Expression And Characterization Of Human Immunodeficiency Virus-1 Membrane Protein Gp140 From Mammalian Cells And Generation Of Neutralizing Monoclonal Antibodies

AIDS is an acquired immunodeficiency syndrome caused by HIV(Human Immunodeficiency Virus)infection.AIDS is prevalent in the world,and type 1 HIV virus mainly poses a serious threat to human health.As reported by World Health Organization,there are more than 70 million people who have been infected since the discovery of HIV-1.By the end of 2015,there were still more than 36 million AIDS patients worldwide.HIV-1 infection rate of 15 to 49 years old adults is as high as 0.8%.AIDS incidence of China was more than 40,000 people per year in 2012-2014.At present,highly active antiretroviral therapy(HAART)is able to inhibit the replication of the virus in people living with HIV and make a significant contribution to extending the life of patients.However,anti-transcription drugs need to be taken every day,and 37.74%of patients get side effects such as gastrointestinal reactions,abnormal liver function and others.HAART can not cut off the root of AIDS epidemic,while the vaccine research and development is the urgent affairs.HIV-1 envelope glycoprotein(Env)gp160 is hydrolyzed to gp120(surface glycoprotrin,SU)and gp41(transmembrane protein,TM)during maturation,and these two proteins are assembled by noncovalent bonds into heterodimers.Then three dimers assemble into mature trimer and rivet on the virus envelope.When the virus invade into the host cells,the CD4 binding site on gp120 will recognize the CD4 cell surface receptor molecule,triggering the close to cell membrane and virus-cell fusion.For this reason,envelope protein is an important point for preventing viral infection and an important target for HIV vaccine.The broadly monoclonal neutralizing antibodies N6,VRC01,b12,2F5 and other antibodies can neutralize multiple types HIV-1,and are directed to the CD4 binding site on gp120 or the MPER region on gp41.However,vaccines designed according to these epitopes have not yet achieved the desired results.RV144 is the first vaccine with the effect of reducing HIV-1 infection.In this experiment,canarypox virus vector expressing subtype E Env and subtype B Gag is used for priming inoculation,subtype B/E gp120 with aluminum adjuvant is used for boost inoculation.The vaccine protection rate maintain 31.2%after three and a half years.As reported,the body with a high level of blocking and binding antibodies may have the protective effect on HIV infection.Through the transformation of BG505 Env,it can be expressed soluble as approximate natural trimer in vitro.Recombinant envelope protein BG505 SOSIP.664 and B41 SOSIP.664 induced strong broad-spectrum neutralizing antibody in rabbits.These reports provide new directions and confidence in further research on HIV vaccines.This study aimed to construct the recombinant clone of HIV-1 envelope glycoprotein gp140,seek efficient expression methods in HEK293 and characterize the recombinant protein.Construct design based on HIV-1 NL4-3 gp140 included codon opimization,signal peptide replacement,flexible linker involvement and foldon utilization,and at last we got the clone of SOSIP(G4S)2 663CO-T4his.Besides,the helpful impact was confirmed that co-transfection with HIV-1 transactivator of transcription(Tat)can enhance the expression of gp140,and the best plasmid ratio of Tat/gp 140 was founded.After purified by Ni NTA chromatography,about 0.5 mg recombinant gp140 with a purify of about 70%could be gotton per liter medium.Afterwards,the antigenicity of this recombinant gpl40 was confirmed by ELISA.In electron microscopy image,the protein was observed as trimer.Purified gpl40 was emulsified with freund’s adjuvant to subcutaneously innoculate Balb/c mice,and the neutralizing capacity of mice serum increased gradually.At last,ten neutralizing antibodies was screened,and two of them could neutralize corss subtypes of HIV-1.In this research,the subtype B HIV-1 virus NL4-3 gp140 was cloned and engineered,transfection methods was optimized,to express envelope protein trimer efficiently in vitro and assess its antigenic activity and immunogenicity.Besides,we explore the feasibility of using HEK293 cells to express gp 140 as a recombinant protein vaccine,and to provide the foundation for the study of HIV-1 envelope protein structure,vaccine and drug design.