副溶血弧菌tlh基因的克隆及序列分析

目的　构建副溶血弧菌不耐热性溶血毒素 (thermolabilehemolysin ,TLH)tlh基因重组质粒。方法　根据已知GenBank中的tlh基因序列 ,设计合成一对引物 ,用PCR方法从副溶血弧菌基因组DNA中扩增编码TLH的基因片段 ,克隆至pET32a+ 质粒 ,转化大肠杆菌DH5感受态细胞 ,经酶切及PCR鉴定 ,而后进行测序。结果　tlh基因体外扩增产物大小约1 30 0bp ,重组质粒经酶切及PCR鉴定表明获得正确重组子 ,测序结果与已知序列基本吻合。 结论　在国内首次克隆了副溶血弧菌tlh基因 ,为研究TLH的功能和探讨TLH作为作为特异性诊断靶抗原的研究奠定了基础

Cloning and sequence analysis of the tlh gene of vibrio parahaemolyticus

Aim To construct a recombinant plasmid containing thermolabile hemolysin (TLH) gene of vibrio parahaemolyticus (VP) Methods A couple of primers were designed for PCR according to the known sequence of tlh gene The tlh gene obtained by amplification from genomic DNA of vibrio parahaemolyticus by PCR technique was cloned into plasmid pET32a + directionally,the constructed recombinant plasmid was transferred into E coli DH 5 The transformants were screened and identified by restriction analysis and PCR Then the cloned genes were sequenced Result The size of amplified tlh gene was 1280 bp The correct recombinant plasmid pET32a + tlh was isolated and confirmed by restruction analysis and PCR DNA sequencing showed the DNA sequence of the cloned gene was the same as the published sequence Conclusion The tlh gene can be successfully amplified and cloned into plasmid pET32a + The study provides the basis material for the further study of the function and diagnosis of tlh gene of VP