cDNA cloning and prokaryotic expression of human pancreatic alpha-amylase
- VernacularTitle:人胰腺?-淀粉酶的cDNA克隆和原核表达的初步研究
- Author:
Xiangdong TV
;
Fei XIE
;
Fenghua LAN
;
Zhongyong ZHU
- Publication Type:Journal Article
- Keywords:
alpha-amylase;
Cloning;
Prokaryotic expression;
Purification
- From:
Chinese Journal of Laboratory Medicine
2003;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To obtain recombinant human pancreatic alpha-amylase protein (AMY2A). Methods Human pancreatic AMY2A cDNA was synthesized by RT-PCR using total RNA from human pancreatic tissues and a couple of primers designed according to the know sequence of human pancreatic alpha-amy lase gene, then digested with BamH Ⅰ and Kpn Ⅰ and inserted into the prokaryotic expression plasmid pGEX-5T vector. Construct The prokaryotic expression vector pGEX-5T-AMY2A was constructed and transformed into E. coli BL21 cell . Protein expressed under the induction of IPTG. The inclusion bodies were isolated and solubilized with urea and washed denatured and refolded. The fusion protein wes purified by affinity chrom atography with glutathione agarose. Results Sequence and restrction analysis revealed AMY2A gene was cloned in frame into pGEX-5T, SDS-PAGE profile showed a clear protein band with a relative molecular weight of 84000 and western blot indicated that the expressed product specifically reacted to polyclonal anti -human pancreatic AMY2A genes. Conclusion Human pancreatic AMY2A gene was successfully cloned,expressed and purification.
