Construction of pcDNA3.1(+)/EMC6 Eukaryotic Expression Vector And Its Expression in Human Liver Cell Line L-02

Abstract

The present is aim to examine the construction of pcDNA3.1(+)/EMC6 eukaryotic expression vector and its expression in human liver cell line L-02. This will also explain various stages including PCR amplification of target gene fragment, Plasmid Extraction and transfection, Detection of EMC6 mRNA expression by fluorescence quantitative PCR (RT-PCR), and EMC6 protein expression was detected by Western blot (Western-blot). Statistical analysis was performed with the help of SPSS17.0 Software. Results for the study have shown that the autophagy is a highly conserved process evolution; eukaryotic cells by autophagy can remove misfolded proteins and damaged organelles and macromolecules to maintain cellular homeostasis, its basic process, autophagy induction, formation, autophagy and autophagy by soluble hydrolases to lysosomes of the autophagy package enzyme fusion, substance. Also, The eukaryotic expression vector pcDNA3.1(+)/EMC6 is successfully constructed, and highly expressed in L-02 cells. To construct a eukaryotic expression vector pcDNA3.1(+)/Endoplasmic reticulum membrane protein complex subunit 6(EMC6) and to overexpress it in the human liver L-02 cell line. The sequence ofEMC6，gene published in PubMed was analysed, a suitable restriction enzyme cutting site was designed. The Human EMC6 gene was amplified by PCR with the cDNA of L-02 cells. The template and the fragment were combined with plasmid pcDNA3. 1(+) by gene recombination technology. After constructing a eukaryotic expression vector pcDNA3.1(+)/EMC6, the accuracy of it 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-blot. After identification by Colony PCR and sequencing, the eukaryotic expression vector pcDNA3.1(+)/EMC6 was successfully constructed. The qPCR and Western-blot results showed the overexpression of EMC6 in L-02 cells. The eukaryotic expression vector pcDNA3.1(+)/EMC6 is successfully constructed, and highly expressed in L-02 cells.