Recombinant Expression Of HIV-1 Main Antigens In Pichia Pastoris And Escherichia Coli

Human immnodeficiency virus (HIV) is the pathogen of acquired immunodeficiency syndrome (AIDS). Since the first report of ADIS in 1981, it has been spread rapidly and widely in the world. The main reason is that the genome of HIV is very complex and highly variable, which leads to great difficulty in the research of diagnosis reagents and vaccines. After the infection of HIV, p24 is the first detectable protein, and then the antibodies stimulated by envelope glycoprotein gp120 and gp41 can be examined. In the new-generation diagnosis kits for detecting HIV antibodies, the coated antigen must be contained more components, including gp120, gp41 and p24 of HIV-1 antibody as well as gp36 of HIV-2. Considering the features of coated antigen, p24 gene was integrated into Pichia pastoris strain GS115 while gp120 and gp41 genes were transformed into E. coli BL21 and M15 respectively for expression using gene recombinant technique.The full-length p24 gene was amplified by PCR from the plasmid pT24, that later was inserted into the secretory expression vector pPIC9 to construct the yeast recombinant vector: pPIC924. Linearized recombinant plasmid by SacI was transformed into the yeast strain GS115 by electroporation. After screening by selective media MM and MD, PCR was performed to confirm the positive transformants, the integration rate is 83% and has good genetic stability. The positive transformants were induced to express the interest protein by adding methanol into the medium, and the culture supernatant was analyzed by SDS-PAGE and Western blotting. As a result, p24 was expressed correctly in the yeast with the expression level of 55.6mg/L. After purification by ion-exchange chromatography, the bio-activity of recombinant p24 was determined by indirect ELISA to examine HIV serums and the result showed that the re-p24 possessed satisfactory antigen specificity.The truncated DNA fragment of gp120 gene, which have strong antigenicity of full-length gp120 gene was amplified from plasmid pT120 and was later inserted into vector pET28. The recombinant vector (p120s) and pT41 were transformed into E. coli BL21 and M15 respectively. After the expression induced by IPTG, the SDS-PAGE and Western blot were performed to check the target proteins. The results demonstrated that gp120s was expressed as soluble form and the yield was up to 17.9% of total cellular proteins. Gp41 was expressed as inclusion bodies with amount of 24.6% of total E. coli proteins. After washing and denaturing inclusion body steps, the refolding rate of denatured inclusion body is 14.1%. The nickel-chelating chromatography was used to purify these two proteins which expressed inbacteria, the purity reach to 63.1% and 89%, respectively. Also, the ELISA results showed that these two recombinant proteins had good reactogenicity.In this study, the important HIV antigens p24, gp120s and gp41 were successfully expressed in yeast and E. coli systems. And all the recombinant proteins showed good antigen specificity. These studies can supply some basis for further research on the properties of main antigens, and also provide evidence for the development of HIV diagnosis reagents and subunit vaccines.