Preparation Of HIV-Tat Protein And Human Anti-Tat ScFv And Some Research On Their Biological Activity

Human immunodeficiency virus(HIV)is pathogens of acquired immune deficiency syndrome(AIDS). HIV-1 encodes 3 structure gene and 6 regulating gene and Tat is one of the positive regulatory protein.The provirus genome could not transcribe completely in the absence of Tat protein. Tat protein in the cytoplasm transduced into the cell nucleus and interacts with transactivating responsive element (TAR),which trans-activated the transcription to product integrated mRNA.TAR was a part of Long Terminal Repeat (LTR) of the new virus RNA. Tat protein could be secreted out of infected cells and suppressed immune system. This promoted the infection of HIV and increased possible diseases correlated to AIDS,such as Kaposi's sarcoma, AIDS dementia and other diseases.Tat protein had become a new target for the research on treating AIDS.In early studies, the monoclonal antibodies (mAbs) against Tat were usually used to block the expression of Tat and control the disease's progression. However, mouse-derived monoclonal antibodies produced Human Anti-Mouse Antibody (HAMA) response after repeated use that limited its clinical use. Single-chain antibody (scFv) significantly reduced the HAMA response and its small molecular weight made scFv with strong organizations penetration, short plasma half-life and other advantages. We prepared Tat fusion protein by genetic engineering technology and obtained human anti-HIV-Tat scFv by screening from Tomlinson(I+J)semi-synthetic phage antibody library.We detected some immune and biological activity of anti-Tat scFv which established foundation for the research of anti-HIV drugs.We synthesized the Tat gene and connected it to the PET-32a vector to build PET-32a-Tat recombinant. Then the plasmid was transducted into E. Coli BL21 (DE3) competent cells. It was successfully induced to express Tat fusion protein. After series of purification, we got the Tat fusion protein in the purity of more than 95 percent.We used Tomlinson (I + J) semi-synthetic phage antibody library to clone human anti-HIV-Tat scFv. Since the basic region of Tat protein is highly conservative, we synthesized short peptide by the amino acid sequence RKKRRQRRR. The synthetic short Tat peptide was coated on 96-well plate as antigen, the phage antibodies against Tat were screened by four times'combining-eluting-amplifying using phage diplay technique.Through retrieval database from Kabat, we found that its light and heavy chain variable region belonged to VκⅠand VHⅢtype respectively.The soluble scFv protein were expressed after induced by IPTG. Besides, we tried different expression conditions and optimized them. At last, we determined the best condition(1mmol/L IPTG,30℃,24h and pH7.2) , which established foundation for mass expression.We used different chromatography ways to purify scFv protein secreted by bacteria. In the end, we got the final purification of the technical line: first, salting-out;second, Protein A; third, Chelating Cu; last, dialysis. The results showed that this method was suitable for purifying the objective protein.The immunologic property and biologic activity of the scFv protein were assessed by ELISA, Western blotting , Immunocytochemistry and Cytotoxic experiments.The results showed that the scFv protein could specially bind the fusion Tat protein outside. The anti-Tat scFv protein could prevent Tat fusion protein entering cells. It also could suppress the cytotoxic effects on Jurkat cells caused by Tat protein. Moreover, this anti-Tat scFv protein had no toxity on EC-304 and HeLa cells.We successfully constructed PET-32a-Tat recombinant plasmid and obtained highly expressed Tat fusion protein in E.Coli BL21 (DE3). After series of purification, we got highly purified Tat fusion protein. Besides, we successfully screened a human anti-HIV-Tat protein scFv from Tomlinson (I + J) semi-synthetic phage antibody library. This scFv protein could be expressed by E.coli HB2151. Purified scFv protein had good biological activity which would lay the foundation for its clinical application.