人3型腺病毒纤突的原核表达及其免疫原性分析  

Expression and immunogenetic analysis of human adenovirus serotype 3 knob

作　　者：蒋再学[1] 王芳[1] 田新贵[1] 朱蔚云[2] 苏晓波[1] 

机构地区：[1]广州医科大学基础学院医学遗传学与细胞生物学教研室,广东广州511436 [2]广州医科大学基础学院生物学实验中心,广东广州511436

出　　处：《热带医学杂志》2016年第1期22-24,30,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金青年科学基金(31200132);广东省自然科学基金(S2013040015594);广东高校优秀青年创新人才培养计划项目(育苗工程2013LYM0072)

摘　　要：目的克隆表达人3型腺病毒（HAdV-3）的纤毛蛋白纤突片段（knob）,分析其免疫原性,为腺病毒临床诊断试剂及新型疫苗的研发奠定基础。方法 PCR扩增knob基因片段,克隆到表达载体pGEX-4T-3中进行诱导表达,SDSPAGE电泳分析表达产物,包涵体溶解复性后亲和层析纯化融合蛋白,Western blot鉴定,纯化的蛋白免疫小鼠制备抗血清,间接ELISA法检测小鼠血清的效价,Western blot鉴定抗血清特异性。结果成功构建了重组原核表达质粒pGEX-Ad3 knob,其在大肠杆菌中高效表达并纯化重组knob融合蛋白,融合蛋白免疫小鼠抗血清效价高达10~7,Western Blot证实此抗血清可识别人3型腺病毒纤维蛋白。结论成功获得了重组HAdV-3 knob蛋白,其具有强免疫原性,可以作为研发人3型腺病毒入侵机制研究、诊断试剂及疫苗研发的原料。Objective To express the recombinant protein of human adenovirus serotype 3 （HAdV-3） fibre knob, and analyze its immunogenecity. Methods Ad3 knob gene was specifically amplified and cloned into pGEX-4T-3 vector.The recombinant knob protein was expressed in E.coli BL21 （DE3）. Inclusion body protein was dissolved and renatured by general way, and purified with GST-Bind resin. The knob protein was used to immunize mice and prepare antiserum.The specificity and titer of antiserum were detected by Western blot and indirect ELISA. Results The recombinant plasmid pGEX-Ad3 knob was constructed and the recombinant GST fusion protein of knob was expressed and purified with high yield. The mouse antiserum was prepared with a high titer of 107 against knob. This antiserum can specifically bind to the fibre protein of HAdV-3. Conclusion The recombinant HAdV-3 fibre knob protein with strong immunogenecity was expressed and purified successfully. This research provides a basis for adenovirus clinical diagnosis and the novel vaccine development.

关 键 词：人3型腺病毒 纤毛蛋白 纤突 重组表达 多克隆抗体 

分 类 号：R511.8[医药卫生—内科学]