犬贾第鞭毛虫α-12贾第素基因的原核表达及纯化  

The prokaryotic expression and purification of the α 12-giardin gene of Giardia canis

作　　者：宋百军[1,2] 赵娜[1] 赵源[1] 宫鹏涛[1] 李建华[1] 杨举[1] 李赫[1] 张西臣[1] 

机构地区：[1]吉林大学动物医学学院,长春130062 [2]吉林农业科技学院,吉林吉林132101

出　　处：《黑龙江畜牧兽医》2014年第11期51-54,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(30970322)

摘　　要：为了原核表达、纯化犬贾第鞭毛虫α-12贾第素蛋白,试验经RT-PCR获得α-12贾第素基因片段,克隆至原核表达载体p ET-28a(+),构建重组原核表达质粒p ET-28a-α-12,再转化入E.coli Rosetta(DE3)后,用IPTG诱导表达,表达的重组蛋白经SDS-PAGE和Western-blot分析验证,并用Ni琼脂糖凝胶纯化融合的α-12贾第素蛋白。结果表明:重组原核表达质粒p ET-28a-α-12经双酶切和测序证实构建正确;表达的重组蛋白相对分子质量约为40 ku,且主要以包涵体形式存在;表达量约占菌体总蛋白的15.7%;纯化的重组蛋白纯度达90%以上。To prokaryotically express and purify α 12 - giardin protein of Giardia canis, a gene fragment of the α - 12 giardin was obtained by RT - PCR and cloned into prokaryotic expression vector pET -28a （ ＋ ） to construct a recombinant plasmid pET -28a - α- 12. And then the recombinant plasmid was transformed into E. coll Rosetta （ DE3 ） for the expression using IPTG. The expressed recombinant protein was validated by SDS -PAGE and Western -blot analysis, and then the fused α -12 protein was purified by Ni -Agarose resin. The results showed that the recombinant prokaryotie plasmid pET -28a - α - 12 was constructed correctly, which was proved by double restriction enzyme digestion and se- quencing. The expressed protein had a relative molecular mass of about 40 ku, and existed in the form of inclusion bodies, it accounted for about 15.7% of total bacterial protein in the expression level. The purity of the purified recombinant protein reached more than 90%.

关 键 词：犬贾第虫 α-12贾第素 原核表达 包涵体蛋白 纯化 

分 类 号：S852.722[农业科学—基础兽医学] Q786[农业科学—兽医学]