The Modification Of HIV-1Surface Antigen And Prokaryotic Expression Of The Broadly Neutralizing Antibody2F5and4E10

AIDS is a kind of serious infectious disease which is caused by humanimmunodeficiency virus. So far, scientists have not developed an effective vaccine, but havefound several broad-spectrum neutralizing monoclonal antibodies (monoclonal antibody,mAb), such as b12, VRC01,2F5,4E10and10E8. The extra cellular membrane proximalregion (membrane proximal external region, MPER) of gp41plays a very important role inthe fusion of viral and hosts cell membranes, and has relatively conserved sequence in thisregion. Broad-spectrum neutralizing antibodies2F5and4E10recognize epitopes located inapproximately30amino acid residues of gp41MPER. In pseudotyped virus neutralizationtests,2F5and4E10can neutralize most of viruses. Therefore, vaccine design based on MPERsite has become very popular.In order to study MPER antigen, we constructed eukaryotic recombinant plasmids,expressed and identified fusion proteins. MPER epitope was expressed in eukaryotic insectcells after plasmids and bacmid DNA being transfected into Sf9cells. Western blottingshowed that the recombinant protein MPER was correctly expressed in insect cells. In orderto detect the activity of MPER, we constructed single-chain antibodies2F5and4E10recombinant plasmids, expressed, purified and identified fusion proteins in this study. Underdenaturing condition, we used nickel-chelating chromatography method to purify inclusionbody proteins. Then, we got soluble single-chain antibodies after refolding. ELISA resultsshowed that single-chain antibodies2F5and4E10had ability to combine antigen GST-MPERand OD (2F5)-Fc. After combining2F5-scFv and4E10-scFv with gp120OD (MPER)-Fc, wefound that gp120OD (MPER)-Fc had binding activity, indicating that MPER had beenexposed in gp120OD. Thus, gp120OD (MPER)-Fc could be used for future immunization,and it laid foundation for getting neutralizing antibodies.In this study, we synthesized gene encoding the MPER antigen in vitro, and expressedMPER epitope with baculovirus/insect cell expression system. Gp120OD (MPER)-Fc hasmany neutralizing epitopes. It can be used as candidate immunogen for studing theimmunogenicity of HIV Env, and can also be used as an antigen to screen neutralizinganbodies. And it lays foundation for further designing recombinant vaccines.