摘要
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 and express the recombinant envelope glycoproteins gp140 and gp140FdFc of China HIV-1 subtypes B/C （CN54） with stable trimer conformation and compare their immunogenicity. MethodsAfter constructing gp140 and gp140FdFc plasmids, the recombinant plasmids were transfected into 293F cells andthe supernatants were collected 120 hours after transfection. After purification and concentration, the proteins ofgp140FdFc and gp140 were identified by SDS-PAGE and ELISA, accurately determined by BCA Protein Assay Kit.The proteins were used to immunize rabbits intramuscularly and blood samples were collected. Then, serum anti-bodies were detected by ELISA and neutralizing antibodies were detected by pseudovirus system to compare the im-munogenicity of gp140 and gp140FdFc. Results SDS-PAGE and Western-blot indicated that the proteins ofgp140FdFc and gp140 were successfully expressed in 293F cells. The proteins of gp140 and gp140FdFc producedstrong humoral immune responses after immunizing rabbits and the titers of gp 160 were 2.24 X l0^5 and 4. 48 x10^5 ,respectively. However, there was no neutralizing antibody in gp140 groups. Neutralizing antibody in gp140FdFcgroup can neutralize 4 pseudoviruses to some degree. Conclusions Gp140FdFc trimer could stimulate strong im-mune response and induce the neutralizing antibody and might be a potential immunogen in research of HIV in vac-cine.
出处
《中国艾滋病性病》
CAS
2014年第3期149-153,共5页
Chinese Journal of Aids & STD
基金
国家科技重大专项(2012ZX10001008-012)
传染病预防控制国家重点实验室(2012SKLID402)~~
