chromosomes.In the result,12(6 pairs) rice lines with similar phenotypic characters but significantly differed in heat tolerance at the milky stage were obtained.Molecular marker analysis indicated that the genomic polymorphism between 703T and 704S was the smallest in the 6 pairs of rice lines,with only 16 polymorphic sites,including 22 different alleles.The application of these two backcross introgression rice lines for future study on the mechanisms of heat tolerance in rice at the milky stage will be theoretically beneficial in reducing the interference caused by genetic differences from experimental materials.展开更多
Objective To assess the severity and reversibility of the chronic toxicity of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and the dose-effect relationship.Methods A total of 100 ...Objective To assess the severity and reversibility of the chronic toxicity of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and the dose-effect relationship.Methods A total of 100 Sprague-Dawley rats (equal numbers of male and female) were randomly divided into five groups (20 rats in each group):four groups were treated with rhG-CSFa at 500,100,10,1 μg/kg,respectively,and one group was treated with vehicle only to serve as the control.The rats were received subcutaneous injections of rhG-CSFa or vehicle daily for 13 weeks.During the course of the chronic toxicity study,the physical status,body weight,and food consumption were monitored.Half of the rats in each group (n=10) were sacrificed after the last rhG-CSFa administration,and the other half were sacrificed at five weeks after the last rhG-CSFa administration.Urinalyses,blood biochemistry,hematological analysis,histopathological examination,and immunological tests were performed for each of the rats.Results The hematological analyses revealed that the mean white blood cells count,neutrophils count,and neutrophils percentage were increased in male rats at the dose of 10 μg/kg or higher,and these were related with the biological activity of rhG-CSFa.Some small abnormalities were observed in the spleen of a few rats when used highest dose (500 μg/kg,a dosage of 200 folds higher than the normal clinical dosage),but these abnormalities were recovered within 5-week recovery period.No other rhG-CSFa-related abnormalities were observed in this chronic toxicity study.Conclusion No significant toxicity and immunogenicity are observed with rhG-CSFa administration to rats in the chronic toxicity studies.展开更多
AIM: To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS: Gateway recombinant cloning technology was used to co...AIM: To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS: Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus (CMV) 〉mutlumican/IRES/EGFP, respectively.RESULTS: The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION: We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.展开更多
BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeu...BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning. SETTING: Central Laboratory of Northern China Coal Medical College. MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT 1080 cells. X-gal stain was used to measure virus titer. MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus titer. RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosarcoma cells (HT1080) and the recombinant virus titer was measured as 1 ×10^12 virus particles per milliliter. CONCLUSION: Total-length cDNA fragment of NT-3 gene, which is obtained from pCDNA3-NT-3 plasmids, is closely matched to polyclone enzyme-shearing sites of adeno-associated viral vectors, while the combination can be used to construct recombinant adeno-associated viral vectors expression in hNT-3 gene.展开更多
Objectives: To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods: The immuno-dominant epitope of Tp0453 gene was amplif...Objectives: To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods: The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions (PCR), subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results: The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% (30/30) and a specificity of 100% (30/30). While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion: The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.展开更多
文摘Objective: This paper aims to investigate the effect of applying recombinant human brain natriuretic peptide in patients with heart failure combined with hypotension. Recombinant human brain natriuretic peptide is a synthetic polypeptide drug that is primarily used to treat acute heart failure. Its mechanism of action closely mimics that of human endogenous brain natriuretic peptide. By binding to receptors on cardiomyocytes, it exerts its pharmacological effects. Methods: For the study, 76 heart failure patients with hypotension were selected from our hospital between May 2022 and June 2023. These patients were divided into two groups: a control group and an observation group, each comprising 38 patients. The control group received dopamine treatment, while the observation group was treated with recombinant brain natriuretic peptide. The objective was to compare the effects of the treatments in both groups by analyzing cardiac function indices and levels of vasoactive substances to identify any significant differences in outcomes. Results: The overall response rate of the patients in the observation group and the control group was 94.74% and 73.68%, significantly higher as compared with the observation group (P 0.05). After the following treatment, BNP, ANNP and urine output in the observation group were significantly different compared with the control group, of the statistical significance (P Conclusion: For the treatment of heart failure patients with hypotension, the clinical application of recombinant human brain natriuretic peptide is the most ideal, and significantly improves the cardiac function of patients, which is worth popularizing.
