摘要
对从含有鸡毒霉形体H3株TM 1基因的大肠埃希氏菌DH5α中提取的质粒,进行EcoRⅠ和SalⅠ双酶切,获得了大约为786bp的目的片段。将此片段与经EcoRⅠ和SalⅠ双酶切的线性原核表达载体pET 30a 1连接,转化宿主菌BL21(DE3),筛选阳性克隆。提取质粒,经酶切、PCR扩增和测序分析确证其插入正确,从而构建成了重组表达载体。阳性克隆用IPTG诱导表达,表达产物经SDS PAGE电泳分析,证明其分子质量约为29ku;Western blotting分析表明,该蛋白可被鸡毒霉形体阳性血清识别,具有生物学活性。
The abstracted recombinant plasmid of TM-1 gene of Mycoplasma gallisepticum(MG) H3 strain was digested with EcoRⅠ and SalⅠ. The obtained fragment 786 bp in length was inserted into the prokaryotic expression vector pET-30a-1 digested by the same restriction endonucleases and transformed into BL21(DE3). The insert position, size and open reading frame (ORF) were all confirmed to be right by PCR, restriction digestion and sequencing analysis. Thus the prokaryotic expression vector was (constructed) successfully. Positive clone was induced with IPTG for expression of TM-1 gene. SDS-PAGE (analysis) showed that molecular weight of the expressed protein was 29 ku. Western-blotting result showed that the recombinant protein can be recognized by positive serum of MG.
出处
《中国兽医科技》
CSCD
北大核心
2004年第4期18-20,共3页
Chinese Journal of Veterinary Science and Technology