Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon a2b

Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.

Use of recombinant human bone morphogenetic protein-2 in spine surgery

Bone morphogenetic proteins are osteoinductive factors which have gained popularity in orthopaedicsurgery and especially in spine surgery. The use of recombinant human bone morphogenetic protein-2 has been officially approved by the United States Food and Drug Administration only for single level anterior lumbar interbody fusion, nevertheless it is widely used by many surgeons with off-label indications. Despite advantages in bone formation, its use still remains a controversial issue and several complications have been described by authors who oppose their wide use.

PURIFICATION OF RECOMBINANT HUMAN INTERFERON-γ BY IMMUNOAFFINITY CHROMATOGRAPHY WITH MONOCLONAL ANTIBODY

E.coli cells expressing recombinant human interferon-γ was disrupted by sonication anddissolved in 7mol·L-1 guanidine hydrochloride.The extract obtained was then renaturated by 70 folddilution with PBS.HulFN γ was purified by affinity chromatography with monoclonal antibody fromthe renaturated crude feed solution.After washing the column with PBS,the adsorbed HulFN γ waseluted with PBS containing 0.5mol·L-1 NaCl.The column was regenerated with 2mol·L-1 GuHClfor reuse.After one step of affinity purification the purity of interferon-γ was over 95%.and thespecific activity of the HulFN-γ reached 1.2×107 IU·mg-1 protein.92.8% of recovery was obtainedin the elution step.Total recovery of HulFN γ activity in the affinity chromatography was 78%.

Neuronal changes in the retinal ganglion cell layer following recombinant human interleukin-2 intravitreal injection in a rat model of chronically elevated intraocular pressure

Intraperitoneal injection of recombinant human interleukin-2（rhIL-2）inhibits neuronal apoptosis in the chronic ocular hypertension retinal ganglion cell layer.Intravitreous injection was performed on retinal ganglion cells in a Wistar rat model of chronically elevated intraocular pressure to observe the effects of LY294002 and AG490 on retinal ganglion cell survival,macrophage activation,and PI3K/Akt and JAK/STAT activation.The number of retinal ganglion cells in the rhIL-2 treatment group was much greater than in the normal control and phosphate-buffered saline groups.Western blot analysis revealed low Akt and STAT3 protein expression in the retina after 3-hour intravitreous injections of rhIL-2.However,protein expression was increased at 12 hours,but decreased again at 24 hours,with very low expression at 96 hours.LY294002 and AG490,which are inhibitors of the PI3K/Akt and JAK/STAT3 signal pathways,prevented upregulation of Akt and STAT3 protein expression in the retina,respectively.Intravitreous injection of rhIL-2 exhibited neuroprotective effects by decreasing retinal ganglion cell layer damage in a rat model of chronic glaucoma.These results suggest that intravitreal injection of rhIL-2 could induce the PI3K/Akt and JAK/STAT3 signaling pathways to protect retinal ganglion cells in chronically elevated intraocular pressure models.

Implantation of xenogeneic bone combined with recombinant human bone morphogenetic protein-2 into bone defect—An scanning electron microscopic study

To determine the ability of a new type of composite xenogeneic bone grafting to repair bone defect. Methods: The new type of composite xenogeneic bone was obtained by combining the chemically treated cance1lous bone with recombinant human bone morphogenetic protein-2 (rhBMP-2). It was implanted on the bone defect of rabbit. Results: There was a large amount of new bone formation within the combined material and the amount was increasing as the time elapsed. In contrast, there was a lot of fibrous tissue with a little new bone formed on the area of the bone defect when the treated cancellous bone was implanted alone. Conclusion: The results imply that the rhBMP-2 plays a very important role in new bone formation and the composite xenogeneic bone appear to be an ideal material for repair of bone defect.