文摘Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare efficacy based on in vitro level was evaluated by detecting the inhibition rate of elastase,the inhibition rate of collagenase,the protein content of type I collagen in human fibroblasts,the inhibition of reactive oxygen species(ROS)with human keratinocytes,and the effects of the recombinant humanized collagen on the expression of hyaluronic acid(HA),filaggrin(FLG)and transglutaminase 1(TGM1)in keratinocytes.The results showed that recombinant humanized collagen was able to maintain stability at temperatures below 70℃.With regard to its skincare efficacy,recombinant humanized collagen could inhibit elastase and collagenase activities and promote the increase of type I collagen content in human fibroblasts.It also showed good inhibition of ROS in keratinocytes in vitro and could increase the expression of HA,FLG,and TGM1 in keratinocytes.In short,the recombinant humanized collagen exhibited a favourable skin care effect in vitro level.This study proved that it has potential firming,anti-wrinkle,moisturizing,and repairing efficacy,and is a valuable cosmetic raw material.
基金Supported by Tianjin Health Research Project,No.TJWJ2023MS062。
文摘BACKGROUND Dry eye syndrome(DES)after diabetic cataract surgery can seriously affect the patient’s quality of life.Therefore,effective alleviation of symptoms in patients with this disease has important clinical significance.AIM To explore the clinical effect of recombinant human epidermal growth factor(rhEGF)plus sodium hyaluronate(SH)eye drops on DES after cataract surgery in patients with diabetes.METHODS We retrospectively evaluated 82 patients with diabetes who experienced DES after cataract surgery at Tianjin Beichen Hospital,Affiliated Hospital of Nankai University between April 2021 and April 2023.They were classified into an observation group(42 cases,rhEGF+SH eye drops)and a control group(40 cases,SH eye drops alone),depending on the different treatment schemes.The therapeutic efficacy,dry eye symptom score,tear film breakup time(TFBUT),basic tear secretion score[assessed using Schirmer I test(SIt)],corneal fluorescein staining(FL)score,tear inflammatory markers,adverse reactions during treat-ment,and treatment satisfaction were compared between the two groups.RESULTS Therapeutic efficacy was higher in the observation group compared with the control group.Both groups showed improved TFBUT and dry eye,as well as improved SIt and FL scores after treatment,with a more pronounced improvement in the observation group.Although no marked differences in adverse reactions were observed between the two groups,treatment satisfaction was higher in the observation group.CONCLUSION rhEGF+SH eye drops rendered clinical benefits to patients by effectively ameliorating dry eye and visual impairment with favorable efficacy,fewer adverse reactions,and high safety levels.Thus,this treatment should be promoted in clinical practice.
文摘Objective:To evaluate the therapeutic effect of recombinant bovine basic fibroblast growth factor(rbFGF)eye gel combined with tobramycin-dexamethasone(TOB-Dex)eye drops on dry eye syndrome(DES)after cataract surgery.Methods:86 patients with DES after cataract surgery,admitted from November 2021 to November 2023,were randomly divided into groups.The observation group included 43 patients treated with rbFGF eye gel combined with TOB-Dex eye drops.The reference group included 43 patients treated with TOB-Dex eye drops alone.Multiple indicators,including total effective rate and clinical symptom scores,were compared between the two groups.Results:The total effective rate in the observation group was higher than in the reference group(P<0.05).Before treatment,there were no differences in clinical symptom scores,serum factors,or disease severity scores between the two groups(P>0.05).Three weeks after treatment,the observation group had lower clinical symptom scores,serum factors,and disease severity scores compared to the reference group(P<0.05).The adverse reaction rate in the observation group was lower than in the reference group(P<0.05).Conclusion:rbFGF eye gel combined with TOB-Dex eye drops can improve the clinical efficacy for patients with DES after cataract surgery,alleviate disease symptoms,reduce inflammatory responses,and have fewer adverse reactions.
