猪瘟病毒保护性抗原E2基因的克隆及在原核细胞中的表达  

CLONING OF PROTECTIVE ANTIGEN E2 GENE OF HOG CHOLERA VIRUS AND ITS EXPRESSION IN PROTOKARYOTIC CELLS

作　　者：李红卫[1] 涂长春[1] 余兴龙[1] 金扩世[1] 吕宗吉[1] 李茂祥 章金钢[1] 殷震[1] 

机构地区：[1]解放军农牧大学

出　　处：《畜牧兽医学报》1999年第2期146-152,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金

摘　　要：应用ＲＴＰＣＲ分两段扩增猪瘟病毒（ＨＣＶ）石门株Ｅ２基因，然后对其进行了克隆。利用两个片段重叠部分的单一ＢｇｌⅡ位点，将它们连接成为完整的Ｅ２基因，并克隆到ｐＵＣ１９质粒中，获得重组质粒ｐＨＣＥ２。另设计一对引物，以ｐＨＣＥ２为模板扩增出不含信号肽的Ｅ２基因，然后将其克隆到表达载体质粒ｐＢＶ２２０和ｐＥＴ２８ａ（＋）中，获得重组质粒ｐＢＶＥ２和ｐＥＴＥ２，用酶切、ＰＣＲ和序列分析鉴定Ｅ２基因插入位置、方向和读码框架的正确性。经Ｗｅｓｔｅｒｎｂｌｏｔ和直接ＥＬＩＳＡ检测表明重组质粒ｐＢＶＥ２和ｐＥＴＥ２转化、诱导的受体菌均能表达Ｅ２抗原。The complete E2 gene of hog cholera virus strain Shimen,the Chinese virulent reference strain,was cloned into pUC19 plasmid.One pair of primers were designed and synthesized to amplify the E2 gene without signal peptide.Then it was cloned into the expression vector pBV220 and pET 28a(+),resulting in the recombinant pBVE2 and pETE2.The recombinants were identified by restriction endonuclease analysis,PCR and sequence analysis,proving that foreign gene was in the correct position and oritention.The recombinant pBVE2 and pETE2 plasmids expressed E2 protein in E.coli BL21(DE3)which could be detected by Western blot and direct ELISA.

关 键 词：猪瘟病毒 E2基因 克隆 表达 原核细胞 猪瘟 诊断 

分 类 号：S858.285.3[农业科学—临床兽医学] S852.53[农业科学—兽医学]