Construction of pcDNA3.1(+)/EMC6 eukaryotic expression vector and its expression in human liver cell line L-02
Syed Hameed Ali Shah 1,2 , HE Da-wei 3, HE Ao-lin 3, LI Sha-sha 3, WANG Bing-fang 2
(1. School of Medicine, Jiangsu University, Zhenjiang Jiangsu 212013; 2. Department of Gastroenterology， 3. Department of Laboratory, Kunshan Hospital Affiliated to Jiangsu University, Kunshan Jiangsu 215300, China)
Abstract：Objective: To construct an eukaryotic expression vector pcDNA3.1(+)/endoplasmic reticulum membrane protein complex subunit 6(EMC6) and to transfect it into human liver L-02 cell line. Methods: The sequence of EMC6 gene published in Pubmed was analyzed and suitable restriction enzyme cutting site was designed. Human EMC6 gene was amplified by PCR with the cDNA of L-02 cells. The template and the fragment was combined with plasmid pcDNA3.1(+) by gene recombination technology. After constructing an eukaryotic expression vector pcDNA3.1(+)/EMC6, the accuracy of construction was verified by colony PCR and sequencing. The recombinant expression vector pcDNA3.1(+)/EMC6 was transfected into L-02 cells by lipofectamine. The mRNA and protein expression of EMC6 in L-02 cells was respectively detected by RT-PCR and Western blotting. Results: After identification by colony PCR and sequencing, the eukaryotic expression vector pcDNA3.1(+)/EMC6 was successfully constructed. The RT-PCR and Western blotting results showed the overexpression of EMC6 in L-02 cells. Conclusion: The eukaryotic expression vector pcDNA3.1(+)/EMC6 was successfully constructed, and can be highly expressed in L-02 cells. ［Key words］endoplasmic reticulum membrane protein complex subunit 6; recombinant plasmid; vector construction; transfection; L-02 cells
Syed Hameed Ali Shah 1, 2, 何大伟3, 何敖林3, 李莎莎3, 王炳芳2. 含EMC6基因重组真核表达载体的构建及其在人肝L-02细胞中的异位表达[J]. 江苏大学学报:医学版, 2017, 27(06): 466-469.
Syed Hameed Ali Shah 1, 2 , He-Da-Wei-3, He-Ao-Lin-3, Li-Sha-Sha-3, Wang-Bing-Fang-2. Construction of pcDNA3.1(+)/EMC6 eukaryotic expression vector and its expression in human liver cell line L-02. Journal of Jiangsu University(Medicine Edition), 2017, 27(06): 466-469.
2017, Vol. 27 