Study on pharmacodynamics of recombinant interferon α-2b suppository

Objective To investigate the antiviral activity of recombinant interferonα-2b suppository(IFNα-2b)in vivo and in vitro.Methods The cytopathic-effect inhibition assay was applied in this study to investigate the antiviral activity of this drug as well as yingtelong and axiluowei as positive control.The guinea pig model of vaginitis and skin infection caused by HSV-2 infection were established,treated with IFNα-2b suppository at dosages of 60000、180000、540000 IU,using IFNα-2b injection 180000 IU·kg-1 as controls.Score the pathological changes of appearance and skin,the virus activities of vaginal secretion and tissue sections of viginae were assayed after treatment.Results The TD50 of IFN α-2b and yingtelong for Vero cells was(>100)μg·mL-1 and(>100000)IU·mL-1,respectively.The IC50 of IFN α-2b and yingtelong and axiluowei for Herpes virus type 1 was(0.29±0.08)μg·mL-1 and(185.0±28.8)IU·mL-1 and(0.19±0.03)μg·mL-1,respectively.The mean scores for vaginal and skin lesion of the treated groups were lower than those of untreated group.Among these concentrations,the IFNα-2b suppository of 540000 IU·kg-1 group.Showed highest anti-viral activity.The virus activity in vaginal secretion of treated group was lower than that of untreated group too(P<0.01 or P<0.05).Tissue sections of viginae after treatment with IFNα-2b suppository showed significantly therapeutical effects on the degrees of vaginal lesion.At the same dosage,The anti-HSV activity of IFNα-2b suppository was also compared with IFNα-2b injection,the results showed that the activity of suppository of 540000 IU·kg-1 group was similar to that of the injection.Conclusions The IFNα-2b suppository has anti-viruses function both in vivo and in vitro.

SYNTHESIS OF INTERFERON-α_A MONOCLONAL ANTIBODY PACKING MATERIAL IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PURIFICATION OF RECOMBINANT HUMAN INTERFERON-α_A

A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.

CORAL AS A CARRIER FOR RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2

By combining coral with recombinant human bone morphogenetic protein-2 (rhBMP-2), rhBMP-2/coral composite was obtained in this study. Following implantation of the composite into the muscle pouches of mice, cartilage growth was induced in the pores or on the surface of the implants at one week, woven bone at three week and lamellar bone with bone marrow at six week, and coral was absorbed partially. The induced formation of endochondral bone was time-related and rhBMP-2 dose-related. The results of this study indicate that the composite possesses a superior ability of osteogenesis, and coral acts as one of the most suitable rhBMP-2 slowrelease carriers currently available. The composite will be a new type of bone substitute to be used in orthopaedics and maxillofacial surgery.

SYNERGIC CYTOTOXICITY TO GASTRIC CANCER CELLS BY COMBINED USE OF TRICHOSANTHIN ANDRECOMBINANT INTERFERON α-2B

Objective To investigate a new approach of the combined use of trichosanthin (TCS) andrecombinant interferon alpha - 2b (rIFN α- 2b) against digestive system cancer cells. Methods Detect separatelythe cytotoxicity of TCS, rIFN α- 2b and their combination against digestive system cancer cell SGC- 7901.Results In the experiment in vitro, TCS, rIFN α- 2b both had direct, dose dependent cytotoxicity againstSGC - 7901. Their combined use demonstrated a toxicity signijicantly higher than that of the two drugs used alone,showing a signilicant synergic effect. This synergic cytotoxicity was confirmed in the animal experiment.Conclusion Combined use of TCS and rIFN α - 2b decreases the therapeutic dose of TCS and its toxic adverseellect, and this synergic effect is favorable to the clinical use of TCS protein against gastric cancer.

