Construction of pEGFP-C1-CXCL1 plasmid and detection of its expression in eukaryotic cells
10.7644/j.issn.1674-9960.2017.10.002
- VernacularTitle:重组pEGFP-C1-CXCL1真核表达载体构建及其对内质网应激下人肝癌细胞HepG2的增殖作用
- Author:
Rui-Tao MAO
1
;
Wei CHEN
;
Zi-Hui LI
;
Ling ZOU
;
Jia-Zhou YE
;
Tao BAI
;
Jie CHEN
;
Jian-Kang CHEN
;
Cui WANG
;
Ning LIU
;
Xiao-Li YANG
;
Cong-Wen WEI
;
Hui ZHONG
;
Fei-Xiang WU
Author Information
1. 吉林大学中日联合医院
- Keywords:
chemotactic factors;
CXCL1;
recombinant plasmid;
cell proliferation;
carcinoma;
hepatocellular
- From:Military Medical Sciences
2017;41(10):792-795
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the pEGFP-C1-CXCL1 eukaryotic expression vector and to investigate the effect of CXCL1 on the proliferation of HepG2 cells under endoplasmic reticulum stress ( ERS).Methods Fragments of CXCL1 were obtained from the cDNA library of HepG2 cells before CXCL1 was cloned into a pEGFP-C1 vector for a recombinant plasmid pEGFP-C1-CXCL1 which was screened and identified by PCR and sequence alignment .Then,the recombinant plas-mid of pEGFP-C1-CXCL1 was transfected into human 293 T cell line and the expression of CXCL 1 was detected by fluores-cence microscopy and Western blotting.pEGFP-C1-CXCL1was furhter transfected into HepG2 cells, and CCK8 was used to detect the inhibitory effect of CXCL1 on tumor proliferation induced by TM in hepatocellular carcinoma .Results pEGFP-C1-CXCL1 was vertified by sequencing analysis .Fluorescence microscopy showed that pEGFP-C1-CXCL1 was transfected into 293T.CXCL1 expression was detected by Western blotting .CCK8 showed that TM inhibited tumor proliferation , while overexpression of CXCL1 decreased the inhabitory rate on cell proliferation of HepG 2 cells under ER stress compared to pEGFP-C1 group and the control group .Conclusion A recombinant pEGFP-C1-CXCL1 plasmid is successfully constructed that can be expressed stably in human 293T cells.Overexpression of CXCL1 can effectively reduce the inhabitory rate of HCC cells induced by the ER stress.
