Construction of rat CnA/EGFP recombinant eukaryotic gene coexpression plasmid
- VernacularTitle:大鼠CnA/EGFP基因共表达的真核表达质粒的构建
- Author:
Xiaomei SHEN
;
Juyan ZHANG
;
Bei CHENG
- Publication Type:Journal Article
- Keywords:
calcineurin;
gene expression;
vector;
enhanced green fluorescent protein;
cardiomyocytes
- From:
Journal of Jilin University(Medicine Edition)
2006;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct rat calcineurin catalytic subunit ?(CnA)/enhanced green fluorescent protein EGFP gene coexpression plasmid for exploring the effect of calcineurin on the myocardium apoptosis induced by ischemia-reperfusion.Methods Total RNA was isolated from the heart of the adult Wistar rat,and CnA CDS segment of approximate 1 590 bp size was amplified by reverse transcription PCR method.CnA cDNA segment was cloned into pMD18-T Simple vector for sequencing,and the right clone was named T-CnA.CnA cDNA segment excised from plasmid T-CnA was ligated with pShuttle2 which had inserted IRES-EGFP segment into before.The DNA of the recombinant plasmid was extracted and was identified by double digesting with Nhe Ⅰ and Not Ⅰ.The right clone was named pShuttle2-CnA-IRES-EGFP.Results Sequencing result verified that the PCR product of CnA gene was identical to GenBank(NM_017041).1% agarose electrophoresis showed the bands of recombinant plasmid pShuttle2-CnA-IRES-EGFP digested by Nhe Ⅰ and Not Ⅰ were in the right range corresponding with expectation(1 590 bp and 5 240 bp).Conclusion Recombinant eukaryotic expression plasmid carrying rat CnA cDNA as well as a report gene-EGFP gene is successfully constructed in this experiment.
