HIV-1 CRF07_BC毒株gp41重组DNA-蛋白疫苗的制备及免疫  

Preparation and application of HIV-1 CRF07_BC recombinant gp41 DNA-protein vaccine

作　　者：王妮丹[1] 罗振武[1] 孙红岩[1] 何彦平[1] 邵一鸣 郝彦玲[1] 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京102206

出　　处：《中国生物制品学杂志》2014年第9期1113-1117,共5页Chinese Journal of Biologicals

基　　金：十二五重大专项"创新抗病毒重组蛋白和病毒样颗粒(VLP)疫苗孵化基地建设"(2011ZX09401-019);十二五重大专项"预防性艾滋病疫苗研究"(2012ZX10001008-001-001)

摘　　要：目的制备中国HIV-1流行株CRF07_BC膜蛋白gp41重组DNA-蛋白疫苗,并观察其在BALB/c小鼠中的免疫效果。方法从CRF07_BC毒株（CN54）中扩增gp41胞外段,删除抗原决定环loop区（the immune dominant loop region）,引入T569A和I675V两个突变位点,制备含N-端七价重复序列（N-terminal heptad repeat,NHR）、C-端七价重复序列（C-terminal heptad repeat,CHR）和近膜区（membrane proximal external region,MPER）的重组疫苗,以未改造的gp41相应区域为对照免疫原。使用DNA疫苗初免-蛋白疫苗加强的方式免疫BALB/c小鼠,最后一针免疫后4周,经小鼠眼眶采血,分离血清,检测结合抗体水平、中和抗体水平、线性表位抗体水平。结果 CN54 gp41野生型及改造型重组DNA疫苗质粒和原核蛋白表达质粒经双酶切及测序鉴定均构建正确;CN54 gp41W和CN54 gp41M表达蛋白相对分子质量约在12 000-15 000之间,纯化的重组蛋白可被豚鼠抗gp140阳性血清识别。CN54M组（CN54 gp41改造抗原组）诱导出较高的结合抗体水平,而线性表位抗体水平明显低于CN54W组（CN54 gp41未改造组）,两组均未检测出具有保护性的中和抗体水平。结论 CN54 gp41改造抗原经DNA疫苗初免-蛋白疫苗加强免疫小鼠,可产生较高的结合抗体,且主要是针对空间构象表位,但不具有中和能力。Objective To prepare recombinant DNA-protein vaccine with membrane protein gp41 of epidemic strain CRF07_BC of HIV-1 and observe its immune effect in BALB / c mice.Methods The extracellular domain of gp41 was amplified from CRF07_BC strain,in which the immune dominant loop region was deleted and two site mutations T569 A and I675 V were introduced,to prepared a recombinant vaccine composed of N-terminal heptad repeat（NHR）,C-terminal heptad repeat（CHR）and the membrane-proximal external region（MPER）,using the corresponding regions unmodified in gp41 as control immunogen.BALB / c mice were immunized by DNA prime-protein boost strategy,of which serum samples were collected 4 weeks after the last immunization,and determined for binding antibody,neutralizing antibody and linear epitope antibody levels.Results Recombinant DNA vaccine plasmids with wild and modified CN51 gp41 genes as well as the plasmid for protein expression were constructed correctly as proved by restriction analysis and sequencing.The relative molecular masses of expressed recombinant CN54 gp41 W and CN54 gp41 M proteins were12 000 - 15 000.The purified recombinant proteins were recognized by guinea antisera against gp140.CN54 M induced high binding antibody level.However,the linear epitope antibody level in CN54 M group was lower than that in CN54 W group.No protective neutralizing antibodies were induced in the two groups.Conclusion Modified CN54 gp41 antigen induced high binding antibody level probably targeting to the conformational epitopes but without neutralizing ability in mice by DNA prime-protein boost strategy.

关 键 词：人类免疫缺陷病毒 CRF07_BC GP41 疫苗 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]