猪肝羧酸酯酶1基因的克隆和在原核细胞中的功能性表达

doi:10.11843/j.issn.0366-6964.2015.04.020

ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2015, Vol. 46 ›› Issue (4): 650-656.doi: 10.11843/j.issn.0366-6964.2015.04.020

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Cloning and Functional Expression of Pig Liver Esterase 1 Gene in Prokaryotic Cells

XIAO Qi-ling^1，2，LI Peng-hui^1，2，YAN Bing-fang³，YANG Lu^1，2，WANG Xi-liang^1，2，XIAO Yun-cai^1，2，LI Zi-li^1，2，LIU Mei^1，2,BI Ding-ren^1，2，SHI De-shi^1，2*

（1． State Key Laboratory of Agricultural Microbiology,College of Veterinary Medicine，Huazhong Agricultural University，Wuhan 430070，China；2.Key Laboratory of Development of Veterinary Diagnostic Products of Ministry of Agriculture,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;3．Department of Biomedical Sciences，Center for Pharmacogenomics and Molecular Therapy，University of Rhode Island，Kingston 02881，USA）

Received:2014-07-04 Online:2015-04-23 Published:2015-04-20
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Abstract

Abstract:

In order to obtain active pig liver carboxylesterase 1 (PLE1) and study its function，PLE1 were obtained by RT-PCR and normal PCR from large white pig liver. PLE1 ORF was linked into the prokaryotic expression vector pET15b，and was functional expressed in E.coli Origami^TM 2(DE3) cells which were transformed ahead with pGro7.The target protein was purified and its enzyme activity was tested.The sequencing results showed that total length of PLE1 ORF is 1 686 bp，which coding 562 aa.The SDS-PAGE and Western Blotting results demonstrated that recombinant protein PLE1 was expressed successfully，and the highest amount was acquired after culture in 30 ℃ for 6 h，and most of the protein is soluble，and the recombinant PLE1 hydrolysis activity for p-NPA is 1.24 U.The research results not only provide high purity active PLE1 for the study of PLE1 hydrolysis of veterinary medicine，but also provide the theory basis，technical methods for the function study of other important PLE isoenzymes.

Key words: pig liver carboxylesterase 1, functional expression, activity measurement

CLC Number:

S852

XIAO Qi-ling，LI Peng-hui，YAN Bing-fang，YANG Lu，WANG Xi-liang，XIAO Yun-cai，LI Zi-li，LIU Mei,BI Ding-ren，SHI De-shi. Cloning and Functional Expression of Pig Liver Esterase 1 Gene in Prokaryotic Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(4): 650-656.

References

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Cloning and Functional Expression of Pig Liver Esterase 1 Gene in Prokaryotic Cells

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