原代HIV-1包膜糖蛋白修饰体诱导小鼠产生高滴度gp120抗体  

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

作　　者：凌虹[1] 王甲业[1] 王开利[2] 李妍[1] 陈文江[1] 杨丹[1] 田丹[1] 焦娜[1] 周海舟[3] 

机构地区：[1]哈尔滨医科大学微生物学教研室,黑龙江省感染与免疫重点实验室,黑龙江省教育厅病原生物学重点实验室,150081 [2]黑龙江省疾病预防控制中心病毒病研究所 [3]哈尔滨医科大学第一临床医学院检验科

出　　处：《国际免疫学杂志》2011年第3期234-237,共4页International Journal of Immunology

基　　金：黑龙江省教育厅重点项目（11531206）

摘　　要：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-106044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10d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10d，gp120免疫血清中结合抗体滴度大于1：1000，gp120／W427S免疫血清中结合抗体滴度大于1：10000，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。Objective To investigate the effect of the mutation W427S within human immunodefieieney virus type 1 （HIV-1） envelope glyeoprotein （Env） on the Env immunogenicity in BALB/e mice. Methods The gp120 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 gp120 trimer-expression vectors, peTgp120 and pcT-gp120/W427S. HEK293T cells were transfected with these recombinant plasmids. Gpl20 proteins in the culture supernatants were detected by SDS-PAGE and western blot. BALB/c mice were inoculated intramuscularly with pcT-gp120 or peT-gp120/W427S at week 0, 2, respectively, and intraperltoneally with trimerle 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 immuniza- tion, the antibody titers in the sera were measured by using an enzyme immunoassay （EIA）. Results Two gp120 expression vectors, peT-gp120 and pcT-gp120/W427S were constructed and confirmed by DNA sequencing. The trimerie gp120 proteins secreted into the transfected culture supematants were observed. On day 10 post-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, immunogenieity than its naive 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.

关 键 词：Ⅰ型人类免疫缺陷病毒 GP120 BALB/C小鼠 免疫原性 

分 类 号：R3[医药卫生—基础医学]