猪瘟病毒石门株E2基因的克隆及在大肠杆菌中的表达

CLONING AND EXPRESSION OF E2 GENE OF CLASSICAL SWINE FEVER VIRUS (CSFV) SHIMEN STRAIN IN CHINA

采用ＲＴ－ＰＣＲ技术扩增了猪瘟病毒石门株的Ｅ２基因．扩增片段大小为１１８４ｂｐ，与预期大小一致．经ＳａｃＩ酶切和巢式ＰＣＲ证实了Ｅ２基因的特异性．将Ｅ２基因扩增片段首先克隆至ｐＧＥＭ－Ｔ载体中，进行全序列测定，将该基因再克隆到表达载体ｐＢＶ２２１．采用温控诱导，ＳＤＳ－ＰＡＧＥ结果显示Ｅ２基因获得表达．ＥＬＩＳＡ表明Ｅ２基因表达产物可与猪瘟阳性血清起特异性反应．

The E2 gene fragment of classical swine fever virus(CSFV)shimen strain was amplified with RT PCR method from RNA extracted from blood of infected pig. The specificity of the amplified E2 gene fragment was confirmed by nested PCR and internal digest of restriction endonuclease. The amplified E2 gene fragment was directly inserted into pGEM T Vector. Then the recombinant plasmid pGEME2 was double digested with NaeI and PstI to release the inserted E2 fragment,the resultant E2 fragment was subcloned into expressing vector pBV221.The recombinant expressing plasmid pBVE2 was induced by heat. SDS PAGE assay showed E2 protein was expressed as expected. The result of ELISA showed that the expressed E2 protein could bind CSFV positive serum with specificity. [WTHZ〗