原代HIV-1包膜糖蛋白修饰体诱导小鼠产生高滴度gp120抗体

目的 探索1型人类免疫缺陷病毒(HIV-1)包膜糖蛋白修饰对包膜免疫原性的的影响.方法 通过PCR扩增获得原代HIV-1 06044株包膜gp120基因及其突变体gp120/W427S基因,并构建gp120三聚体蛋白真核表达载体pcT-gp120和pcT-gp120/W427S,重组表达载体体外瞬时转染人胚肾HEK2...

High-titer antibody induced by an envelope glycoprotein mutant of a primary HIV-1 isolate in BALB/c mice

Objective To investigate the effect of the mutation W427S within human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) on the Env immunogenicity in BALB/c mice. Methods The gpl20 gene derived from the HIV-1 primary isolate 06044 and its mutant gp120/W427S were PCR amplified and then inserted into a trimer motif-bearing plasmid to make gpl20 trimer-expression vectors, pcTgp120 and pcT-gp120/W427S. HEK293T cells were transfected with these recombinant plasmids. Gp120 proteins in the culture supernatants were detected by SDS-PAGE and western blot. BALB/c mice were inoculated intramuscularly with pcT-gp120 or pcT-gp120/W427S at week 0, 2, respectively, and intraperitoneally with trimeric gp120 protein-containing PAGE gel at week 6, 8 and 14 respectively. The naive mice were inoculated with PBS and blank PAGE gel instead of plasmid and gp120 respectively. On day 10 after the final immunization, the antibody titers in the sera were measured by using an enzyme immunoassay (EIA). Results Two gp120 expression vectors, pcT-gp120 and pcT-gp120/W427S were constructed and confirmed by DNA sequencing. The trimeric gp120 proteins secreted into the transfected culture supernatants were observed. On day 10post-immunization, the binding antibody titer in the serum of gp120- and gp120/W427S-immunized mice were higher more than 1: 1 000 and 1: 10 000, respectively, indicating that mutant W427S within gp120 showed stronger immunogenicity than its na(i)ve protein. And compared with gp120-immunized sera, a much stronger binding activities were observed in the gp120/W427S-immunized sera. Conclusion The substitution of Tryptophan on the position 427 of envelope by Serine improved the immunogenicity of HIV-1 06044 gp120. This modification strategy may provide a new clue for designing and optimizing Env immunogen.