Eukaryocyte expression and characterization of HIV-1 envelope trimer gp140

Objective To construct the recombinant plasmid that for express gp140 of HIV-1 CRF_07 subtypes B/C(CN54)and the mutations,two amino acids on gp140 protein was mutated through Q567K and K588R,named gp140 ST in order to make the gp140 become stable trimer conformation produced to be used as a candidate of HIV-1 vaccine.Methods HIV-1 gp140 and gp140 ST plasmids were concstructed by PCR technology. Recombinant plasmids of gp140and gp140 ST were transfected into 293F cells,respectively,and the supernatants were collected 120 hours after transfection.After purified and concentrated,the proteins of gp140 and gp140 ST were identified by SDS-PAGE,Western-blot,and ELISA were performed to analyze and determine the bioreactivity of the gp140 and gp140 ST proteins. Results SDS-PAGE,western-blot suggested that gp140 ST was a stable trimer compared with gp140. ELISA indicated that the recombinant proteins of gp140 ST could strongy bind with the sera of Chinese HIV-1 positive patients than that with gp140.Conclusions Gp140 ST trimer was more stable with more immunogenic and can be an excellent immunogen for HIV-1 vaccine design.