摘要
目的构建、表达不同片段1型人类免疫缺陷病毒(human immunodeficiency virus type 1,HIV-1)的膜蛋白三聚体,并检测其免疫原性。方法以中国主要流行株HIV-1CRF_07 BC亚型基因为模板构建pFUSE-gp160重组质粒,以pFUSE-gp160为模板构建pFUSE-gp140FF、pFUSE-gp120FF、pFUSE-gp41FF、pFUSE-N55FF、pFUSE-N28FF重组质粒,将重组质粒分别转染293F细胞,转染120 h后,收获上清,经Ni-NTA superflow、nProtein A SepharoseTM4 Fast Flow及Superose 12 Prep Grade分子筛纯化后,分别进行SDS-PAGE及Western blot鉴定。将各重组蛋白联合MF59佐剂经大腿肌肉注射免疫豚鼠,100μg/只,分别于第1、30及60天各免疫1次,初次免疫前7 d及末次免疫后第90天心脏采血,分离血清,ELISA法及假病毒系统分别检测血清特异性抗体效价及中和抗体滴度。结果质粒pFUSE-gp160、pFUSE-gp140FF、pFUSE-gp120FF、pFUSE-gp41FF、pFUSE-N55FF、pFUSE-N28FF经测序鉴定构建正确。各纯化蛋白的SDS-PAGE及Western blot鉴定均可见目的条带,gp120FF和gp140FF均可高通量表达,其表达量分别为6和2.5 mg/L,gp41FF、N55FF和N28FF蛋白的表达量均低于0.5 mg/L,纯化后的纯度均较高。gp140FF组和gp120FF组豚鼠血清抗体效价分别为3.2×105和5.33×105、中和抗体滴度分别为405和462,均显著高于其他组(P<0.05)。结论 gp140FF和gp120FF能诱导豚鼠产生较强的体液免疫反应和中和抗体,本实验为HIV-1疫苗的研发提供了参考。
Objective To construct and express the recombinant envelope glycoprotein trimers of human immunodeficiency virus type 1（HIV-1）,at various lengths,compare determine their immunogenicity. Methods Recombinant plasmid pFUSE- gp160 was constructed using the gene of prominent HIV-1 CRF_07 strain of subtypes BC in China as a template,based on which recombinant plasmids pFUSE-gp140FF,pFUSE-gp120FF,pFUSE-gp41FF,pFUSE-N55FF and pFUSE-N28FF were constructed and transfected to 293F cells respectively. The supernatant of transfected cells were collected 120 h lat- er,purified by Ni-NTA superflow,nProtein A SepharoseTM4 Fast Flow and Superose 12 Prep Grade molecular sieve chro- matography,and identified by SDS-PAGE and Western blot. Guinea pigs were injected i.m. with three doses of various recombinant proteins combined with MF59 adjuvant in legs,100 μg for each,on days 1,30 and 60 respectively. Heart blood samples were collected 7 d before the first injection and on day 90 after the last injection,from which sera were separated and determined for antibody potency and neutralizing antibody titer by ELISA and pseudovirus system. Results Sequencing proved that recombinant plasmids pFUSE-gp160, pFUSE-gp140FF, pFUSE-gp120FF, pFUSE-gp41FF, pFUSE-N55FF and pFUSE-N28FF were constructed correctly. The target bands of various purified proteins were observed on SDS-PAGE and Western blot profiles. The expression levels of gp120FF and gp140FF were 6 and 2. 5 mg / L respectively,while those of gp41FF, N55FF and N28FF were less than 0. 5 mg / L. The expressed products reached highpurities after purification. The serum antibody potencies of guinea pigs in gp140FF and gp120FF groups were 3. 2 × 105 and 5. 33 × 105,while the neutralizing antibody titers were 405 and 462, respectively, which were significantly higher than those in other groups（P〈 0. 05）. Conclusion Both gp140FF and gp120FF induced strong humoral immune response and neutralizing antibody in guinea pigs, which provided a reference for development of HIV-1 vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第2期145-150,共6页
Chinese Journal of Biologicals
基金
国家十二五科技重大专项-艾滋病和病毒性肝炎等重大传染病防治-DNA疫苗(2012ZX10001008-012)
传染病预防控制国家重点实验室留学归国人员项目资助(2012SKLID402)
关键词
1型人类免疫缺陷病毒
膜蛋白
三聚体
免疫原性
Human immunodeficiency virus type 1（HIV-1）
Envelope glycoprotein
Trimer
Immunogenicity
