基于IRES序列的多基因共表达载体构建  

Construction of the IRES-based Vector for Multiple Gene Co-expression

作　　者：田聪慧 谢雪梅[1] 李英[1] 尹晓东 韩军[1] 李军 TIAN Cong-hui XIE Xue-mei LI Ying YIN Xiao-dong HAN Jun LI Jun(School of Pharmacy and Institute of BioPharmaceutical Rescarch, Liaocheng University, Liaocheng 252000, China Yixing Cell Biotechnology Limited Company, Yixing 214200, China)

机构地区：[1]聊城大学药学院生物制药研究院,聊城252000 [2]宜兴市赛尔生物科技有限公司,宜兴214200

出　　处：《中国生物工程杂志》2017年第7期97-104,共8页China Biotechnology

基　　金：国家自然科学基金青年项目(81402512);聊城市科技发展计划项目(2014GJH11);泰山学者工程专项资助项目

摘　　要：目的:构建一个IRES序列介导的多基因共表达载体,实现两个目的基因和筛选标记基因共用一个启动子高效表达,提高多基因稳定共表达细胞株的筛选效率。方法:以实验室前期构建的载体pLV-MCS-Puro为骨架,设计并全基因合成双基因克隆表达元件,连接到骨架载体,构建多基因共表达载体pLV-2MCS-Puro,以DsRed2和EGFP荧光蛋白基因验证该载体用于多基因稳定共表达细胞株筛选的效率。结果:成功构建了pLV-2MCS-Puro载体以及DsRed2和EGFP共表达重组质粒pLV-DsRed2-EGFP-Puro。瞬时转染实验证明该载体能介导多基因共表达。抗性筛选获得了MDCK和HeLa两种细胞的多基因稳定共表达细胞池。细胞池涂片荧光显微镜观察和计数表明抗性细胞池DsRed2和EGFP双阳率接近100%。基因组和转录水平PCR及蛋白质免疫印迹实验表明,DsRed2和EGFP稳定整合到抗性细胞基因组,并且两种蛋白质表达水平较为一致。结论:成功构建了多基因共表达载体pLV-2MCS-Puro,实现了两个目的基因和抗性基因串联共表达,并且具有高效的多基因稳定共表达细胞株筛选效率。该载体在研究蛋白质相互作用及工程细胞构建等方面具有一定的应用前景。Objective： An IRES-based vector was constructed to achieve co-expression of two target genes with the screening marker gene promoted by the single promoter, and to improve the screening efficiency of multiple genes co-stable expression cell lines. Methods： A bicistronic expression element BamHI-MCSI-IRES- MCS2-IRES-BsiWI which has two multiple cloning sites was designed and synthesized. The vector named pLV- 2MCS-Puro was constructed by inserting the element into the skeleton vector pLV-MCS-Puro which was constructed previously in lab. The DsRed2 and EGFP genes were inserted simultaneously into the vector to test the screening efficiency of multiple genes co-stable expression cell lines. Results： The vector pLV-2MCS-Puro and the recombinant plasmid pLV-DsRed2-EGFP-Puro were constructed successfully. Transient transfection experiment showed that the vector can mediate co-expression of multiple genes. MDCK and HeLa cell pools resistant to puromycin were obtained through transfection of the recombinant plasmid. The fluorescent inverted microscope showed that DsRed2 gene at the upstream of the IRES sequence and EGFP gene at the downstream of IRES sequence were co-expressed in cells, and the double positive rate was close to 100%. It indicated that this vector has high screening efficiency. The results of genomic PCR, RT-PCR and Western blot showed that DsRed2 and EGFP genes were stably integrated into cell genome and the two proteins were expressed consistently. Conclusion： The IRES-based vector pLV-2MCS-Puro was successfully constructed and proved to be efficiently in screening multiple genes co-stable expression cell lines. This vector will have certain application prospects in studying protein interactions and constructing engineering cell lines.

关 键 词：IRES 载体构建 基因共表达 

分 类 号：Q782[生物学—分子生物学]