Expression of Human Interferon α 2b Gene in Ginseng Cells

The human interferon α 2b（hIFN-α2b） gene was cloned into binary vector pBI121 to obtain plant expression vector pBIFN. The recombinant plasmid pBIFN was transferred into Agrobacterium tumefaciens strain LBA4404. Then the hIFN-α2b gene was introduced into ginseng callus cells via Agrobacterium-mediated transformation and the ginseng cell line carrying hIFN-α2b gene was selected on G418-containing medium. The presence of target gene in transformed cells was confirmed by PCR and RT-PCR. The results indicate that hIFN-α2b gene has been integrated into the ginseng cells＇ genome, with transcription products, hIFN-α2b expressed by the transgenic ginseng cells was detected by Western blot. It was shown that a specific protein band at 19000 could be observed. Cytopathic effect（CPE） inhibition assay using the W1SH-VSV system shows that the mean antiviral activity of expressed hlFN-a2α was 6.0× 10^4 IU/mL.

The Concept Study of Recombinant Human Soluble Thrombomodulin in Patients with Acute Respiratory Distress Syndrome

Background: Recombinant human soluble thrombomodulin (rhTM) was approved for the treatment of disseminated intravascular coagulation in Japan, and rhTM has anti-inflammatory effects. Disordered coagulation is a part of the acute respiratory distress syndrome (ARDS) pathophysiology and thus we hypothesize that anticoagulant therapy may help. This preliminary study was to observe the safety of rhTM administration and the improvement on biomarker levels after the therapy for ARDS-patients. Objectives: Case series of ARDS-patients. Methods: Seventeen ARDS-patients that required ventilatory management were treated with rhTM and clinical and laboratory data were collected including platelets, thrombin-antithrombin complex (TAT), fibrinogen degradation products, oxygen saturation/the fraction of inspired oxygen (SpO2/FIO2), and high-mobility group-1 (HMG-1). The administration of rhTM was started during 6 days at a bolus dose of 0.06 mg/kg/day immediately after the diagnosis of ARDS. Results: Eleven of the 17 ARDS-patients were alive at 28 days after the beginning of the administration of rhTM. The serial pattern of the SpO2/FIO2 showed remarkable differences between the survivors and nonsurvivors from day 5 to day 7. The TAT in the survivors significantly decreased after treatment, and there were significantly lower levels in the TAT on day 7 in comparison to that of the nonsurvivors. The serial changes of HMG-1 showed increased levels in the nonsurvivors until day 5 after the administration of rhTM. Conclusions: Additional rhTM administration can safely improve the parameters in survival ARDS-patients, as demonstrated by significant improvements in the SpO2/FIO2, HMG-1 and TAT.

Regulating the bioactivity of non-glycosylated recombinant human bone morphogenetic protein-2 to enhance bone regeneration

Recombinant human bone morphogenetic protein-2(rhBMP-2)is the predominant growth factor that effectively induces osteogenic differentiation in orthopedic procedures.However,the bioactivity and stability of rhBMP-2 are intrinsically associated with its sequence,structure,and storage conditions.In this study,we successfully determined the amino acid sequence and protein secondary structure model of non-glycosylated rhBMP-2 expressed by an E.coli expression system through X-ray crystal structure analysis.Furthermore,we observed that acidic storage conditions enhanced the proliferative and osteoinductive activity of rhBMP-2.Although the osteogenic activity of non-glycosylated rhBMP-2 is relatively weaker compared to glycosylated rhBMP-2;however,this discrepancy can be mitigated by incorporating exogenous chaperone molecules.Overall,such information is crucial for rationalizing the design of stabilization methods and enhancing the bioactivity of rhBMP-2,which may also be applicable to other growth factors.

