摘要
目的构建靶向新生隐球菌MIS1基因的siRNA重组表达载体质粒,并进行鉴定。方法根据GenBank的MIS1基因序列,按照载体要求设计单链引物,克隆到空载体psilencer4.1-CMV neo中,经过LiAc化学法将重组质粒转染到新生隐球菌细胞中并用G418筛选,利用real time PCR鉴定阳性细胞的MIS1基因水平。结果重组表达质粒Psilencer4.1-CMV-si-MIS1经PCR、双酶切及测序鉴定,结果证明重组表达载体构建成功,其能在mRNA水平显著抑制MIS1的表达。结论已成功构建新生隐球菌MIS1基因的siRNA表达载体,为深入研究MIs1在隐球菌相关疾病的发生及发展中的作用提供了技术手段。
Objective To construct siRNA expression vector targeting MIS1 gene in Cryptococcus neoformans.Methods Genome sequences of MIS1 gene were retrieved from Genebank and siRNA sequences were designed by Ambion online software.The cDNA was synthesized and inserted into Psilencer4.1-CMV neo,then identified through sequencing and real time PCR.Results The recombinant plasmid of siRNA targeting MIS1 in Cryptococcus neoformans completely coincided with the design,and the mRNA level of MIS1 was inhibited significantly.Conclusion The siRNA expression vector was constructed successfully,and laid a foundation for exploring the function of MIS1 in Cryptococcus neoformans.
出处
《中国真菌学杂志》
2012年第1期17-19,23,共4页
Chinese Journal of Mycology
