托佩克猪SLA-2基因克隆与表达  

Cloning and Expression of TOPIGS Pig SLA-2 Gene

作　　者：刘腾飞[1] 冯磊[1] 田永发[1] 刘畅[1] 姜巍[1] 严婷婷[1] 高凤山[1] 

机构地区：[1]大连大学生命科学与技术学院,辽宁大连116622

出　　处：《动物医学进展》2013年第1期19-23,共5页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31172304);辽宁省大学生创新创业训练计划项目(201211258006);大连大学本科生创新教育基金重点项目(2011018)

摘　　要：为提高托佩克猪SLA-2-TPK基因胞外区在pET-21a的表达量,对其5′端进行密码子优化,并设计表达引物,PCR扩增SLA-2-TPK胞外区,然后克隆入pMD○R19-T Simple Vector,经酶切鉴定后连接至pET-21a载体,转化BL21(Rosseta)进行诱导表达,SDS-PAGE检测目的蛋白。PCR结果显示,SLA-2-TPKe大小约为850bp,并成功克隆入pMD○R19-T Simple Vector,双酶切后大小为834bp。酶切后的SLA-2-TPKe成功与pET-21a链接,重组菌经诱导后目的蛋白大小为30.9ku,与密码子优化前重组菌相比,目的蛋白相对表达含量提高约40%。研究证明,密码子优化可明显提高蛋白的表达量,为进行其他蛋白表达研究提供了参考。In order to improve the expression content of the extr＇acellular domain of SLA-2-TPK in TOPIGS pig, codons in 5＇ end of SLA-2-TPK were optimized and a pair of primers for expression of SLA-2-TPK were designed. Then, the extracellular dorfiain of SLA-2-TPK was amplified by PCR followed by cloning the gene into pMD19-T Simple Vector. After identification by digestion with NdeI and Xho I , the SLA-2-TPK was inserted into pET-21a and the recombinant plasmids were transformed into BL21（Rosse- ta）. After induction, the 0bjective protein was detected by SDS-PAGE. The PCR results showed that SLA-2-TPK was about 850 bp and it was successfully cloned into pMD^19-T Simple Vector. After diges- tion, the SLA-2-TPK with 834 bp was ligated with pET-21a. The recombinant BL21 （Rosseta） was in- duced to express the objective proteins with 30.9 ku. Compared with recombinant BL21（Rosseta） before condon-optimization, the relative expression content for codon-optimized SLA-2-TPK was improved with 40%. The research proved that optimization of codons sited at 5r end of SLA-2-TPK can improve the ex- pression content of the genes,and the research will supply a reference for studying other genes.

关 键 词：托佩克猪 SLA-2基因 密码子优化 表达 

分 类 号：S852.4[农业科学—基础兽医学] Q786[农业科学—兽医学]