动物营养学报2015，Vol.27Issue(5)：1485-1491,7.DOI:10.3969/j.issn.1006-267x.2015.05.019
硒蛋白X基因克隆、定点突变、原核表达及多克隆抗体制备
Gene Cloning, Site?Directed Mutagenesis, Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Selenoprotein X
摘要
Abstract
The objective of this experiment was to clone, site?directed mutate, prokaryotic express porcine sel?enoprotein 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 se?quence 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 suc?cessfully prepared in the present study.关键词
猪SelX基因/克隆/定点突变/原核表达/多克隆抗体Key words
porcine SelX gene/cloning/site?directed mutagenesis/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
赵华,汤加勇,曹蕾,周继昌,贾刚,刘光芒,陈小玲,王康宁..硒蛋白X基因克隆、定点突变、原核表达及多克隆抗体制备[J].动物营养学报,2015,27(5):1485-1491,7.基金项目
国家自然科学基金面上项目(31072043,31272468) （31072043,31272468）
四川农业大学双支计划 （）
