小鼠白细胞介素5真核表达质粒pVAX-1mIL-5的构建及其体外表达

目的构建能表达小鼠白细胞介素5(mIL-5)的质粒并鉴定其蛋白的体外瞬时表达。方法以含有mIL-5cDNA序列的质粒为模板,通过聚合酶链反应(PCR)扩增获得小鼠IL-5基因片段后,定向插入真核表达载体pVAX1中,获得重组表达质粒pVAX1mIL-5。通过双酶切、PCR及插入片段序列测定对质粒进行鉴定。采用间接免疫荧光技术检测pVAX1mIL-5在HeLa细胞的瞬时表达。结果酶切、PCR及测序鉴定证实,插入片段正确,间接免疫荧光检测表明其可以在HeLa细胞中瞬时表达。结论构建的真核表达质粒pVAX1mIL-5可以在HeLa细胞中瞬时表达小鼠IL-5的蛋白,为下一步利用IL-5作为DNA疫苗黏膜免疫佐剂研究奠定了基础。

Construction of eukaryotic expression plasmid pVAX1-mIL-5 and its expression in vitro

Objective: To construct the eukaryotic expression plasmid pVAX1-mIL-5 and examine its expression in vitro.Methods: The DNA fragment encoding signal peptide and mIL-5 core peptide was cloned from the plasmid containing total mIL-5 cDNA by polymerase chain reaction (PCR) and inserted into the vector pVAX1. This recombinant plasmid pVAX1-mIL-5 was identified by digestion of endonuclease, PCR and sequencing. Then, it was transfected into HeLa cell with lipofectin. Expression of mIL-5 in HeLa cell was checked by immunofluorescence with rabbit anti-mIL-5 antibody as the first antibody.Results: mIL-5 DNA was correctly inserted into the vector plasmid pVAX1, which was conformed by the endonuclease digestion, PCR and sequencing. mIL-5 can be detected in the plasmid pVAX1-mIL-5 transfected HeLa cells. These cells showed light-green fluorescence.Conclusion: Eukaryotic expression plasmid pVAX1-mIL-5 was constructed successfully. mIL-5 can be expressed in transfected HeLa cell in vitro. These studies are believed to lay a foundation for the study of IL-5 as mucosal immunity adjuvant for DNA vaccines.