The Production Of Monoclonal Antibody Of SHIV P27

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are classified to Lentivirus of Retroviridae. The genome of HIV and SIV is highly homologous and most closely in Lentivirus group.SHIV, SIV/HIV chimeric virus, was constructed by replacing the corresponding region of SIV and HIV.Now,SHIV/rhesus monkey model system is widely used in the research of acquired immunodeficiency syndrome (AIDS). Capsid protein p27 is one of the main antigen of SHIV and it's the necessary material in preparing p27 antibody .The p27 protein was used broadly and of great significance. The present study is intended to obtain highly purified protein of p27 by gene engineering and produce SHIV detection kit for anti-p27 antibody.A pair of PCR primers with two enzyme digested sites (EcoR I and Xho I) were designed according to the sequence of gag gene of SHIV89.6P reported by GenBank. The full length gene fragment of SHIV p27 was amplified by PCR, cloned into the pMD18-T vector and sequenced.Under the control of the bacteriophage T7 promoter ,the gag gene fragment of p27 was inserted into the pET32a vector in order to construct the pET32a-p27 recombined vector. After highly expressing in E.coli, the purified p27 protein was prepared by metal-ligand affinity chromatography(IMAC).The accuracy of the inserted gene and activity of the SHIV p27 protein were detected by SDS-PAGE and Western Blot.The result indicated that the fusion p27 protein with a single expected band in SDS-PAGE. Immunized Balb/c mice with purified p27 protein and fused the immunized spleen cells with SP2/0 cells by PEG. Hybridoma cell lines secreting anti-SHIV p27 antibody were established by limiting dilution. The monoclonal antibodies (mAbs) were produced in ascites of mice by injecting 5×106 hybridoma cells.The following results were obtained in this study:1. Detected with agarose gel electrophoresis,an apparent stripe in the PCR product was identified,which coincide with the anticipation of 763 bp of SHIV p27 gene fragment anticipated.2. By agarose gel electrophoresis of products of restriction endonuclease analysis for the recombinant clone pasmid T-27 and pET32a-p27 respectively,the results are correct. The fragment was coincided with the anticipated SHIV p27 by DNA sequencing.The above results indicated that the recombinant expression plasmid was constructed successfully.3. SDS-PAGE analysis showed that there was an apparent stripe between 43Ku and 66.2Ku.The stripe was the product of E.coli transformed by the recombinant expression plasmid pET32a-p27 and induced by IPTG, The result indicated that the fusion p27 protein with a single expected band about 46Ku coincided with the anticipation.4. After purifying by Ni2+ affinity chromatography, the final p27 protein purification could reach 90%.It shows that the high purity intended protein had obtained.5. By using the purified recombinant p27 as antigen, five hybridoma cell lines anti-p27 antibodies were obtained(named 1A5,1C7,3C2,2D12 and 3D12 ). Western Blot and IFA detection showed that five monoclonal antibody strains have good specificity.Above all, five hybridoma cell lines anti-SHIV p27 antibodies were established in this study. It demonstrated that five hybridoma cell lines anti-SHIV p27 antibodies can detect SHIV virus in vitro. This study lays a firm foudation for developing confirmation test kit and the research about AIDS.