GPx6过表达慢病毒载体和稳定转染细胞系的构建

Construction of GPx6 Over-Expression Lentiviral Vector and Stably Transfected Cell Line

为了建立稳定表达谷胱甘肽过氧化物酶6(Glutathione peroxidase 6,GPx6)的真核表达细胞系,对GPx6进行基因克隆,构建慢病毒表达载体,建立表达猪源GPx6的CHO-K1细胞系.根据NCBI数据库中的猪GPx6基因的c DNA序列,利用Primer5设计PCR扩增引物.在猪附睾组织中克隆出6His-GPx6基因,构建含有6His-GPx6的慢病毒过表达载体,利用三质粒包装系统进行慢病毒包装,慢病毒侵染CHO-K1细胞,进一步筛选获得阳性单克隆细胞系,通过免疫荧光、蛋白印迹和实时荧光定量确定目的基因表达效果.试验成功克隆出6His-GPx6基因,构建了含有6His标签的GPx6过表达慢病毒载体,并成功获得了含有GPx6的CHO-K1细胞系.为下一步研究GPx6在猪繁殖力的提高和精液稀释液添加剂的应用提供新思路.

In order to establish a eukaryotic expression cell line stably expressing glutathione peroxidase6(GPx6),the study carried out gene cloning,construction of lentiviral expression vector,and establishment of expression CHO-K1 cell line with porcine GPx6 gene.PCR amplification primers were designed by Primer5 according to the cDNA sequence of porcine GPx6 gene in the NCBI database.6His-GPx6 was cloned from porcine epididymis,a lentiviral over-expression vector containing 6His-GPx6 was constructed,the three-plasmid packaging system was used for lentiviral packaging,and the lentivirus infected CHO-K1 cells were further screened to obtain positive monoclonal cell lines.The expression effect of GPx6 was determined by immunofluorescence,western blotting and real-time fluorescence quantification.Results showed that GPx6 gene was successfully cloned,a lentivirus over-expression vector containing 6His-GPx6 was constructed,and CHO-K1 cell line containing GPx6 was successfully obtained by infected with lentiviral particles.GPx6 over-expression lentiviral vector was successfully constructed,CHO-K1 cell line expressing the target protein was established,which can provide new ideas for further studies in the improvement of porcine fertility and the application of semen diluent additives.