Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium Tuberculosis Heat Shock Protein 65

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69 % in total bacterial protein and 74.09 % in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.

Recombinant IgM expression in mammalian cells: A target protein challenging biotechnological production

For many years, the potential of immunoglobulin M (IgM) antibodies was not fully understood because of characteristics different to the well-known immunoglobulin G type like low target affinity, cross reactivity and complex protein structure. In the meanwhile IgMs have been positively evaluated for their use as therapeutic agent in the mucosal environment but also in serum to eradicate upcoming tumor cells and invading antigens. Therefore IgM class of antibodies will play a significant role in clinical applications but also diagnosis in the future. To evaluate the full potential of this kind of antibody molecules large amounts of high quality product will be needed. In this review the focus is set on the biotechnological aspect of producing IgM class antibodies recombinantly in mammalian cells. Current achievements in expression and purification of this molecule are highlighted and compared.

Expression of fluorescent tagged recombinant erythroferrone protein

Objective: To produce fluorescent tagged recombinant erythroferrone protein(ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone(ERFE) gene was fused to green fluorescent protein(eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5α and the eGFP-fused ERFE(ERFE_eGFP) protein was expressed in human embryonic kidney(HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE_eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE_eGFP fusion protein(approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE_eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.

Soluble Expression and Rapid Quantification of GFP-hepA Fusion Protein in Recombinant Escherichia coli

To establish a rapid quantification method for heparinase I during its production in recombinant Es-cherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C ter-minus of a green fluorescent protein mutant (GFPmut1). As a result, not only was the functional recombinant ex-pression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluores-cence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.

Susceptibility to AcMNPV and Expression of Recombinant Proteins by a Novel Cell Clone Derived from a Trichoplusia ni QAU-BTI-Tn9-4s Cell Line

It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.

Potential bone-inducing activity in vitro of recombinant human bone morphogenetic protein-7 from a CHO expression system

Objective: To express the recombinant human bone morphogenetic protein-7(rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine 2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results: rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.

An Exploration of the Optimal Induction Conditions for Recombinant Fusion Protein Expression

[ Objective] This study aimed to investigate the optimal induction conditions for expression of recombinant fusion protein. [ Method] The optimal ini- tial OD600, induction dose, induction duration, induction temperature, ampicillin concentration, liquid volume and other culture conditions were explored by using parallel fermentation experiments with different technical indicators, to improve the expression level of the recombinant fusion protein of IMPACT-CN pTYB11 vector. [Result] SDS-PAGE and western-blotting analysis show that the recombinant fusion protein has a high immunogenicity. [Conclusion] This study laid the foundation for further protein purification in diagnosis, production of diagnostic kits with protein and clinical application.

Construction of Recombinant Expression Plasmids Containing H and F Protein Genes of Canine Distemper Virus Isolated from a Mink and Their Expression in Prokaryotic Cells

[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prekaryotic cells as well as to study the reactogenieity of the expressed products. [ Method ] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subclonlng techniques were used to construct the cloning plasmids（pMD-18T-H and pMD-18T-F） and recombinant expression plasmids（pET28a-H and pET28a-F） ; SDS-PAGE and Western-blotting were adopted to verify whether the target proteins were successfully expressed. [ Result] The recombinant expression plasmids pET28a-H and pET28a-F containing H and F protein genes of Canine distemper virus isolated from a mink were successfully constructed, and both the expressed H and F proteins with respectively relative molecular mass of 31 400 and 38 200 produced positive reac- tion with the CDV standard positive serum. [ Conclusion] The H and F proteins expressed in prokaryotic cells were the same with the natural ones in terms of reac- togenicity, which can be utilized for diagnosis of a CDV＇s infection or for an epidemiological investigation. Meanwhile, they also provide a basis for developing ge- netically engineered subunit vaccines.

Expression and purification of the recombinant outermembrane protein Tp0453 of Treponema pallidum and its characterization of immuno-competence

To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

Astrocytic endothelin-1 overexpression impairs learning and memory ability in ischemic stroke via altered hippocampal neurogenesis and lipid metabolism

