CAJ | 학술논문

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HEK293T细胞,表达gp120三聚体蛋白.采取DNA初免-蛋白凝胶加强的策略免疫BALB/c小鼠,末次免疫后10 d检测免疫血清中结合抗体的水平.结果获得真核表达载体pcT-gp120和pcT-gp120/W427S及gp120三聚体蛋白;末次免疫后10 d,gp120免疫血清中结合抗体滴度大于1∶1 000,gp120/W427S免疫血清中结合抗体滴度大于1∶10 000,而且,gp120/W427S免疫组血清抗体结合活性显著高于gp120免疫组血清.结论 HIV-1包膜第427位色氨酸突变为丝氨酸,改善了包膜的免疫原性,为包膜免疫原的设计和优化提供了新的线索.
목적 탐색1형인류면역결함병독(HIV-1)포막당단백수식대포막면역원성적적영향.방법 통과PCR확증획득원대HIV-1 06044주포막gp120기인급기돌변체gp120/W427S기인,병구건gp120삼취체단백진핵표체재체pcT-gp120화pcT-gp120/W427S,중조표체재체체외순시전염인배신HEK293T세포,표체gp120삼취체단백.채취DNA초면-단백응효가강적책략면역BALB/c소서,말차면역후10 d검측면역혈청중결합항체적수평.결과 획득진핵표체재체pcT-gp120화pcT-gp120/W427S급gp120삼취체단백;말차면역후10 d,gp120면역혈청중결합항체적도대우1∶1 000,gp120/W427S면역혈청중결합항체적도대우1∶10 000,이차,gp120/W427S면역조혈청항체결합활성현저고우gp120면역조혈청.결론 HIV-1포막제427위색안산돌변위사안산,개선료포막적면역원성,위포막면역원적설계화우화제공료신적선색.
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.