文摘BACKGROUND Chronic heart failure(CHF)is a serious and prevalent condition characterized by impaired cardiac function and inflammation.Standard therapy for CHF has limitations,prompting the exploration of alternative treatments.Recombinant human brain natriuretic peptide(BNP)has emerged as a potential therapy,with evidence suggesting that it can improve cardiac function and reduce inflammation in patients with CHF.However,further research is required to determine the efficacy and safety of lyophilized recombinant human BNP in CHF patients and its impact on microinflammatory status.This study aimed to investigate the effects of lyophilized recombinant human BNP therapy on CHF patients’cardiac function and microinflammatory status.AIM To investigate the effects of freeze-dried recombinant human BNP therapy on cardiac function and microinflammatory status in patients with CHF.METHODS In total,102 CHF patients admitted to our hospital from January 2021 to January 2022 were randomly assigned to control and observation groups(n=51 patients/group).The control patients were treated with standard HF therapy for 3 d,whereas the observational patients were injected with the recombinant human BNP for 3 d.Clinical efficacy,inflammatory factor levels,myocardial damage,cardiac function before and after the treatment,and adverse reactions during treatment were compared between the two groups.RESULTS The overall clinical efficacy was higher in the observation group than in the control group.Compared with baseline,serum hypersensitive C-reactive protein,N-terminal proBNP,and troponin I level,and physical,emotional,social,and economic scores were lower in both groups after treatment,with greater reductions in levels and scores noted in the observation group than in the control group.The overall incidence of adverse reactions in the observation group was not significantly different compared with that in the control group(P>0.05).CONCLUSION Freeze-dried recombinant human BNP therapy can improve heart function and enhance microinflammatory status,thereby improving overall quality of life without any obvious side effects.This therapy is safe and reliable.
文摘Interferon-gamma(IFN-γ)plays a dual role in cancer;it is both a pro-and an antitumorigenic cytokine,depending on the type of cancer.The deregulation of the IFN-γcanonic pathway is associated with several disorders,including vulner-ability to viral infections,inflammation,and cancer progression.In particular,the interplay between lung adenocarcinoma(LUAD)and viral infections appears to exist in association with the deregulation of IFN-γsignaling.In this mini-review,we investigated the status of the IFN-γsignaling pathway and the expression level of its components in LUAD.Interestingly,a reduction in IFNGR1 expression seems to be associated with LUAD progression,affecting defenses against viruses such as severe acute respiratory syndrome coronavirus 2.In addition,alterations in the expression of IFNGR1 may inhibit the antiproliferative action of IFN-γsignaling in LUAD.
基金The European Union-Next Generation EU,through the National Recovery and Resilience Plan of the Republic of Bulgaria,Project,No.BG-RRP-2.004-0008.
文摘In this editorial,we comment on an article published in a recent issue of the World Journal of Clinical Cases.There is a pressing need for reliable tools for diagnosing tuberculosis(TB)of the gastrointestinal tract.Despite advancements in the diagnosis and treatment,TB remains a global health challenge.Ali et al demon-strated that TB may mimic gastrointestinal conditions,such as gastric outlet obstruction,causing a delay in the diagnosis.Furthermore,the latter complication is frequently observed during infections,including Helicobacter pylori,and rarely is related to TB,as in the presented case.In line with this,we think that laboratory tests based on interferon-gamma release assays can be a helpful tool for diagnosing latent TB paced in the gastrointestinal tract.Innovative strategies and approaches for diagnosing latent/active extra pulmonary TB are crucial for establishing the diagnosis early and enhancing treatment strategies to mitigate the global burden of TB.
