1.High expression of the foot-and-mouth disease's structural protein P1 in Escherichia coli and analysis of its biology activity.
Xiao-Lan YU ; Shao-Bo XIAO ; Liu-Rong FANG ; Meng-Yu HU ; Lin YAN ; Xiao-Hui DONG ; Huan-Chun CHEN
Chinese Journal of Biotechnology 2005;21(1):163-166
Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.
Capsid Proteins
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biosynthesis
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genetics
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immunology
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Escherichia coli
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genetics
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metabolism
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Foot-and-Mouth Disease Virus
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genetics
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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immunology
3.Construction, expression and immunogenicity analysis of a Tat N-terminus-deleted mutant fusion protein of human immunodeficiency virus type 1.
Hua-Qun ZHANG ; Wen-Ting LIAO ; Qiu-Li CHEN ; Yi-Bing GE ; Jie YANG ; Ping-Ping ZHANG ; Pei-Pei QI ; Chao LIU ; Ting HE ; Jin-Hong WANG ; Wei PAN ; Jie CAO
Chinese Journal of Virology 2011;27(6):580-586
In the study, a gene encoding Tat protein N terminal 1- 21 amino acid residues-deleted mutant (Tat22-101) was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21(DE3) and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni(2+)-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the anti-sera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.
Animals
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Female
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Gene Products, tat
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biosynthesis
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genetics
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immunology
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HIV-1
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genetics
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immunology
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Humans
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Mice
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Mice, Inbred BALB C
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Mutant Proteins
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genetics
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immunology
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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immunology
4.Construction of the recombinant integrating shuttle plasmid with cfpl0-esat6 fusion gene of Mycobacterium tuberculosis and its expression in BCG.
Xiaoying WANG ; Lang BAO ; Mingcai ZHAO ; Huidong ZHANG ; Yang LONG
Journal of Biomedical Engineering 2006;23(6):1298-1302
This study was conducted to amplify the cfpl0-esat6 fusion gene by SOE and insert into the integrating shuttle plasmid pMV361 to form the recombinant plasmid. Then another recombinant plasmid was constructed by insertinga-A g signal sequence of BCG. The two recombinant plasmids were introduced into BCG and the induced products from recombinant BCG were analyzed. In conclusion,the successful construction of rBCG expressing the fusion protein CFP10-ESAT6 will be the base of the development of novel Mycobacterium tuberclosis vaccines.
Antigens, Bacterial
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biosynthesis
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genetics
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Bacterial Proteins
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biosynthesis
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genetics
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Mycobacterium bovis
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genetics
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metabolism
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Mycobacterium tuberculosis
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genetics
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Plasmids
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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immunology
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Tuberculosis Vaccines
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biosynthesis
5.Cloning, expression and purification of human nuclear autoantigenic sperm protein (hNASP) and preparation of its polyclonal antibody.
Min WANG ; Jian-Li SHI ; Guo-Yan CHENG ; Yan-Qin HU ; Chun-Meng LIU ; Chen XU
National Journal of Andrology 2006;12(10):867-871
OBJECTIVETo acquire the purified human nuclear autoantigenic sperm protein (hNASP) and its polyclonal antibody for investigating the possible functions of hNASP involved in fertilization.
METHODSThe coding sequence of hNASP gene was amplified from human testis RNA with specific primers, and the PCR product was cloned first into pMD-18T and then into pET-28a ( + ) after restriction digestion with BamH I and Hind III. The fusion protein was expressed in E. coli BL21 (DE3) after induction with IPTG. The recombinant protein NASP was purified from the supernatant with Ni2 -NTA His-bind resin under native conditions.
RESULTSThe results of DNA sequencing and SDS-PAGE analysis showed the protein to be what we had hoped to acquire. ELISA showed that we had acquired rabbit anti-hNASP serum with high titer.
CONCLUSIONHigh purity recombinant hNASP protein could be obtained with the above-mentioned prokaryotic expression method, and so could the rabbit anti-hNASP serum with high titer and high specificity.
