荷包猪SLA-1重链四聚体前体链的构建及表达研究  

Construction and Expression of the Heavy Chain Tetramer Precursor Chain of SLA-1 in Hebao Pig

作　　者：高花[1] 郭杨 翟晓鑫 张宗辉 高凤山[1] GAO Hua;GUO Yang;ZHAI Xiao-xin;ZHANGZong-hui;GAO Feng-shan(College of Life Science and Technology,Dalian University,Dalian 116622,China)

机构地区：[1]大连大学生命科学与技术学院,大连116622

出　　处：《中国畜牧兽医》2017年第12期3453-3458,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31172304;31672525);2015年大连大学大学生创新创业训练计划项目(2015075)

摘　　要：试验旨在构建荷包猪猪白细胞抗原1(swine lymphocyte antigen 1,SLA-1)重链四聚体前体链并研究其在表达载体pET-21a(+)中蛋白的表达情况。以SLA-1全基因序列为模板,结合表达载体的特点设计引物,利用PCR扩增技术在SLA-1-HB01胞外区序列C-末端加载上可生物素酰化序列(BirA substrate peptide,BSP)。将PCR扩增产物SLA-1-HB01-BSP克隆到pEASY T1载体上,筛选出阳性克隆菌SLA-1-HB01-BSP/pEASY T1,经双酶切后进一步与表达载体pET-21a(+)连接,再转化到E.coli BL21感受态细胞中构建pET-21a(+)/SLA-1-HB01-BSP重组表达菌,通过IPTG诱导蛋白表达,SDS-PAGE检测蛋白的大小及表达情况,提取包涵体检测蛋白的纯度。PCR扩增结果显示,SLA-1-HB01-BSP大小为898bp,与理论值相符,扩增片段成功克隆至pEASY T1载体构建克隆菌,经NdeⅠ和XhoⅠ双酶切筛选及测序,成功获得阳性克隆菌株SLA-1-HB01-BSP/pEASY T1,插入的目的基因片段大小为876bp。阳性克隆菌株与pET-21a(+)表达载体经NdeⅠ和XhoⅠ双酶切后进行连接,转化E coli BL21感受态细胞后获得pET-21a(+)/SLA-1-HB01-BSP重组表达菌,经IPTG诱导表达,SDS-PAGE检测目的蛋白大小为31.4ku。进一步检测分析发现,目的蛋白以包涵体形式存在,且蛋白纯度达到80%以上。本研究成功构建了荷包猪SLA-1-HB01重链四聚体前体链的pET-21a(+)重组表达体系,为下一步进行猪SLAⅠ类分子四聚体的检测奠定基础。To construct tetramer precursor chain of swine lymphocyte antigen1(SLA-1)heavy chain in Hebao pig and study its protein expression in the pET-21a(+)vector,the SLA-1complete genome sequence was referenced with the characteristics of the expression vector,and a pair of primers was designed to integrate the BirA substrate peptide(BSP)sequence at the C-terminus of the SLA-1-HB01and the SLA-1-HB01-BSP was amplified by PCR.Then,the products were cloned into the pEASY T1vector and the positive clones of SLA-1-HB01-BSP/pEASY T1was selected.After the double digestion,the interest of the gene in positive clone was further ligated into the pET-21a(+)expression vector and transformed into E.coli BL21to construct the recombinant strain of pET-21a(+)/SLA-1-HB01-BSP.After induction with IPTG,the target protein were detected by SDS-PAGE.Finally,the inclusion body of the SLA-1-HB01-BSP was isolated and detected to evaluate its purity.The PCR results showed that the length of SLA-1-HB01-BSP was about898bp,which was consistent with the theoretical value.The amplified fragment was successfully cloned into pEASY T1vector,and the positive clones were identified by NdeⅠand XhoⅠdigestion.The size of inserted fragment was876bp.The interest of gene was also inserted into pET-21a(+)and transformed into E.coil BL21successfully.After induction,SDS-PAGE detection results showed that the target protein was31.4ku.Further detection showed that the target protein was mainly expressed as inclusion bodies,and the purity of the protein was about80%.In this study,the recombinant tetramer precursor of SLA-1-HB01heavy chain was constructed in pET-21a(+)expression line successfully,which would lay a foundation to detect the tetramer of SLA classⅠmolecular.

关 键 词：荷包猪 猪白细胞抗原1 四聚体 可生物素酰化序列 

分 类 号：Q786[生物学—分子生物学]