Recombinant Hiv-1 Envelope Glycoprotein Gp120 And Gp41 Expression, Purification And Preliminary Applications

Human Immunodeficiency Virus (HIV) is the pathogen that causes acquired immunodeficiency syndrome in human. It can be categorized into two types: HIV-1 and HIV-2. HIV-1 is the main type. It's different from other viruses for its complicated genome related to the latency,activation,transcription and replication and for its variation in host cells.Envelope glycoproteins gp120 and gp41 are the main antigens of HIV-1 and are important components of diagnosis kits for detecting HIV antibodies. They can be used to produce gp120 and gp41 monoclonal antibodies. Considering their importance in applications, we have tried to express gp120 and gp41 in Pichia Pastoris and E. coli, respectively.Full length DNA of gp120 and DNA fragment of gp120 (called gp120L and gp120S) were obtained by PCR from the plasmid pHXB2-gpl60.The yeast recombinant plasmids pPICZ α A-gp120L and pPICZ α A-gp120S that contain the alcohol oxidase promoter (A0X1) and zeocin resistant gene were constructed. The recombinant plasmids were linearized before homologous recombination within the genome of yeast GS115 via electroporation. After the screening by a selective medium and the antibiotic zeocin, we obtained some positive transformants. These transformants were induced to express the target protein by adding alcohol into the medium. The expression product was detected by SDS-PAGE and Western blot. As a result, gp120S was expressed intracellularly at a low level.The truncated DNA fragment of gp41(called gp41s) was amplified by PCR and constructed into plasmid pET32a.The recombinant plasmid was transformed into E.coli BL21(DE3)plys. After the induction by IPTG, the expressed protein was detected by SDS-PAGE and Western blot. It's shown that the 35.5 kD fusion protein was mainly expressed in inclusion bodies with an amount that accounts to at least 10% of all E.coli proteins. We obtained very pure gp41s proteins (>90%) by washing,dissolving and refolding the inclusion bodies. Primary applications of gp41s in detecting antibodies in positive HIV serums by indirect ELISA showed that it can react with HIV antibodies. After immuning the mice three times with gp41s, the titer could reach 1:10000. Therefore the recombinant protein has some immunocompetence.In this study we tried to express gpl20 in Pichia Pastoris, and succeeded in expressing gp41 in E.coli. This study has laid some ground work for further researches and development of HIV diagnosis reagents and subunit vaccines for AIDS. It's also important for studying the properties of the main antigens of HIV.