新疆出血热病毒截短糖蛋白的原核表达及抗体制备  

Prokaryotic expression and antibody preparation of truncated glycoprotein of Crimean-Congo hem-orrhagic fever virus

作　　者：俞欢[1] 汪梅芳[1] 张渝疆 彭倩[1] 寇春 达尔肯· 托肯 李阳[1] 李轶杰[1] 孙素荣[1] 

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源与基因工程重点实验室,乌鲁木齐830046 [2]新疆疾病预防与控制中心

出　　处：《中华微生物学和免疫学杂志》2015年第5期348-352,共5页Chinese Journal of Microbiology and Immunology

基　　金：科技基础性工作专项重点项目计划（2013FY113500）;国家自然科学基金（81460303）;病毒学国家重点实验室开放研究基金项目（2015IOV003）

摘　　要：目的：原核表达纯化新疆出血热病毒79121毒株糖蛋白截短片段（ Gn、Gc1和Gc2）并分别制备其多克隆抗体。方法采用反转录PCR的方法克隆79121毒株Gn、Gc1及Gc2基因片段，并将其构建到原核表达载体pET-28a（＋）中，转化到E．coli BL21（DE3）中进行诱导表达，经镍柱亲和层析法纯化Gn-His、Gc1-His及Gc2-His融合蛋白，SDS-PAGE分析蛋白相对分子质量（ Mr ）。用纯化蛋白免疫新西兰白兔制备抗血清，ELISA和Western blot法检测血清效价和特异性。结果双酶切鉴定和测序证实构建的pET-28a-Gn、pET-28a-Gc1和pET-28a-Gc2重组表达载体正确，表达的融合蛋白Gn-His、Gc1-His和Gc2-His其Mr 分别约为25×103、33×103和34×103。 ELISA法检测抗体效价分别为1∶25600、1∶6400及1∶25600。 Western blot结果表明兔多克隆抗体均能特异性识别相应的重组蛋白。结论新疆出血热病毒截短糖蛋白的表达纯化和效价高、特异性好的兔抗血清的制备，为新疆出血热病毒的检测和糖蛋白生物学功能的深入研究提供资料。Objective To express and purify truncated glycoprotein（Gn, Gc1, Gc2） of Crimean-Congo hemorrhagic fever virus（ CCHFV） strain 79121 from E.coli cells, and to prepare their polyclonal anti-bodies respectively.Methods The genes encoding Gn, Gc1 and Gc2 from CCHFV strain 79121 were am-plified by RT-PCR, and cloned into prokaryotic expression vector pET-28a （＋） to construct recombinant plasmids.The three recombinant plasmids were then transformed into E.coli BL21（ DE3） to obtain the Gn-His, Gc1-His and Gc2-His fusion proteins through IPTG induction.The fusion proteins were purified by Ni-NTA purification system, and then analyzed by using SDS-PAGE.To prepare antisera, the purified Gn-His, Gc1-His and Gc2-His proteins were employed to immunize New Zealand White Rabbit （ NZW） , respective-ly.The titer and specificity of the antisera to each truncated glycoprotein were analyzed by using ELISA and Western blot, respectively.Results The recombinant expression vectors of pET-28a-Gn, pET-28a-Gc1 and pET-28a-Gc2 were constructed successfully by restriction enzyme analysis and DNA sequencing.The relative molecular mass（ Mr ） of Gn-His, Gc1-His and Gc2-His were approximately 25 000, 33 000 and 34 000, and the titers of the corresponding polyclonal antibodies were above 1 ∶25 600, 1 ∶6400 and 1 ∶25 600, re-spectively.The antisera were able to bind to the corresponding fusion protein specifically.Conclusion The recombinant expression of truncated glycoprotein （Gn, Gc1, Gc2） of CCHFV strain 79121 and the prepara-tion of their high titer polyclonal antibodies laid foundation for further studying the biological function of gly-coprotein and the rapid detection of CCHFV.

关 键 词：新疆出血热病毒 糖蛋白 原核表达 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]