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目的构建表达中国流行株HIV-1CRF_07B/C亚型(HIV-1CN54)膜蛋白gp140和gp140FdFc三聚体疫苗,比较其免疫原性。方法构建gp140和gp140FdFc重组质粒,将质粒分别瞬时转染293F细胞,120小时后收获上清液,经纯化、浓缩、PBS置换、BCA试剂盒检测蛋白质浓度后,利用SDS-PAGE电泳检测蛋白分子量大小及纯度,酶联免疫吸附试验(ELISA)检测其与阳性病人血清的反应性。通过肌肉注射的方式分别免疫兔子,取兔子血清,ELISA检测血清抗体,并通过假病毒系统检测血清中和抗体滴度,比较gp140和gp140FdFc的免疫原性。结果成功获得基因重组gp140和gp140FdFc蛋白。免疫兔子后,gp140和gp140FdFc蛋白质均有很强的体液免疫应答,抗gp160抗体滴度分别为2.24×105和4.48×105,但gp140组未诱导出中和抗体,gp140FdFc组对4种假病毒具有一定的中和活性。结论 Gp140FdFc三聚体能诱导兔子产生较强的结合抗体和一定的中和抗体反应,可作为潜在的HIV-1免疫原进行疫苗研究。
Objective To construct a vaccine expressing gp140 and gp140FdFc, a membrane protein of HIV-1CRF_07B / C subtype (HIV-1CN54) in China, and compare its immunogenicity. Methods Recombinant plasmids gp140 and gp140FdFc were constructed and transiently transfected into 293F cells respectively. After 120 hours, the supernatants were harvested, purified, concentrated and replaced by PBS. After the protein concentration was detected by BCA kit, the size of the protein was determined by SDS-PAGE. And purity, ELISA test to detect the reactivity with serum of positive patients. The rabbits were immunized by intramuscular injection, serum of rabbits was taken, serum antibody was detected by ELISA and serum neutralizing antibody titers were detected by pseudovirus system. The immunogenicity of gp140 and gp140FdFc were compared. Results The recombinant plasmids gp140 and gp140FdFc were successfully obtained. After immunized rabbits, both gp140 and gp140FdFc protein had strong humoral immune response, anti-gp160 antibody titers were 2.24 × 105 and 4.48 × 105 respectively, but no neutralizing antibody was induced in gp140 group, and the gp140FdFc group had the same effect on four pseudovirions A certain degree of neutralization activity. Conclusion Gp140FdFc trimer can induce rabbits to produce strong binding antibody and some neutralizing antibody reaction, which can be used as potential HIV-1 immunogens for vaccine research.