Adult ; Animals ; Antibody Formation ; Autoantigens ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Humans ; Male ; Nuclear Proteins ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Reverse Transcriptase Polymerase Chain Reaction
6.Soluble expression and characterization of disulfide bond-rich subdomains of membrane protein p185 in Escherichia coli.
Liang-Wei LI ; Hai-Bo LIU ; Si-Yi HU ; Dun LIANG ; Lian-Sheng CHENG ; Jing LIU
Chinese Journal of Biotechnology 2005;21(4):590-596
Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain I-II and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E. coli Origami B (DE3) pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10-15 mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain I-II and domain IV of p185 ECD and this method may also be used to express other disulfide bond rich proteins.
Antibodies, Monoclonal
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immunology
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Disulfides
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immunology
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Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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genetics
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Humans
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Receptor, ErbB-2
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biosynthesis
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genetics
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immunology
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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immunology
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Solubility
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Transfection
7.Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus.
Yun-Bo JIANG ; Liu-Rong FANG ; Shao-Bo XIAO ; Hui ZHANG ; Huan-Chun CHEN
Chinese Journal of Biotechnology 2005;21(6):858-864
Pseudorabies virus (PRV), an alpha-herpesvirus, has been used as a vector for live-viral animal vaccines. The recombinant PRV TK- / gE- / GP5+, which expressing GP5 of PRRSV, is developed based on the PRV genetic-depleting vaccine-virus strain, TK- / gE- /LacZ+. However, this strain stimulated poorly the vaccinated animals to produce neutralizing antibodies against PRRSV. In order to develop a booster specific immunized response of the PRV recombinant, the ORF5 gene of PRRSV TK- / gE- / LacZ+ was substituted by a modified ORF5 gene, ORF5m. The resultant recombinant PRV, TK- /gE- / GP5m+, was verified by PCR, Southern blotting and Western blotting. TK- / gE- / GP5m+ and TK- / gE- / GP5+ expressed GP5 proteins were inoculated into balb/c mice to evaluate their immunogenicity. The results demonstrated that the amount of neutralization antibodies and cell-immunity responses induced by TK- / gE- /GP5m+ against PRRSV were higher than that of TK- / gE- / GP5+. This study indicated that the new recombinant PRV expressing the modified GP5m protein is a candidate for the development of bivalent genetic engineering vaccines against PRRSV and PRV.
Animals
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Genetic Vectors
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Herpesvirus 1, Suid
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genetics
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immunology
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Mice
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Mice, Inbred BALB C
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Porcine respiratory and reproductive syndrome virus
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genetics
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immunology
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Recombinant Fusion Proteins
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genetics
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immunology
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Recombinant Proteins
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biosynthesis
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genetics
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immunology
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Swine
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Viral Envelope Proteins
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biosynthesis
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genetics
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Viral Vaccines
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genetics
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immunology
8.Preparation and bioactivity evaluation of streptavidin-tagged human interleukin-15 fusion protein.
Hua SU ; Yan-Li CHEN ; Su-Yun CHEN ; Bo WEN ; Hong-Sheng YU ; Zhi-Ming HU ; Ji-Min GAO
Journal of Southern Medical University 2009;29(3):397-401
OBJECTIVETo obtain streptavidin-tagged human interleukin-15 (SA/hIL15) fusion protein and evaluate its bioactivity.
METHODSpET24a-6His-SA-hIL-15 and pET32a-hIL-15-SA-6His plasmids were constructed and expressed in BL 21(DE3) host bacteria to generate the fusion protein. The recombinant fusion protein IL-15/SA was purified using Ni-NTA affinity chromatography and refolded, and the efficiency of surface modification of the fusion protein on biotinylated cells was examined by fluorescence-activated cell sorting. CCK-8 method was used to evaluate the effect of IL-15/SA fusion protein in inducing the proliferation of human peripheral-blood lymphocyte (PBL) cells stimulated by PHA.
RESULTSThe recombinant SA-hIL-15 and hIL15-SA fusion proteins were highly expressed in BL21(DE3) at about 20% of the total bacterial proteins. The purified hIL15-SA fusion protein exhibited a bifunctionality by promoting the proliferation of PBL cells activated by PHA and high-affinity binding to biotinylated cell surface mediated by SA, with a cell surface modification efficiency exceeding 95%. SA-hIL-15 showed a 4-fold higher hIL15 bioactivity than hIL15-SA.
