Immune response and monoclonal antibody screening of Human Immunodeficiency Virus V3 loop fused to CRM197 domain A
Objective To evaluate the potential of CRM197-HIV-V3 fusion protein as an epitope vaccine,to screen monoclonal antibodies and provide guideline for the study of HIV vaccine and therapeutic antibodies.Methods NL4-3-299-328 polypeptide was fused with CRM197 domain A and expressed in E.coli.The 6～8 weeks old Balb/c female mice were immunized with the purified fusion proteins.After 3 times of immunization,following spleen immunization,the immune stimulated spleen cells were hybridized with mouse hybridoma ones.Neutralization monoclonal antibody(NmAb)was screened by neutralization screening platform based on ELISPOT approach.The monoclonal antibody were characterized by HIV neutralization assay and ELISA.ResultsConstruct CRM197-A-V3 was successfully expressed in E.coli,and the resultant fusion protein elicited about 10.3 HIV neutralization titers in mice.The reaction titers of anti-sera was 10.5 as shown in ELISA,and 8 NmAbs were raised.ELISA results showed these NmAbs have good binding activity against NL4-3-gp120 proteins.Neutralization assay showed these NmAbs possess good neutralizing capacity against HIVNL4-3 strain,with NC50 of about 0.02μg/mL.Conclusion The V3 epitope was fused to CRM197 domain A,and significantly evoked the HIV specific immune response in mice.The neutralization monoclonal antibodies against NL4-3 were acquired.
human immunodeficiency virusV3 loopCRM197 domain Afusion proteinneutralizing monoclonal antibody
NETL
NSTL
万方数据
维普
