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

作　　者：赵华[1] 汤加勇[1] 曹蕾[1] 周继昌[2] 贾刚[1] 刘光芒[1] 陈小玲[1] 王康宁[1] 

机构地区：[1]四川农业大学动物营养研究所,成都611130 [2]深圳市慢性病防治中心分子生物学实验室,深圳518020

出　　处：《动物营养学报》2015年第5期1485-1491,共7页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金面上项目(31072043;31272468);四川农业大学双支计划

摘　　要：本试验旨在克隆鉴定猪硒蛋白X(Sel X)基因,将其定点突变后进行原核表达,获得重组蛋白,并制备猪Sel X多克隆抗体,为以猪为模型Sel X功能研究奠定基础。利用c DNA末端快速克隆(3'-RACE)技术从猪肝脏总RNA扩增出含开放阅读框(ORF)至poly(A)共1 215 bp的Sel X基因c DNA片段,其348 bp的ORF编码区和对应的氨基酸残基与人相应序列分别有88%和91%序列同源性,猪Sel X基因提交NCBI Gen Bank数据库,序列号为EF113597。ORF编码氨基酸残基中硒代半胱氨酸(Sec)位于C-端第95个残基,其编码密码子TGA经定点突变为半胱氨酸(Cys)的TGT后,将其插入载体p ET30,转入大肠杆菌BL21(DE3)中,在0.5 mmol/L异丙基硫代-β-D-半乳糖苷(IPTG)诱导2 h获得融合表达产物,经His-tag亲和层析后,获得分子质量为18.0 ku的纯化蛋白。将纯化获得的Sel X基因突变体融合蛋白(Sel Xm)免疫兔子,得到效价高达1∶20 000的多克隆抗体,免疫印迹(Western-blot)结果显示该抗体能特异性识别纯化的Sel Xm和猪肝脏中相应Sel X。本试验成功克隆并鉴定了猪Sel X基因,并制备了其多克隆抗体。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-[3-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 SeIX 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Sel X基因 克隆 定点突变 原核表达 多克隆抗体 

分 类 号：S828[农业科学—畜牧学]