猪RUVBL2真核表达载体的构建及其在CHO细胞中的表达

Construction of RUVBL2 Eukaryotic Expression Vector of Pig and Its Expression in CHO Cells

RUVBL2(RuvB-like 2)蛋白是进化上高度保守AAA+(ATPases Associated With Diverse Cellular Activities,AAA)家族成员之一,CHO(Chinese Hamster Ovary)细胞被广泛地用于表达重组DNA蛋白。为探究猪睾丸组织的RUVBL2基因是否能够在CHO细胞中表达,本实验以猪睾丸组织的cDNA为模板,PCR扩增RUVBL2目的基因,并将其克隆至pIRES2-EGFP(Mammalian Expression Vectors pIRES2-EGFP)载体上,进一步转化到DH5α中,再进行PCR、酶切及测序鉴定;将重组质粒转染到CHO细胞中,再进行荧光、RT-PCR、Western blot检测。结果显示:PCR、酶切及测序都证实了RUVBL2基因正确地插入到了载体质粒pIRES2-EGFP的多克隆位点;荧光、RT-PCR、Western blot也证实了RUVBL2基因在CHO细胞中的成功表达。本实验成功构建了猪的pIRES2-EGFP-RUVBL2真核表达载体,并证实了猪睾丸组织中的RUVBL2基因能够在CHO细胞中表达。

RUVBL2(RuvB-like 2)protein is one of the evolutionarily highly conservative AAA+(ATPases associated with diverse cellular activities,AAA)family members,and CHO(Chinese hamster ovary)cells are widely used to express recombinant DNA proteins.To investigate whether the RUVBL2 gene of pig testis tissue can be expressed in CHO cells,using the cDNA of porcine testis tissue as a template in this experiment,the RUVBL2 target gene was amplified by PCR,and it was cloned into pIRES2-EGFP(Mammalian Expression Vectors pIRES2-EGFP)vector,and further transformed into DH5α,then identified by PCR,restriction enzyme digestion and sequencing.The recombinant plasmid was transfected into CHO cells and detected by fluorescence,RT-PCR and western blot.Results showed that PCR,restriction enzyme digestion and sequencing all confirmed that the RUVBL2 gene was correctly inserted into the multiple cloning site of the vector plasmid pIRES2-EGFP,and fluorescence,RT-PCR and western blot all confirmed that the RUVBL2 gene was successfully expressed in CHO cells.In conclusion,this experiment constructed the pIRES2-EGFP-RUVBL2 eukaryotic expression vector of pig,and confirmed that the RUVBL2 gene of pig testis tissue can be expressed in CHO cells.