摘要
应用ＲＴＰＣＲ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ｐＥＴ２８ａ（＋）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Ｗｅｓｔｅｒｎｂｌｏｔ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.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
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
