Construction of eukaryotic expression vector for IL-37 and EGFP fusion protein and its expression in 293T cells
10.3969/j.issn.1006-5725.2015.09.005
- VernacularTitle:IL-37融合EGFP真核表达载体的构建及其在293T细胞中的表达
- Author:
Qian LIU
;
Zhuangyan LIANG
;
Suhui HE
;
Sen WANG
;
Zhangquan CHEN
- Publication Type:Journal Article
- Keywords:
Interleukin-37;
Green fluorescent protein;
Expression vector;
293T cells
- From:
The Journal of Practical Medicine
2015;(9):1399-1402,1403
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression vector for IL-37 fused with the enhanced green fluorescent protein (EGFP) and express the fusion protein IL-37-EGFP in 293T cells. Methods The IL-37 and EGFP gene were amplified by PCR, respectively. Then the gene fragments of IL-37 and EGFP were in-serted into the pcDNA3.1 vector to construct the recombinant eukaryotic expression vector pcDNA3.1/IL-37-EGFP. The pcDNA3.1/IL-37-EGFP recombinant expression vector was identified by enzymatic digestion , DNA sequencing, PCR identification. Then the recombinant plasmid pcDNA3.1/IL-37-EGFP was used to transfect 293T cells , the expression of EGFP in 293T cells was detected by fluorescence microscope , the expression of IL-37 in 293T cells was detected by RT-PCR and Western blot. Results The results of enzymatic digestion and DNA sequencing showed that the fusion gene of IL-37 and EGFP linked with (G4S)3 was cloned correctly into pcDNA3.1 vector. The green fluorescence was observed in 293T cells transfected with pcDNA3.1/IL-37-EGFP by fluorescence microscope. RT-PCR and Western blot showed that the expression with high level of IL-37 mRNA and IL-37 protein in 293T cells transfected with pcDNA3.1/IL-37-EGFP , respectively. Conclusion The recom-binant eukaryotic expression vector for IL-37 fused with EGFP, pcDNA3.1/IL-37-EGFP, was constructed suc-cessfully and expressed effectively in 293T cells.