文摘Lyophilized recombinant brain natriuretic peptide(BNP)is an exogenous peptide synthesized by artificial recombination technology,with a similar structure and similar physiological effects with the endogenous natriuretic peptide secreted by the human body.It’s main mechanism of action is to increase cyclic guanosine monophosphate by binding with its corresponding receptor in the body,regulating,thus,the imbalance of the vascular system and cardiac hemodynamics,improving the heart’s pumping capacity,and inhibiting sympathetic excitability and myocardial remodeling.Moreover,it can promote mitochondrial metabolism and enhance the use of adenosine triphosphate in cardiomyocytes.In the present study,102 chronic heart failure(HF)patients were randomly assigned to a control and an observation group consisting of 51 patients each.Patients of the control group were treated with standard HF therapy for 3 d including oral metoprolol tartrate tablets,spironolactone,and olmesartanate while patients of the observation group were administered the recombinant human BNP injection for the same time-period,plus the standard HF therapy.The recombinant human BNP group(observation group)demonstrated better physical,emotional,social,and economic scores,as well as cardiac and inflammatory biomarkers such as serum hypersensitive C-reactive protein,N-terminal pro BNP and troponin I levels,compared to the control group.Moreover,cardiac function was also improved,as left ventricular ejection fraction and stroke volume were significantly higher in the observation group than in the control group.Interestingly,adverse reactions were not different between the 2 groups.However,these results are not generalizable and the need of large multicenter randomized controlled trials examining the safety and efficacy of recombinant human BNP in HF patients is of major importance.
基金Supported by the Shaanxi Provincial Department of Science and Technology(No.2021SF-331)。
文摘AIM:To characterize changes of corneal nerve morphology and tear indices in patients with neurotrophic keratitis(NK)treated with recombinant human nerve growth factor(rhNGF).METHODS:In a prospective observational study,six patients(nine eyes)were locally treated with rhNGF.Visual acuity,corneal fluorescein staining score,the heights of the tear river,lipid layer thickness(LLT),tear ferning(TF)test,conjunctival impression cytology(CIC)examination,the densities of cornea subbasal nerve fibers were determined before and after treatment.RESULTS:Compared with baseline,there was a significant difference in corneal fluorescence staining scores(P<0.01);all patient corneal epithelial defects recovered completely within 8wk,but there was no significant improvement in the height of the tear river(P=0.202).LLT was significantly increased when compared with baseline(P=0.042);however,the function of conjunctival goblet cells and mucin content did not significantly improve using the TF test and CIC examination(P=0.557,P=0.539).After 8wk of treatment,the average corneal subbasal nerve fiber density increased significantly(P<0.01),as did the number of corneal nerve fiber branches(P=0.001).CONCLUSION:RhNGF can increase the density of corneal subbasal nerve fibers,promote the healing of persistent corneal epithelial defects and corneal ulcers in patients with NK,also improving tear function partially.
文摘Lymphatic filariasis (LF) remains a public health concern as it can cause permanent morbidity and disability to those infected. While the global elimination of LF in these endemic areas is ongoing through mass drug administration, there is the need to develop diagnostic tools that would be utilized to track the progress of total global eradication as well as perform surveillance for the recurrence of lymphatic filariasis transmission. Currently, approved LF diagnosis tools are faced with lack of specificity, low sensitivity, and periodicity dependence. Recombinant filarial antigen-based assays can address these drawbacks and offer practical instruments for LF diagnosis and surveillance. This present study, evaluated rWb-SXP-1 and rWb-123 antigens as potential diagnostic biomarker tools for Wuchereria banchrofti in human sera using microspheres-based multiplex serological assay. Based on statistical analysis using XLSTAT 2019 (Addinsoft) on data generated from multiplex technology assay, generated ROC curves for both rWb-SXP-1 and rWb-123 demonstrated 87.1% sensitivity to Wuchereria banchrofti human sera with rWb-SXP-1 antigens having the highest specificity of 96%. Indication that rWb-SXP-1 and rWb-123 antigens are capable of detecting immunoglobulin G4 (IgG4) antibodies in human sera synthesized specifically against W. banchrofti infections. Therefore, rWb-SXP-1 and rWb-123 antigens can be utilized to detect W. banchrofti infections by antibody profiling with excellent diagnostic sensitivity and specificity using microsphere-based multiplex serological tests. This method can be particularly practical for screening a large number of sera samples and/or for quick, extensive field-testing due to the high-throughput and quick formats applied.
基金The authors wish to express their gratitude to the National Natural Science Foundation of China(Nos.51208262,51778300)Key Research and Development Project of Jiangsu Province(No.BE2020703)+2 种基金Natural Science Foundation of Jiangsu Province(No.BK20191390)Six Talent Peaks Project of Jiangsu Province(JZ-017)Qinglan Project of Jiangsu Province for financially supporting this study.
