副溶血性弧菌耐热直接相关溶血素基因的克隆与序列分析

Cloning and sequence analysis of thermostable direct hemolysin-related hemolysin gene of Vibrio parahaemolyticus

目的探讨克隆副溶血弧菌(Vp)耐热直接相关溶血素基因的方法。方法用PCR技术扩增目的基因,双酶切载体pGEX-3X和目的基因DNA,连接并转化大肠杆菌DH5琢。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Vp14-91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GenBank中的序列有99.1%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的保护性疫苗以及Vp的致病机制研究奠定了基础。

Objective To establish a method for cloning thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus(Vp). Methods After enrichment by PCR, the trh gene, along with the plasmid DNA of pGEX-3X, was digested with two enzymes followed by linkage of the gene and the plasmid, the product of which was transferred into E.coli DH5琢. Identification of the recombinant plasmid was performed by means of PCR, digestion and sequence analysis. Results A fragment about 600 bp was identified in the PCR product of Vp14-91, which was also seen in the product of PCR and enzyme digestion of the recombinant fragment. Sequence analysis demonstrated a homology of 99.1% between the trh gene and the reference gene available in the GenBank. Conclusion trh gene has been successfully cloned with its sequence analyzed, which prepares the ground for developing gene probe and protective vaccine against Vp.