Gene Cloning, Site-Directed Mutagenesis, Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Selenoprotein X

ZHAO Hua, TANG Jiayong, CAO Lei, ZHOU Jichang, JIA Gang, LIU Guangmang, CHEN Xiaoling, WANG Kangning

doi:10.3969/j.issn.1006-267x.2015.05.019

2015 , Vol. 27 >Issue 5: 1485 - 1491

DOI: https://doi.org/10.3969/j.issn.1006-267x.2015.05.019

Molecular Nutrition

Gene Cloning, Site-Directed Mutagenesis, Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Selenoprotein X

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1. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China;
2. Molecular Biology Lab, Shengzhen Center for Chronic Disease Control, Shenzhen 518020, China

Received date: 2014-11-30

Online published: 2015-05-13

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Abstract

The objective of this experiment was to clone, site-directed mutate, prokaryotic express porcine selenoprotein X(SelX) and prepare its polyclonal antibody for further study of its roles using pig models. Total RNA was extracted from porcine liver for 3'-RACE, and a 1 215 bp cDNA fragment of the SelX containing sequence from the open reading frame (ORF) till to its poly (A) tail was isolated. The 384 bp ORF share an 88% identity to that of human, while their amino acid sequences had 91% identity. The sequence of porcine SelX was submitted to NCBI GenBank with accession number of EF113597. The selenocysteine (Sec) encoded by TGA codon was located in the 95th amino acid position near the C-terminal of SelX. The codon TGA for Sec was site-directed mutated into TGT for cysteine (Cys), then the mutant was cloned into pET30 vector and expressed in E.coli BL21(DE3) induced by 0.5 mmol/L isopropy-β-D-thiogalactoside (IPTG) for 2 hours. The expressed recombinant protein exhibited a molecular weight of approximately 18.0 ku after purified using Ni Sepharose affinity column. The purified SelX mutant protein (SelXm) was used to immunize rabbit and the obtained polyclonal anti-sera showed a 1:20 000 titer. Western-blot assay showed the rabbit anti-sera exhibited a specific immune recognization against the purified SelXm fusion protein and SelX in porcine liver tissue. In conclusion, the porcine SelX gene is successfully cloned and identified, and also its polyclonal antibody is successfully prepared in the present study.

Key words： porcine SelX gene; cloning; site-directed mutagenesis; prokaryotic expression; polyclonal antibody

Cite this article

ZHAO Hua, TANG Jiayong, CAO Lei, ZHOU Jichang, JIA Gang, LIU Guangmang, CHEN Xiaoling, WANG Kangning . Gene Cloning, Site-Directed Mutagenesis, Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Selenoprotein X[J]. Chinese Journal of Animal Nutrition, 2015 , 27(5) : 1485 -1491 . DOI: 10.3969/j.issn.1006-267x.2015.05.019

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