文摘The destruction of recombinant bamboo depends on many factors,and the complex ambient temperature is an important factor affecting its basic mechanical properties.To investigate the failure mechanism and stress–strain relationship of recombinant bamboo at different temperatures,eighteen tensile specimens of recombinant bamboo were tested.The results showed that with increasing ambient temperature,the typical failure modes of recombinant bamboo were flush fracture,toothed failure,and serrated failure.The ultimate tensile strength,ultimate strain and elastic modulus of recombinant bamboo decreased with increasing temperature,and the ultimate tensile stress decreased from 154.07 to 96.55 MPa,a decrease of 37.33%,and the ultimate strain decreased from 0.011 to 0.008,a decrease of 26.57%.Based on the Ramberg-Osgood model and the pseudo‒elastic design method,a predictive model was established for the tensile stress–strain relationship of recombinant bamboo considering the temperature level.The model can accurately evaluate the tensile stress–strain relationship of recombinant bamboo under different temperature conditions.
文摘A recombinant skin model is a model in which skin cells are grown in vitro on a bioactive scaffold and provided with adequate nutrition to promote cell proliferation and differentiation to produce a mock skin structure and biological features. The development of recombinant skin models allows for effective and scientifically sound in vitro evaluation tests. This review briefly summarizes the overview of recombinant skin models and the progress of their application in in vitro evaluation which focuses on three aspects: skin irritation, skin corrosivity, and anti-skin aging. Moreover, an outlook on the future development of recombinant skin models is also provided in this review.
基金supported by the Natural Sciences Foundation of China (NSFC, No.30660043)the National Basic Research Program of China (2010CB534912)+1 种基金the Doctoral Program of the Ministry of Education of China (No.200806570003)the Governor Talents Foundation of Guizhou Province (No.200822)
文摘Objective Bloom’s syndrome is an autosomal recessive disorder characterized by genomic instability and a predisposition to many cancers. Mutations of the BLM gene (encoding a BLM helicase) may form a structure of the etiology of this disease. As a global pollutant, mercury poses a major threat to human health. The current study was conducted to elucidate the effects of Hg^2+ on the structure and activity of BLM642‐1290 recombinant helicase, and to further explore the molecular mechanisms of mercury toxicity to the DNA helicase. Methods The effects of Hg^2+ on biological activity and structure of BLM642‐1290 recombinant helicase were determined by fluorescence polarized, ultraviolet spectroscopic, and free‐phosphorus assay technologies, respectively. Results The helicase activity, the DNA‐binding activity, and the ATPase activity of BLM642‐1290 recombinant helicase were inhibited by Hg^2+ treatment. The LMCT (ligand‐to‐metal charge transition) peaks of the helicase were enhanced with the increase of the Hg^2+ level. The LMCT peaks of the same concentration of helicase gradually increased over time. Conclusions The biological activity of BLM642‐1290 recombinant helicase is inhibited by Hg^2+ treatment. The conformation of the helicase is significantly altered by Hg^2+ . There exist two binding sites between Hg^2+ and the helicase, which are located in the amino acid residues 1063‐1066 and 940‐944 of the helicase, respectively.
文摘Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column chromatography steps with macroporous resin,DEAE cellulose DES2 and preparative RP-HPLC, respectively, and the optimal conditions were obtained.Purity of the product was determined by SDS-PAGE and analytical RP-HPLC. The molecular weight wasdetermined by mass spec-trometry. The structure of the product was analyzed by peptide map.ResultsThe product with purity of 95.4786% was obtained after three purification steps in the optimumconditions with a total yield of 39%. The molecular weight of the product was 6 913.32 ± 6.55 Da,coincident to the theoretical molecular weight of r-hirudin 3. The structure of the product wascoincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfullyemployed for purification of r-hirudin 3 from E. coli using batch-type approaches. The productobtained with high purity was confirmed to be r-hirudin 3.