重组人干扰素α2b和拉米夫定序贯治疗用于乙肝免疫耐受期患儿的临床研究

目的观察重组人干扰素α2b(recombinant human interferon-α2b,rhIFN-α2b)和拉米夫定序贯治疗用于乙肝免疫耐受期患儿的效果。方法选择西安医学院第三附属医院2020年4月至2023年4月收治的96例乙肝免疫耐受期患儿为研究对象,按随机数字法分为研究组与对照组各48例。研究组男29例,女19例;年龄(7.54±1.36)岁;病程(2.25±0.54)年;有乙肝家族史28例。对照组男26例,女22例;年龄(7.63±1.41)岁;病程(2.44±0.51)年;有乙肝家族史26例。对照组口服拉米夫定片,0.1 g/次,1次/d,治疗24周。研究组采用rhIFN-α2b和拉米夫定序贯治疗:前4周单用rhIFN-α2b肌内或皮下注射,5 mIU/(m^(2)·次),1次/2 d,4周后加用拉米夫定,0.1 g/次,1次/d,持续治疗8周后停用rhIFN-α2b仅用拉米夫定,继续治疗12周。比较两组疗效;治疗4周、12周、24周后,记录两组乙肝病毒E抗原(hepatitis B virus E antigen,HBeAg)、乙肝病毒脱氧核糖核酸(hepatitis B virus DNA,HBV-DNA)转阴率和乙肝病毒E抗体(hepatitis B virus E antibody,HBeAb)转换率;治疗前及治疗24周后,比较两组肝功能[谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspertate aminotransferase,AST)、总胆红素(total bilirubin,TBil)]及肝纤维化指标[透明质酸(hyaluronic acid,HA)、层黏蛋白(laminin,LN)、Ⅲ型前胶原肽(type Ⅲ procollagen peptide,PⅢP)、Ⅳ型胶原(type Ⅳ collagen,CⅣ)];记录两组不良反应发生情况。采用t检验、χ^(2)检验进行统计学分析。结果研究组总有效率高于对照组[77.08%(37/48)比52.08%(25/48)](P<0.05)。治疗4周及12周后,研究组与对照组HBeAg、HBV-DNA转阴率比较差异均无统计学意义(均P>0.05),治疗24周后,研究组HBeAg、HBV-DNA转阴率均高于对照组(均P<0.05);两组治疗后不同时间点HBeAb转换率比较差异均无统计学意义(均P>0.05)。治疗24周后,两组ALT、AST、TBil均降低,且研究组[(39.16±7.51)U/L、(41.92±8.26)U/L、(15.13±4.29)μmol/L]均低于对照组[(47.38±8.02)U/L、(52.36±8.73)U/L、(18.67±4.86)μmol/L](均P<0.05)。治疗24周后,两组HA、LN、PⅢP、CⅣ均降低,且研究组[(113.57±30.16)μg/L、(96.41±29.05)μg/L、(78.14±20.96)μg/L、(90.26±26.31)μg/L]均低于对照组[(208.34±64.72)μg/L、(124.27±32.19)μg/L、(104.37±22.48)μg/L、(143.75±33.49)μg/L](均P<0.05)。两组不良反应发生率比较差异无统计学意义(P>0.05)。结论rhIFN-α2b和拉米夫定序贯治疗在乙肝免疫耐受期患儿中疗效确切,能有效减少病毒含量,阻止肝功能恶化及肝纤维化,且安全性较高。

Expression and Significance of SHP-2 in Human Papillomavirus Infected Cervical Cancer

This study investigated the expression and prognostic value of SHP-2 in cervical cancer caused by human papillomavirus (HPV) infection. Forty-five specimens from patients with cervical cancer (stageⅠ-Ⅲ), 32 specimens from patients with cervical intraepithelial neoplasia (CIN) (Ⅰ, Ⅱ) and 20 normal cervical samples from patients with hysteromyoma were collected in Department of Pathology for comparison. The expression levels of SHP-2 and IFN-β proteins were detected by using immunohistochemistry. The mRNA expression level of SHP-2 was detected by using quantitative real-time polymerase chain reaction (PCR). HPVs were detected by HPV GenoArray Test. The Spearman correlation was used to compare the expression level of SHP-2 in HPV infected cervical cancer vs non-HPV infected normal cervix. The level of SHP-2 protein expression in the cancer tissues (88.8%) was significantly higher than in CIN tissues (62.5%) and normal cervixes (45%) (P<0.05 and P<0.05, respectively). The SHP-2 mRNA levels in the cancer tissues were upregulated as compared with those in the normal cervixes (P<0.05). Twenty-one (46.7%) cervical cancers, 25 (78.1%) CINs and 17 (85%) normal cervixes showed IFN-β positive staining in cytoplasm. There was statistically significant difference in the expression rate of IFN-β between cervical cancer and normal cervix (χ2=8.378, P<0.05) as well as between cervical cancer and CIN (χ2=7.695, P<0.05). HPV16/18 infections could be found in normal cervixs (15%), CINs (68.7%) and cervical cancers (84.4%). There was a correlation between HPV infection and SHP-2 expression in cervical cancer (rs=0.653, P<0.05). SHP-2 may be a useful prognostic and diagnostic indicator for HPV infected cervical cancer. In cervical cancers, SHP-2 mRNA and protein overexpression was associated with IFN-β lower-expression.

Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations (】 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION :Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Preparation and in vitro studies of microencapsulated cells releasing human tissue inhibitor of metalloproteinase-2

Objective： To prepare microencapsulated cells releasing human tissue inhibitor ofmetalloproteinase-2 （TIMP-2）, and investigate their biological characteristics in vitro. Methods： Chinese hamster ovary （CHO） cells were stably transfected with a human TIMP-2 expression vector, encapsulated in barium alginate microcapsules and cultured in vitro. Morphological appearance of the microcapsules was observed under a light microscope. Cell viability was assessed using MTT （3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide） assay. Enzyme linked immunosorbent assay （ELISA） and reverse zymography were used to confirm the release of biologically active TIMP-2 from the microcapsules. Cryopreservation study of the microencapsulated cells was carried out using dimethyl sulfoxide （DMSO） as preservative agent. Results： The microcapsules appeared like a sphere with diameter of 300-600 ~tm. The surface of the capsule wall was clearly smooth. The microencapsulated cells survived well and kept proliferating over the 6 weeks observed. No significant difference in TIMP-2 secretion was found between encapsulated and unencapsulated cells. Reverse zymography confirmed the bioactivity of MMP （matrix metalloproteinase） inhibition of TIMP-2. The cryopreservation process did not damage the microcapsule morphology nor the viability of the cells inside. Conclusion： Microencapsulated engineered CHO cells survive at least 6 weeks after preparation in vitro, and secrete bioactive TIMP-2 freely from the microcapsules.

Chimeric oncogenic interferon regulatory factor-2 (IRF-2): Degradation products are biologically active

Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immunomodulation, hematopoietic differentiation, cell cycle regulation and oncogenesis. We have done molecular sub-cloning and expression of recombinant murine IRF-2 as GST (Glutathione-S-Transferase)- IRF-2 fusion protein in E. coli/XL-1blue cells. Recombinant IRF-2 with GST moiety at N-terminus expressed as GST-IRF-2 (~66 kd) in E. coli along with different low molecular mass degradation products revealed approximately 30, 42, 60 and 62 kd by SDS-PAGE and Western blot, respectively. We further confirm that degradation takes place at C-terminus of the fusion protein not at N-terminus as anti-GST antibody was detecting all bands in the immunoblot. The recombinant IRF-2 was biologically active along with their degradation products in terms of their DNA binding activity as assessed by Electrophoretically Mobility Shift Assay (EMSA). We observed three different molecular mass DNA/protein complexes (1 - 3) with Virus Response Element (VRE) derived from human Interferon IFNβ gene and five different molecular mass complexes (1 - 5) with IRF-E motif (GAAAGT)4 in EMSA gel. GST only expressed from empty vector did not bind to these DNA elements. To confirm that the binding is specific, all complexes were competed out completely when challenged with 100-X fold molar excess of IRF-E oligo under cold competition. It means degradation products along with full-length protein are able to interact with VREβ as well as IRF-E motif. This means degradation products may regulate the target gene (s) activation/repression via interacting with VRE/IRF-E.

