传染性鼻气管炎病毒gD基因表位的原核表达与纯化  

Expression and Purification of Gene gD of Infectious Bovine Rhinotracheitis Virus

作　　者：王欣[1] 王琦[1] 齐晓雪[1] 毕莹[1] 倪宏波[1] 

机构地区：[1]黑龙江八一农垦大学食品学院,黑龙江大庆163319

出　　处：《安徽农业科学》2015年第35期231-233,共3页Journal of Anhui Agricultural Sciences

基　　金：黑龙江省农垦总局攻关项目(HNK125B-11-02;HNK125B-11-10A)

摘　　要：[目的]对牛传染性鼻气管炎病毒(IBRV)g D基因表位进行原核表达,并对表达产物进行纯化鉴定。[方法]利用PCR扩增出g D基因3段表位即g D-A、g D-B、g D-C,并构建原核表达重组质粒p ET-28a-g D。将其转入BL21(DE3)表达菌中,IPTG诱导表达。表达的g D重组蛋白经亲和层析纯化后,进行Western blot分析。[结果]重组表达质粒p ET-28a-g D经PCR、双酶切及测序证明构建正确。SDSPAGE表明,g D蛋白在大肠埃希菌中高效表达,表达的重组蛋白相对分子量约48 ku,与预期的蛋白分子量一致。纯化后的g D重组蛋白浓度为0.128 mg/ml,免疫印迹结果显示纯化后的g D重组蛋白能与IBR标准阳性血清发生特异性反应,说明其免疫原性良好。[结论]成功表达了g D基因表位蛋白,该蛋白具有良好的反应原性,可作为预防IBR基因工程亚单位疫苗的候选抗原。[ Objective ]To construct a fusion gene of Enterotoxigenic Escherichia coli （ETEC） adhesion K99,987P and F41 ,and purify the fusion expressed protein. [ Method] A pair of primers was designed according to the IBRV gD sequences in GenBank to amplify the complete open reading frame. PCR product of gD, containing three epitope： gD-A,gD-B,gD-C,was then inserted into the prokaryotic expression vector pET-28a-gD respectively,then transform the recombinant plasmid into competent cells of BL21 （DE3）. After induced by IPTG, the expressed recombinant protein gD was purified by nickel ion affinity chromatography and analyzed by Western blot. [ Result] The recombinant plasmid was confirmed by PCR,restriction analysis and sequencing. The expressed recombinant protein with a relative molecular mass of about 48 ku. The concentration of the purified recombinant protein Was 0. 128 mg/ml for gD. The fusion protein specifically reacted with IBRV polyclonal antibody from mice,indicating good reactionogenicity. [ Conclusion ] Recombinant plasmid pET-28a-gD is constructed and expressed in E. coli BL21 （DE3）, indicating that protein gD is potential of candidate antigen for prevention of IBR.

关 键 词：IBR GD 表达 纯化 亚单位疫苗 

分 类 号：S858.23[农业科学—临床兽医学]