CONCLUSIONSA/hIL-15 bifunctional fusion protein has been successfully obtained to facilitate the future development of hIL-15-surface-modified cancer cell vaccine.
Cancer Vaccines ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Interleukin-15 ; biosynthesis ; genetics ; Lymphocyte Activation ; drug effects ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Streptavidin ; biosynthesis ; genetics
9.Cloning and efficient prokaryotic expression of soluble stage-specific antigen cC1 from Cysticercus cellulosae.
Qiang FANG ; Jiang-kun LUO ; Zhuo CUI ; Wen-juan QI ; Yuan-sheng HU ; Ji-long SHEN
Journal of Southern Medical University 2010;30(2):206-209
OBJECTIVETo clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli.
METHODSThe cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD18-T vector, followed by subcloning into the prokaryotic expression plasmid pET28a. The recombinant plasmid was transformed into E.coli BL21(DE3) and the expression conditions were optimized. The expressed product was purified by Ni(+)-affinity chromatography, analyzed by high-performance liquid chromatography (HPLC), and identified with SDS-PAGE and Western blotting.
RESULTSThe fragment length of the amplification product by RT-PCR was 1056 bp. Comparison of the amplified gene sequence with the cC1 gene in Genbank identified a samesense point mutation at 423 position in the gene cloned into the expression plasmids. After a 6-h induction with 0.05 mmol/L IPTG at 37 degrees celsius;, the expression of the 40 kd soluble fusion protein exceeded 60% of the total bacterial protein, and the fusion protein was recognized by Cysticercus-infected human sera. The purity of the fusion protein was about 94% after purification by affinity chromatography.
CONCLUSIONThe stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, which provides the basis for its further study and application.
Animals ; Antigens, Helminth ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Cysticercus ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Swine ; Taenia solium ; immunology
10.Evaluation of three in-vitro refolding methods for human-derived anti-CTLA4 scFv expressed in E. coli.
Qiang HUANG ; Lihong CHEN ; Lingyu ZENG ; Lin WAN ; Shengfu LI ; Xiaofeng LU ; Jingqiu CHENG
Journal of Biomedical Engineering 2006;23(2):388-391
To evaluate the efficiency of three in vitro refolding methods for a humanized single-chain Fv antibody against human CTLA4(CD152) expressed in E. coli, the denatured and purified inclusion bodies (IBS) were refolded by dilution, dialysis and in situ refolding via Immobilized Metal-Ion-Affinity Chromatography (IMAC), respectively. The concentration of refolded scFvs was examined by Bradford method. And the antigen binding activity of the refolded scFvs was analyzed by indirect cell-ELISA. The highest and lowest refolding yields could be obtained by dialysis and in situ refolding via IMAC, respectively. The binding activity of the refolded scFv by dialysis was 1.95-fold higher than that by dilution, 4.13-fold higher than that by in situ refolding via IMAC (GSH/GSSH excluded) and 3.63-fold higher than that by in situ refolding via IMAC (GSH/GSSH included), respectively. In conclusion, a high refolding yield and binding activity of scFv with natural conformation could be obtained by dialysis in the condition of 0. 15 mol/L sodium chloride, 50 mmol/L Tirs-HCl, pH 8. 0 buffer containing 3 mmol/L reduced glutathione and 1 mmol/L oxidized glutathione for 48 hours at 4 degrees C.
Antigens, CD
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biosynthesis
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genetics
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immunology
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Antigens, Differentiation
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biosynthesis
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genetics
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immunology
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CTLA-4 Antigen
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Cloning, Molecular
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Escherichia coli
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genetics
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metabolism
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Humans
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Immunoglobulin Fc Fragments
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biosynthesis
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genetics
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immunology
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Immunoglobulin Variable Region
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biosynthesis
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genetics
;
immunology
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Recombinant Fusion Proteins
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biosynthesis
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genetics
;
immunology