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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。
OBJECTIVE: To construct a mutant strain of Yersinia pestis attenuated vaccine strain of toxins and to establish a gene knockout method for Yersinia pestis, so as to lay the foundation for the study of Yersinia pestis. Methods One Yersinia pestis attenuated vaccine strain (strain No. 0614F) was selected as the experimental strain. Using Artemis software to read Yersinia pestis 91001 strain gene sequence, find and export the toxin (Ymt) and its 100 gene sequences on both sides. The homology region mutation primer and primers were designed. The 0614F mouse toxin encoding gene was knocked out by λ-Red system and identified. Results Plasmid extraction kit Escherichia coli DH5α culture pKD46 plasmid 50μl, the concentration of 121.1ng / μl; homologous region of the mutation cassette primer amplified DNA containing chloramphenicol resistance gene, PCR products were 1% agarose gel electrophoresis After gel electrophoresis, 50 μl of chloramphenicol linear mutant cassette was extracted with a gel recovery kit at a concentration of 724 ng / μl. The strains with ampicillin and chloramphenicol resistance were obtained by two electroporation of strain 0614F. PCR identification, the strain was identified as a toxin gene deletion strain 0614F- △ Ymt. Conclusion The targeted mutagenesis of Yersinia pestis toxin gene was successfully carried out by gene knockout technique, which laid the experimental foundation for the research of novel vaccine against plague strains in Qinghai-Tibet Plateau.