The Second Immunodominant Epitopes Of HIV-1 Gp21: Effcient Expression In E.coli

Objective It is the most important way to provide HTV-1 gp41 antigens of first-rate quality in the detection of HTV-1 infection, which can improve the quality of HTV-1 screening as well as the secificity and sesitivity in the application as reagent for anti-gp41 antibody test. The main obejective is to construct an efficiently expressive prokaryotic clone of the second immunodominant epitopes in HTV-1 gp41, detect its products and evaluate it in testing the anti-HTV-1 antibody.Methods Accoding to the principle of the codon degeneracy, we designed a pair of primers .Two base of the upper primers were subsituted , so as to mutate two rare codons into major codons for E.coli. Of course, we dont change the amino acids of the target protein and the specificity of the upper primer. Then, DNA fragments of HTV-1 gp41 (nt 9153-9233) encoding 27 amino acids ( gp160 aa 649-675) were obtained by PCR. The fragments and the pGEX-4T-2 were digested by EcoRI and Sail, recombined by T4 ligase, and then the recombinant plasmid pGEX-4T-2/SE was transformed into E.coli DH5 a . After being iduced with IPTG, the pGEX-4T-2/SE/DH5 a efficiently expressed the fusion protein, which was indentified by SDS-PAGE and Westen-blot.Results We succeeded in construct an expressive clone pGEX-4T-2/ SE/DH5 a , which can efficiently produce the second immunodominant epitopes -GST fusion protein after being induced with IPTG. The gel scan system reported that the target protein represented about 30% of the total bacterial proteins. Western- blot assay showed that the expressed products could react specificly with the antibody in the HTV-1 positive sera., noactivity was detected with the negative sera.Conclusion The second immunodominant epitopes of HIV-1 gp41 can be expressed efficiently in E.coli. The fusion protein has good antigenicity and could be a good candidate antigen for HTV-1 ELISA. Therefore, this research is useful for further discussion about how to improve expression of exogenous genes in E.coli.Postgraduate: Li wenli (Microbiology ) Directed by : Prof. Wang Bin...