Preparation Of Envelope Gp120 Of Human Immunodeficiency Virus Type 1 And Screening Of Neutralizing Monoclonal Antibody

Acquired immunodeficiency syndrome(AIDS),it is caused by the human immunodeficiency virus(HIV),which causes the body’s immune system to malfunction,leading to various complications.Since HIV was found in 1981,it has been nearly 40 years,but there is still no effective vaccine or drug can completely prevent and cure AIDS,and the global HIV infected cases are still growing every year.By the end of 2016,about 36.7 million people all around the worldwere infected with HIV,and about 1 million people worldwide died of HIV-related diseases in 2016.Therefore,the development of HIV vaccine or drugs becomes an urgent task.As HIV vaccine research,people’s viewpoint about HIV,from the initial pure tend to rely on the cellular immune response or rely on humoral immunity to produce broad-spectrum neutralizing antibodies,the two direction for HIV vaccine research,base on the people in the experience of failure before now,people recognize that the necessarity of combined cellular immunity with humoral immunity at the same time to have hope to develop safe and effective HIV vaccine.With the rapid development of modern biotechnology,more and more new techniques and methods have been applied to HIV research.Recent studies have also shown that HIV can be eliminated in human body,in addition to cellular immunity,it is more important that the body can produce broad spectrum neutralizing antibodies.It is also recognized that the HIV-1 membrane protein gp120 is the main neutralizing epitope of the broad-spectrum neutralizing antibody.In this study,we loaded 7 different types protein gene sequences in insect cell baculovirus expression vector pAcgp67B,including NL4-3-gp120,89.6 gp120,3074-gp120,MJ4-gp120,94 UG144-gp120,3088-gp120,3096-gp120,get the pAc-gp120 recombinant expression plasmid.And then using the insect baculovirus expression system,we get a successful and productive protein,then using SDS-PAGE,Western Blot,ELISA,AUC,HPSEC,etc,to identify characterof gp120.Then,using the NL4-3-gp120 protein,with the adjuvant,with a dose of 100 g,and two weeks as the immune cycle,the 6-week Balb/C female mice were immunized.At the same time,the serum of mice was collected before each immunization,and the.combination and neutralization titer of antibodies in serum of mice were monitored by ELISA and the neutralization experiment of Tzm-bl cells,the fusion screening of monoclonal antibodies was carried out after the neutralization of the mice with higher titer.In this study,9 neutralizing antibodies were successfully screened,8 of which were antibodies against the laboratory adapted strain NL4-3,and 10G6 was a relatively broad-spectrum neutralizing antibody that was screened.Then,by using Western Blot,ELISA,neutralization test,immunofluorescence and alanine scanning,the properties of these neutralizing monoclonal antibodies were identified.The results showed that the monoclonal antibody of 10G6 was affected by glycoprotein glycosylation type and identified the broad spectrum neutralizing antibody to gp120 V3 region.To sum up,this research use insect cell baculovirus expression system,successfully get protein gp120 of HIV-1,and through the protein immune mice,successful screening 8 strains specific neutralizing antibody and 1 strain of relatively broad spectrum mNAbs of 10G6.This indicates that gp120 protein has good immunogenicity,which can induce the body to produce antibodies with broad spectrum neutralization.This has laid a foundation for the future research of vaccines based on broad-spectrum and epitope and the design of immunogen.