摘要
目的探讨克隆副溶血性弧菌（Ｖｐ）耐热直接溶血毒素基因的方法。方法用ＰＣＲ技术扩增目的基因，双酶切载体 ｐＵＣ１９和目的基因 ＤＮＡ，连接并转化大肠杆菌 ＪＭ１０９。对重组质粒进行 ＰＣＲ鉴定、酶切鉴定和序列分析。结果凝胶电 泳显示，标准株Ｖｐｌ４－９０基因组ＤＮＡ的ＰＣＲ产物长约６００ ｂｐ。阳性克隆的ＰＣＲ产物也为一长约６００ ｂｐ）的条带，经双 酶切可切出一约 ６００ ｂｐ的条带。 ＤＮＡ 测序结果表明与 Ｇｅｎｅｂａｎｋ中的序列有 ９９．６５％的同源性。结论利用该方法成功 克隆了目的基因，为制备用于现场检测的基因探针和Ｖｐ的保护性菌苗以及阐明Ｖｐ的致病机理奠定了基础。
Objective To clone thermostable direct hemolysin (TDH)gene (tdh gene) of Vibrio parahaemolyticus. Methods PCR technique was adopted to amplify the target fragments of tdh gene from the genomic DNA of Vp14-90. Digestion with the restriction endonucleases EcoRI and BamHI was performed and the digested fragments were ligated with plasmid pUC19 followed by digestion in the same manner. The recombinant plasmid was subsequently transformed into E.coli JM109, and the positive clone was screened and identified by PCR and restriction analysis. Sequencing of the recombinant plasmid was con- ducted. Rasults DNA agarose gel electrophoresis showed that the PCR product of Vp14-90 was about 600 bp, confirmed by PCR and double restriction analysis of the positive clone. The homogneity between the positive clone and the reported tdh gene in Genebank was 99.65%. Conxlusion The positive recombinant clone were successfully cloned. which has laid preliminary foundation for studying the pathogenesity of Vibrio parahaemolyticus and also for preparing specific gene probe for the bacterial detection.
出处
《第一军医大学学报》
CSCD
北大核心
2001年第2期136-137,139,共3页
Journal of First Military Medical University
