杆状病毒AcMNPV包膜蛋白GP64胞外区的原核表达、纯化及多克隆抗体的制备  

Prokaryotic expression,purification and preparation of polyclonal antibody of extracellular domain of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64

作　　者：王奔[1] 王宁[1] 周思杭[1] 李静[1] 佟雪莲[1] 唐玉龙[1] 王玉琳[1] 

机构地区：[1]北京生物制品研究所有限责任公司第七研究室,北京100024

出　　处：《中国生物制品学杂志》2013年第9期1209-1213,共5页Chinese Journal of Biologicals

摘　　要：目的原核表达、纯化苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)包膜糖蛋白GP64胞外区,并制备多克隆抗体。方法根据GenBank中登录的AcMNPV GP64基因全长序列,采用DNAStar软件设计去除GP64基因信号肽和跨膜区的引物,PCR扩增GP64胞外区基因,插入原核表达载体pET-21b(+)中,转化大肠埃希菌Rosetta(DE3),IPTG诱导表达。表达的重组蛋白经His Trap FF crude纯化后,进行SDS-PAGE和Wester blot分析。将纯化的重组蛋白经背部皮下免疫家兔,制备多克隆抗体,采用免疫染色法检测多克隆抗体对AcMNPV的中和作用。结果重组表达质粒pET-21b(+)-GP64经双酶切及测序证实构建正确;表达的重组蛋白相对分子质量约为52 000,表达量占菌体总蛋白的46.8%,主要以包涵体形式表达;纯化的重组蛋白纯度可达95%以上,可与鼠抗AcMNPV GP64蛋白单克隆抗体特异性结合;制备的兔抗GP64蛋白多克隆抗体能与纯化的重组蛋白和杆状病毒发生特异性反应,抗体效价高于1∶1 000 000,其可中和100 TCID50/ml的AcMNPV,使AcMNPV无法感染昆虫细胞Sf9,中和效价为1∶8。结论成功原核表达了AcMNPV GP64蛋白胞外区,并制备了对AcMNPV有完全中和能力的多克隆抗体,为昆虫细胞/杆状病毒表达系统的应用研究及杆状病毒毒株的检定等提供了材料。Objective To express the extracellular domain of envelope protein GP64 of Autographa californica multiple nueleopolyhedrovirus （AcMNPV） in prokaryotic cells, purify the expressed product and prepare its polyelonal antibody. Methods According to the full-length GP64 gene sequence of AcMNPV reported in GenBank, the primers without signal peptide and transmembrane domain were designed by using DNAStar software, based on which GP64 gene was amplified by PC R and inserted into prokaryotic expression vector pET-2 l b （＋）. The constructed recombinant plasmid pET-2 l b （＋）- AcMNPV-GP64 was transformed to E. coli Rosetta （DE3） for expression under induction of IPTG. The expressed protein was purified with His Trap FF crude, and identified by SDS-PAGE and Western blot. Potyclonal antibody was prepared by immunizing rabbits with the purified protein, and determined for neutralization to AcMNPV. Results Restriction analysis and sequencing proved that recombinant plasmid pET-21b （＋）-AeMNPV-GP64 was constructed correctly. The expressed recombinant GP64 protein, with a relative molecular mass of about 52 000, contained 46. 8% of total somatic protein and mainly existed in a form of inclusion body. The purified recombinant protein reached a purity of more than 95%, and showed specific binding to mouse anti-AcMNPV GP64 monoclonal antibody. The prepared rabbit anti-GP64 polyclonal antibody showed specific reaction with purified recombinant GP64 protein and AcMNPV, of which the titer reached more than 1 ： 1 000 000, and the neutralization titer reached 1 ： 8. The AcMNPV at a concentration of 100 TCIDso/ml was neutralized with the polyclonal antibody, which lost the infectivity to St9 cells. Conclusion The ex- tracellular domain of AcMNPV GP64 protein was successfully expressed in prokaryotic cells, and the polyclonal antibody with completely neutralizing ability to AcMNPV was prepared, which provided a material for application of insect ceils/ baculovirus expression system and control tests on baculovi

关 键 词：杆状病毒 苜蓿银纹夜蛾核型多角体病毒 GP64蛋白 原核细胞 基因表达 多克隆抗体 

分 类 号：Q786[生物学—分子生物学] R392.33[医药卫生—免疫学]