Vascular etiology is the second most prevalent cause of cognitive impairment globally.Endothelin-1,which is produced and secreted by endothelial cells and astrocytes,is implicated in the pathogenesis of stroke.However,the way in which changes in astrocytic endothelin-1 lead to poststroke cognitive deficits following transient middle cerebral artery occlusion is not well understood.Here,using mice in which astrocytic endothelin-1 was overexpressed,we found that the selective overexpression of endothelin-1 by astrocytic cells led to ischemic stroke-related dementia(1 hour of ischemia;7 days,28 days,or 3 months of reperfusion).We also revealed that astrocytic endothelin-1 overexpression contributed to the role of neural stem cell proliferation but impaired neurogenesis in the dentate gyrus of the hippocampus after middle cerebral artery occlusion.Comprehensive proteome profiles and western blot analysis confirmed that levels of glial fibrillary acidic protein and peroxiredoxin 6,which were differentially expressed in the brain,were significantly increased in mice with astrocytic endothelin-1 overexpression in comparison with wild-type mice 28 days after ischemic stroke.Moreover,the levels of the enriched differentially expressed proteins were closely related to lipid metabolism,as indicated by Kyoto Encyclopedia of Genes and Genomes pathway analysis.Liquid chromatography-mass spectrometry nontargeted metabolite profiling of brain tissues showed that astrocytic endothelin-1 overexpression altered lipid metabolism products such as glycerol phosphatidylcholine,sphingomyelin,and phosphatidic acid.Overall,this study demonstrates that astrocytic endothelin-1 overexpression can impair hippocampal neurogenesis and that it is correlated with lipid metabolism in poststroke cognitive dysfunction.

Identification,evolution,expression and protein interaction analysis of genes encoding B-box zinc-finger proteins in maize

The B-box(BBX)family of proteins consists of zinc-finger transcription factors with one or two highly conserved B-box motifs at their N-termini.BBX proteins play crucial roles in various aspects of plant growth and development,including seedling photomorphogenesis,shade avoidance,flowering time,and biotic and abiotic stress responses.Previous studies have identified many different BBXs from several plant species,although the BBX family members in maize are largely unknown.Genome-wide identification and comprehensive analysis of maize BBX(ZmBBX)expression and interaction networks would therefore provide valuable information for understanding their functions.In this study,36 maize BBXs in three major clades were identified.The ZmBBXs within a given clade were found to share similar domains,motifs,and genomic structures.Gene duplication analyses revealed that the expansion of BBX proteins in maize has mainly occurred by segmental duplication.The expression levels of ZmBBXs were analyzed in various organs and tissues,and under different abiotic stress conditions.Protein–protein interaction networks of ZmBBXs were established using bioinformatic tools and verified by bimolecular fluorescence complementation(BiFC)assays.Our findings can facilitate a greater understanding of the complexity of the ZmBBX family and provide novel clues for unravelling ZmBBX protein functions.

Cloning and expression of human arresten gene and effect of its recombinant protein on endothelial cell proliferation

To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.

Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein

Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

Effects of Astragalus membranaceus on Energy Metabolism and Expression of CNTF Protein in Skeletal Muscle of Exercise-induced Fatigue Rats

[Objectives]This study was conducted to investigate the effects of Astragalus membranaceus in different groups on energy metabolism and CNTF protein expression in skeletal muscle of exercise-induced fatigue rats.[Methods]Thirty-five clean male SD rats were randomly divided into a normal group,and low-,meddle-and high-dose groups of A.membranaceus aqueous solution,with 7 rats in each group.The low-dose,medium-dose and high-dose groups were given by gavage at 0.65,1.3 and 2.6 g/kg,respectively,while the normal group and the model group were given normal food and water.The weight of rats was observed.The contents of serum urea,lactate,muscle glycogen,liver glycogen and CNTF expression were detected.[Results]After modeling,compared with the normal group,the serum lactate and urea contents of rats in the model group significantly increased(P<0.01),while the muscle glycogen content(P<0.01)and liver glycogen content(P<0.05)of the skeletal muscle significantly decreased.Compared with the model group,the low-,meddle-and high-dose groups of A.membranaceus significantly reduced the levels of lactate and urea in serum(P<0.01),while the levels of muscle glycogen and liver glycogen in the skeletal muscle significantly increased(P<0.01,P<0.05).[Conclusions]This study provides a good research foundation for the treatment of exercise-induced fatigue using traditional Chinese herb A.membranaceus in modern clinical practice.

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line

Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZPl was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZPl was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZPl from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

Expression,purification and immunocharacteristics of recombination UreB protein of H.pylori

INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by Western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

The Prokaryotic Expression and Bioactivity of the Recombinant Red Fire Ant Venom Allergen Sol i 4

The sting of red imported fire ant （RIFA） could cause serious allergic response in fraction of people. These allergic reactions are mainly caused by its venom, especially venom allergen Sol i 1-4. To produce large amount of RIFA venom allergen Sol i 4 for diagnosis of RIFA allergy and allergen-specific immunotherapy, the gene encoding this protein was amplified and cloned into the prokaryotic expression vector pET43, la. The recombinant plasmid was used to transform competent cells and the recombinant proteins were expressed in E. coll. SDS-PAGE and Western blotting analysis indicated that high-level expression of Sol i 4 protein was successfully achieved. Allergenic activity analysis of the recombinant allergen Sol i 4 was then performed on rabbit. The result showed that the recombinant protein obtained had significant allergenic activity. It indicated that the recombinant allergen Sol i 4 of RIFA venom was successfully expressed in E. coli, which provided foundation for further developing therapeutic and diagnosis reagents of RIFA allergy.