肠炎沙门氏菌鞭毛蛋白基因快速克隆和鉴定

RAPID CLONING AND IDENTIFICATION OF filC g,m GENE OF SALMONELLA ENTERITIDIS

以提纯的肠炎沙门氏菌染色体ＤＮＡ为材料，在一对含有ＣＵＡＣＵＡＣＵＡＣＵＡ的特殊引物引导下，应用ＰＣＲ扩增出鞭毛蛋白基因ｆｌｉＣｇ，ｍ，约１．８ｋｂ左右大小，然后以ＵＤＧ法快速克隆到质粒ｐＡＭＰ１０中，转化第１宿主大肠杆菌ＴＧ１或大肠杆菌ＤＨ５α，在氨苄青霉素平板上筛选转化子，小量制备重组质粒ＤＮＡ，再转入第２宿主大肠杆菌ＬＣ－２ａ（ｈａｇ－，ｒｅｃＡ－），所有转化子均出现动力。其中的一个克隆ｐＡＭＰ·ＧＭ经动力和动力抑制试验、血清学试验、鞭毛电镜观察及部分序列测定，均证实ｐＡＭＰ·ＧＭ中载有肠炎沙门氏菌鞭毛蛋白基因ｆｌｉＣｇ，ｍ。ｆｉｌＣｇ，ｍ克隆成功为寻求肠炎沙门氏菌防制新方法奠定了基础，研究中建立的快速克隆策略为广泛、深入地开展ｆｌｉＣ研究提供了便捷的新途径。

The fliC g,m gene was amplified from purified chromosomal DNA of Salmonella enteritidis by PCR reaction using a pair of particular primers, derived from the upstream and downstream regions of the fliC gene, with the addition of CUACUACUACUA to their 5′ends. After amplification the PCR product was purified, and originally cloned into the plasmid pAMP10 by using the UDG method with DH5α or TG1 as the initial host for recombinant plasmids, then recombinant plasmid DNA of pAMP. GM containing the filC g,m was isolated and transformed into Lc 2a as the second host, a nonflagellated strain of E. Coli. The results of motility/inhibition test, seroagglutination assay, microscope screening of these transformants, and partial sequence of pAMP. GM showed that the filC g,m gene of S. enteritidis has been successfully cloned and expressed. This cloned filC g,m is helpful for the exploration of new approach in the control of S. entertidis. And the established cloning method is very useful in determining the variation and evolution of different fliC genes in Salmonella .