荷包猪SLA-3-HB 01基因四聚体前体链的构建及表达  

Construction and Expression of Tetramer Precursor Chain of SLA-3-HB01 Gene in Hebao Pig

作　　者：高花[1] 翟晓鑫 姜平[1] 甘慧 张宗辉 许崇波 高凤山[1] GAO Hua;ZHAI Xiaoxin;JIANG Ping;GAN Hui;ZHANG Zonghui;XU Chongbo;GAO Fengshan(College of Life Science and Technology,Dalian University,Dalian 116622,China;North Guangdong Collaborative Innovation and Development Center for Swine Farming and Disease Control,Yingdong College of Life Sciences,Shaoguan University, Shaoguan 512005,China)

机构地区：[1]大连大学生命科学与技术学院,大连116622 [2]韶关学院英东生命科学学院,粤北生猪生产及疫病防控协同创新发展中心,韶关512005

出　　处：《中国畜牧兽医》2018年第10期2691-2699,共9页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学CRISPR/Cas9技术构建sT2细胞系筛选猪源病毒SLA-Ⅰ限制性CTL表位的研究(31672525);辽宁省教育厅重点实验室项目(LZ2015003);辽宁省大学生创新创业训练计划项目(201611258008)

摘　　要：为了构建荷包猪SLA-3-HB01基因四聚体前体链原核表达载体,并获得SLA-3-HB01表达蛋白,试验以SLA-3-HB01/pMD18-T为模板进行PCR扩增四聚体前体链SLA-3-HB01-BSP,并克隆至pMD19-T载体中,经NdeⅠ和XhoⅠ双酶切筛选阳性克隆并测序,目的基因连接至表达载体pET-21a(+),转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达,SDS-PAGE检测目的蛋白大小及表达情况,提取包涵体并进行检测。结果显示,PCR成功扩增得到SLA-3-HB01-BSP,大小为896bp左右。酶切鉴定证实,目的基因成功克隆至pMD19-T载体中,插入片段大小为876bp,阳性克隆经测序后所获序列与原序列一致,并在3′端带有BSP标签序列。酶切鉴定进一步证实成功构建SLA-3-HB01-BSP/pET-21a(+)重组表达载体,经转化及诱导表达,SDS-PAGE检测显示目的蛋白分子质量在33.5ku左右。包涵体蛋白分子质量约33.5ku,与菌体中目的蛋白大小一致,经凝胶成像系统UVP扫描分析,包涵体蛋白纯度接近于90%,符合进行相关结构和功能研究的要求。本研究成功构建了荷包猪SLA-3-HB01基因四聚体前体链的pET-21a(+)重组表达系统,并获得了一定纯度的包涵体蛋白。In order to construct the prokaryotic expression vector of tetramer precursor chain of SLA-3-HB 01 gene in Hebao pig and express the SLA-3-HB01 protein,using SLA-3-HB01/pMD18-T as a template,one pair of primers was designed to amplify the tetramer precursor chain named as SLA-3-HB01-BSP by PCR.The SLA-3-HB01-BSP was cloned into pMD19-T simple vector,the positive clones were double digested by NdeⅠand XhoⅠfollowed by sequencing.The target gene was further ligated into the expression vector pET-21a(+)and transformed into Escherichia coli BL21(DE3).After induction with IPTG,the size of expressed protein was detected by SDS-PAGE,and then inclusion bodies of SLA-3-HB01-BSP protein were extracted and detected.The results showed that SLA-3-HB01-BSP was amplified successfully,the size of product was about 896 bp.The double digestion with NdeⅠand XhoⅠdemonstrated that the target gene was successfully cloned into pMD19-T simple vector with the inserted size of 876 bp.After sequencing and analyzing,it was proved that the target gene was consistent with the primary gene in sequence and a BSP tag was identified on the 3′end of the target gene.The recombinant expression vector SLA-3-HB01-BSP/pET-21a(+)was successfully constructed.SDS-PAGE detection result showed that the molecular weight of the target protein was about 33.5 ku.The molecular weight of inclusion body was about 33.5 ku,which was consistent with that of the target protein in mycoproteins.Scanning by UVP in Gel Imaging system,it was shown that the purity of inclusion body of protein was about 90%,which was suited for the requirements to study the related structure and function of the protein.In this study,the recombinant expression vector pET-21a(+)for SLA-3-HB01 tetramer precursor chain was successfully constructed,and inclusion body of protein with a certain purity was obtained.

关 键 词：猪白细胞抗原(SLA) 原核构建 密码子优化 表达 包涵体 

分 类 号：Q786[生物学—分子生物学]