Construction and expression of prokaryotic expression vector for Rana catesbeiana ribonuclease gene
- VernacularTitle:牛蛙核糖核酸酶基因原核表达载体的构建及表达
- Author:
Yong FU
;
Yanfang LIU
;
Qin SU
;
Jun ZHAO
;
Jingme ZHANG
- Publication Type:Journal Article
- Keywords:
RC-RNase;
Escherichis coli;
Clone;
Expression
- From:
Journal of Medical Postgraduates
2004;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To clone the RC-RNase gene and prepare its recombinant prokaryotic construct, and then to express RC-RNase protein using Escherichia coli system. Methods: RC-RNase cDNA was obtained by RT-PCR from liver of Rana catesbeiana, and cloned into pUCm-T plasmid for nucleotide sequencing. Its expression construct was prepared using the 6?His vector pRSET-A, and induced to express by IPTG in Escherichia coli BL21(DE3). Western blotting identified the expression product. Results: A 380 bp long cDNA was obtained from liver of Rana catesbeiana, restriction sites and sequence being consistent to those reported for RC-RNase. After introducing the gene into Escherichia coli and through the induction by IPTG, it was observed a new peptide at the expected position (Mr 16000) on SDS-PAGE gel. This product was proved to be the target protein via Western blotting. It existed in a form of inclusion body and its efficiency reached 12.5% of total bacterial proteins. Conclusion: RC-RNase gene was cloned and expressed in Escherichia coli. The protein could be used for characterizing the biological activities and function of RC-RNase.