摘要
目的构建鼠疫耶尔森氏杆菌减毒疫苗株鼠毒素基因缺失的突变菌株,建立鼠疫耶尔森氏杆菌的基因敲除方法,为研究鼠疫耶尔森氏杆菌基因奠定基础。方法选择1株鼠疫耶尔森氏杆菌减毒疫苗株(菌株号为0614F)为实验菌株。利用Artemis软件阅读鼠疫菌91001株基因序列,查找并导出鼠毒素(Ymt)及其两侧100个基因序列。设计同源区突变盒引物和鉴定引物,采用λ-Red系统一步法敲除0614F鼠毒素编码基因并进行鉴定。结果质粒提取试剂盒提取Escherichia coli DH5α培养物pKD46质粒50μl,浓度为121.1ng/μl;同源区突变盒引物扩增含有氯霉素抗性基因菌株DNA,PCR产物进行1%琼脂糖电泳凝胶电泳鉴定后切胶,用胶回收试剂盒提取氯霉素线性突变盒50μl,浓度为724ng/μl;0614F菌株经两次电转化获得具有氨苄青霉素和氯霉素抗性的菌株,用相应鉴定引物进行PCR鉴定,获得菌株鉴定为鼠毒素基因缺失株0614F-△Ymt。结论应用基因敲除技术对鼠疫耶尔森氏杆菌鼠毒素基因定向突变成功,为青藏高原型鼠疫菌株新型疫苗研究奠定了实验基础。
Objectives To construct a murine toxin deletion mutant of Yersinia pestis and to establish a method of knocking out Y. pestis genes to lay the foundation for the study of the genes of Y. pestis. Methods An attenuated vaccine strain （0614F） was used as the experimental strain. A pair of PCR primers was designed to amplify chloramphenicol resistance genes. The software Artemis was used to read the gene sequences of 91,001 strains to identify toxin genes of Y. pestis with homologous arms. ）,-red based mutagenesis was used to knockout the routine toxin gene of 0614F. Results Fifty μl of the plasmid pKD46 was extracted from Escherichia coli DH5a cultures with a plasmid extraction kit. The concentration of the plasmid was determined to be 121.1 ng/μl. A kit and primers were used to generate desired mutations containing DNA of ehloramphenicol resistance genes. This DNA was amplified with PCR, and PCR products were identified using 1 % agarose gel electrophoresis. A DNA gel recovery kit was used to isolate 50 μl of DNA of chloramphenicol resistance genes. The concentration of the isolated DNA was determined to be 724 ng/μl. After two rounds of electroporation, 0614F was resistant to ampicillin and chloramphenicol. The strain was identified with PCR using corresponding primers, revealed that a Y. pestis strain with a knocked-out murine toxin gene, 0614F-△Ymt, was obtained. Conclusion This study used a gene knockout technique to knock out the murine toxin gene in a strain of Y. pestis. This technique allows genetic manipulation at the chromosomal level. This technique is a powerful tool with which to study the structure and function of Y. pestis genes and it provides a basis for the further study of vaccines against Y. pestis from the Qingha Tibet Plateau.
出处
《中国病原生物学杂志》
CSCD
北大核心
2016年第6期513-516,共4页
Journal of Pathogen Biology
基金
青海省自然科学基金项目(No.2013-Z-906)