基金supported by the National Natural Science Foundation of China(Grant No.30671230)the Natural Science Foundation of Jiangxi Province,China(Grant No.2007GZN0253)
文摘The present study aimed at breeding new rice germplasms with similar genome but significantly differed in heat tolerance during the grain filling stage.A total of 791 BC1F8 backcross recombinant lines,derived from the cross of Xieqingzao B /N22 //Xieqingzao B,were used as materials.Each rice line was separated evenly into two groups,and the heat tolerance of all rice lines were evaluated at natural high temperature in fields.The rice lines with heat tolerant index higher than 90% or lower than 40% were selected to compare the phenotypic characters and further identify heat tolerance at the early milky stage in a phytotron.Rice lines with similar phenotypic characters but significantly differed in heat tolerance at the milky stage were analyzed by 887 simple sequence repeat markers that were evenly distributed on the 12 rice chromosomes.In the result,12(6 pairs) rice lines with similar phenotypic characters but significantly differed in heat tolerance at the milky stage were obtained.Molecular marker analysis indicated that the genomic polymorphism between 703T and 704S was the smallest in the 6 pairs of rice lines,with only 16 polymorphic sites,including 22 different alleles.The application of these two backcross introgression rice lines for future study on the mechanisms of heat tolerance in rice at the milky stage will be theoretically beneficial in reducing the interference caused by genetic differences from experimental materials.
基金Supported by State Scientific Key Projects for New Drug Research and Development (2009ZX09102-250)High-tech Research Project for Medicine and Pharmacology of Jiangsu Province (BG20070605)
文摘Objective To assess the severity and reversibility of the chronic toxicity of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and the dose-effect relationship.Methods A total of 100 Sprague-Dawley rats (equal numbers of male and female) were randomly divided into five groups (20 rats in each group):four groups were treated with rhG-CSFa at 500,100,10,1 μg/kg,respectively,and one group was treated with vehicle only to serve as the control.The rats were received subcutaneous injections of rhG-CSFa or vehicle daily for 13 weeks.During the course of the chronic toxicity study,the physical status,body weight,and food consumption were monitored.Half of the rats in each group (n=10) were sacrificed after the last rhG-CSFa administration,and the other half were sacrificed at five weeks after the last rhG-CSFa administration.Urinalyses,blood biochemistry,hematological analysis,histopathological examination,and immunological tests were performed for each of the rats.Results The hematological analyses revealed that the mean white blood cells count,neutrophils count,and neutrophils percentage were increased in male rats at the dose of 10 μg/kg or higher,and these were related with the biological activity of rhG-CSFa.Some small abnormalities were observed in the spleen of a few rats when used highest dose (500 μg/kg,a dosage of 200 folds higher than the normal clinical dosage),but these abnormalities were recovered within 5-week recovery period.No other rhG-CSFa-related abnormalities were observed in this chronic toxicity study.Conclusion No significant toxicity and immunogenicity are observed with rhG-CSFa administration to rats in the chronic toxicity studies.
基金Supported by the Natural Science Foundation of Guangdong Province(No.2015A030310158No.2014A030313359)+1 种基金the Fundamental Research Funds for the Central Universities(No.21611446)the Scientific and Cultivation Foundation of the First Affiliated Hospital of Jinan University(No.2015201)
文摘AIM: To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS: Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus (CMV) 〉mutlumican/IRES/EGFP, respectively.RESULTS: The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION: We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.
文摘BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning. SETTING: Central Laboratory of Northern China Coal Medical College. MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT 1080 cells. X-gal stain was used to measure virus titer. MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus titer. RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosarcoma cells (HT1080) and the recombinant virus titer was measured as 1 ×10^12 virus particles per milliliter. CONCLUSION: Total-length cDNA fragment of NT-3 gene, which is obtained from pCDNA3-NT-3 plasmids, is closely matched to polyclone enzyme-shearing sites of adeno-associated viral vectors, while the combination can be used to construct recombinant adeno-associated viral vectors expression in hNT-3 gene.
基金Financially supported by Key grant from the Education Committee of Hunan Province (No. 02A046)
文摘Objectives: To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods: The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions (PCR), subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results: The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% (30/30) and a specificity of 100% (30/30). While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion: The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.