摘要
目的 实现HIV 2 RODgp10 5 gag截短体重组蛋白tgp10 5 gag的高效分泌表达 ,对其表达条件进行研究。方法 用PCR方法获得HIV 2 RODtgp10 5截短基因 ,将其与HIV 2 RODgag构建HIV 2 RODtgp10 5 gag嵌合基因并克隆到毕赤酵母 (Pichiapastoris)分泌型表达载体pPIC9中 ,转化GS115酵母菌 ,经MD/MM表型筛选、PCR扩增筛选阳性克隆 ,以甲醇诱导表达后进行SDS PAGE分析及Westernblot证实。结果 HIV 2 RODtgp10 5 gag嵌合截短基因在Pichiapastoris酵母系统中获得了有效分泌表达 ,相对分子质量 (Mr)约为 14 0× 10 3 ,表达量约为 16 % ,表达产物可被HIV 2阳性血清识别。最佳表达条件是大于 85 %的溶解氧 ,摇瓶转速 2 4 0r/min ,1.0 %～ 1.5 %甲醇诱导 ,培养时间 3d。结论 在Pichiapas toris表达系统中实现了HIV 2 RODtgp10 5 gag蛋白的有效分泌性表达 ,表达产物具有良好的抗原特异性。
Objective To carry out the secretive express of HIV 2 ROD tgp105 gag protein and the optimization of expression conditions. Methods HIV 2 gp105 truncated gen (tgp105) was obtained by PCR amplification and was cloned into a secreting expression vector pPIC 9. Recombinant expression vector pPIC9 tgp105 gag was constructed by inserting HIV 2 gag into pPIC9 tgp105 recombinant plasmid, then transformed into GS115 cells. Positive clones were selected with MD/MM plates and confirmed by PCR. Several clones were incubated in BMGY media and induced by 0.5% methanol in BMMY media. The expression product HIV 2 tgp105 gag protein was analyzed by SDS PAGE and confirmed by Western blot. Results The tgp105 gag protein was secreted into media. The molecular weight of the expressed protein, as analyzed by SDS PAGE, was 140kD. The Western blot result showed that the expressed protein could be detected by HIV 2 specific antibody. Conclusion The recombinant plasmid HIV 2 ROD tgp105 gag was successfully expressed in Pichia pastoris and the expressed protein has a good antigen specificity. [
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2003年第5期371-374,共4页
Chinese Journal of Microbiology and Immunology
基金
国家"8 63"高技术计划项目 (No .2 0 0 1AA2 15 0 3 1 3 )
国家杰出青年基金资助项目 (No.3 982 5 119)