含重组人碱性成纤维细胞生长因子和重组人骨形态发生蛋白-2骨水泥在骨质疏松性腰椎压缩性骨折治疗的应用价值

目的:探讨含重组人碱性成纤维细胞生长因子(recombinant human basic fibroblast growth factor,rhbFGF)和重组人骨形态发生蛋白-2(recombinant human bone morphogenetic protein-2,rhBMP-2)骨水泥在骨质疏松性腰椎压缩性骨折(osteoporotic vertebral compression fracture,OVCF)患者经皮椎体后凸成形术(percutaneous kyphoplasty,PKP)治疗的应用价值。方法:回顾性分析2018年1月至2021年1月收治的103例行PKP手术治疗的OVCF患者,男40例,女63例;年龄61~78(65.72±3.29)岁。受伤原因:滑倒33例,跌倒42例,提重物受伤28例。根据填充骨水泥不同分为3组:磷酸钙组34例,男14例,女20例,年龄(65.1±3.3)岁,填充磷酸钙骨水泥;rhBMP-2组34例,男12例,女22例,年龄(64.8±3.2)岁,填充含rhBMP-2的骨水泥;rhbFGF+rhBMP-2组35例,男14例,女21例,年龄(65.1±3.6)岁,填充含rhbFGF和rhBMP-2的骨水泥。比较3组Oswestry功能障碍指数(Oswestry dysfunction index,ODI)、骨密度、椎体前缘丢失高度、伤椎前缘压缩率、疼痛视觉模拟评分(visual simulation score,VAS)及再骨折发生率。结果:所有患者获得12个月随访。3组术后ODI、VAS呈下降(P<0.001),骨密度增高(P<0.001),椎体前缘丢失高度、伤椎前缘压缩率呈先下降后缓慢上升趋势(P<0.001),rhbFGF+rhBMP-2组术后第1、6、12个月ODI、VAS均低于rhBMP-2组和磷酸钙组(P<0.05),术后第6、12个月骨密度大于rhBMP-2组和磷酸钙组(P<0.05)。rhbFGF+rhBMP-2组术后第6、12个月椎体前缘丢失高度、伤椎前缘压缩率均低于rhBMP-2组和磷酸钙组(P<0.05)。3组再骨折发生率比较差异无统计学意义(P>0.05)。结论:含rhbFGF和rhBMP-2骨水泥可更有效地增加OVCF患者骨密度,获得术后满意的临床和放射学效果,显著改善临床症状。

Development of novel interferon alpha2b muteins and study the pharmacokinetic and biodistribution profiles in animal model

Novel human interferon alpha 2b (hIFNα2b) muteins were developed by substituting cysteine residue (C) at positions 2 and 99 with aspartic acid residues (D). The mutein forms were then studied for pharmacokinetic profile. In addition, the influence of charge on the protein structure was tested in vivo for the biodistribution pattern. Codon substitutions were performed by Polymerase Chain Reaction (PCR)-based site-directed mutagenesis on a previously constructed synthetic hIFNα2b open reading frame (ORF) cloned in pET32b expression plasmid. The result of nucleotide sequencing analysis confirmed that all codons were replaced successfully without any additional mutation. Three mutant forms of hIFNα2b ORF were overexpressed in Escherichia coli BL21 (DE3) resulted in three muteins: hIFNα2b C2D, hIFNα2b C99D, hIFNα2b C2D C99D. To follow the kinetic and localization of the mutein interferon after intravenous administration, Tc99m was used to label the proteins. In particular of elimination half-life, it was shown that hIFNα2b C2D C99D > hIFNα2bC2D > hIFNα2bC99D > wild type. hIFNα2b C2D C99D mutein showed highest blood accumulation after 30 minutes administration. Taken together, the charge of hIFNα2b seems to be responsible for the fate of hIFNα2b in vivo.

构建过表达TRPV1基因的Caco-2细胞株

采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.