Sry基因的克隆及其在HeLa细胞中的表达

Construction of Recombinant Vector pCR 3.1-Sry and its Expression in HeLa Cells

目的:构建真核表达重组质粒pCR3.1-Sry,并检验其在真核细胞内的表达.方法:用PCR法从小鼠基因组中扩增出Sry全长基因,重组入pMD18-T载体,酶切鉴定重组质粒,对酶切正确的重组质粒测序.双酶切测序验证的重组质粒,将切下的片段与真核表达质粒pCR3.1相连构建pCR3.1-Sry重组质粒.将其转染HeLa细胞后,RT-PCR法检测重组质粒在细胞中mRNA的转录;免疫荧光法检测重组质粒在细胞中蛋白质的表达.结果:PCR法扩增得到了1.2 kb的特异性Sry基因片段;成功地构建了真核表达重组质粒pCR3.1-Sry;重组质粒pCR3.1-Sry在HeLa细胞中mRNA及蛋白水平均可检测到表达.结论:重组质粒构建成功并可以在体外真核细胞中表达,为进一步研究其作为核酸疫苗的免疫作用提供了可控的实验材料.

Objective： To construct the eukaryotic expression plasmid pCR3.1-Sty and transfect to HeLa cells for its expression. Method： Sry （sex-determining region of the Y chromosome, Sry）gene was amplified from the genomic DNA of C57/6J mouse by polymerase chain reaction （PCR）. The product of Sry gene was inserted into cloning vector pMD18- T. After identification by restrictive enzymes digestion and sequencing, the inserted Sry gene was subcloned into pCR 3.1 vector to construct pCR 3. 1-Sty. The recombinant plasmid was transfected into HeLa cells. The mRNA and protein in HeLa cells were detected successfully by RT-PCR and immunofluorescence assay, respectively. Results： The specific gene Sry about 1.2 kb was obtained. The recombinant plasmid pCR3.1-Sry was constructed correctly. The expressed RNA and protein was identified in HeLa cells after transfected with pCR 3.1-Sry. Conclusion： The plasmid was constructed and can express in eukatyotic cells in vitro, which lay the foundation for studying the immune function of the nucleic acid vaccine.