荷包猪SLA-2-HB01真核表达载体的构建及在PK15细胞中的表达  

Construction of Eukaryotic Expression Vector of SLA-2-HB01 from Hebao Pigs and Its Expression in PK15 Cells

作　　者：翟晓鑫 高花[1] 姜平[1] 许崇波 张宗辉 李文哲[3] 高凤山[1] ZHAI Xiao-xin;GAO Hua;JIANG Ping;XU Chong-bo;ZHANG Zong-hui;LI Wen-zhe;GAO Feng-shan(College of Life Science and Technology,Dalian University,Dalian 116622;College of Basic Medical Science,Dalian MedicalUniversity,Dalian 116044;North Guangdong Collaborative Innovation and Development Center for Swine Farming and Disease Control,Yingdong College of Life Sciences,Shaoguan University,Shaoguang 512005)

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 [3]大连医科大学基础医学院,大连116044

出　　处：《生物技术通报》2018年第12期132-139,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31672525);辽宁省教育厅重点实验室项目(LZ2015003)

摘　　要：为构建荷包猪SLA-2-HB01真核细胞表达载体,观察其在PK15细胞中的表达情况。首先参考SLA-2-HB01基因编码区序列设计了引物对,以SLA-2-HB01-pMD 18-T为模板PCR扩增获得SLA-2-HB01编码区基因片段,经TA克隆及菌落PCR筛选得到阳性克隆菌。测序正确后大量双酶切回收目的基因片段并与真核表达载体pEGFP N3相连接获得重组质粒SLA-2-HB01/pEGFP N3,重组质粒转化至大肠杆菌经大量克隆后,抽提阳性重组质粒并通过脂质体介导转染至PK15细胞,通过荧光显微镜观察转染后PK15细胞的荧光强度,进一步通过Western Blotting检测PK15细胞中SLA-2-HB01蛋白的表达情况。结果显示PCR成功扩增得到SLA-2-HB01,大小为1 104 bp,并成功构建重组质粒SLA-2-HB01/pEGFP N3,酶切鉴定证实插入片段大小为1 092 bp。SLA-2-HB01/pEGFP N3重组质粒转染PK15细胞后具有绿色荧光,Western Blotting显示SLA-2-HB01-EGFP融合蛋白分子量大小为70 kD,与理论值相符。成功构建了SLA-2-HB01/pEGFP N3重组质粒,并初步确认SLA-2-HB01-EGFP融合蛋白成功在PK15细胞中表达,为下一步进行SLA-2-HB01递呈多肽表位的研究奠定基础。This work is to construct a eukaryotic expression vector of SLA-2-HB01in Hebao pigs and observe its expression in PK15cells.Firstly,a pair of primers was designed based on the sequence of coding region of SLA-2-HB01gene,then the gene fragment of codingregion of SLA-2-HB01gene was amplified using SLA-2-HB01-pMD18-T as template.The positive clones were sequenced after TA cloningand screened by colony PCR.The clones with correct sequences were cleaved and the target genes were recovered to ligate with the eukaryoticexpression vector pEGFP N3.The recombinant plasmid SLA-2-HB01/pEGFP N3was transformed into Escherichia coli firstly and to clone ata high copy way,then a large amount of extracted positive plasmids were transfected into PK15cells by liposome method.The expressionof the SLA-2-HB01protein was detected by observing the fluorescence intensity of PK15cells with fluorescence microscopy and by Westernblotting.As results,the SLA-2-HB01with1104bp was successfully amplified,and the recombinant plasmid SLA-2-HB01/pEGFP N3wasconstructed successfully.The inserted fragment was proved to be1092bp by enzymatic cleavage.The SLA-2-HB01/pEGFP N3recombinant plasmid transfected into PK15cells displayed green fluorescence.Western blotting showed that the molecular weight of the SLA-2-HB01-EGFPfusion protein was70kD,which was consistent with the theoretical value.In conclusion,the recombinant plasmid SLA-2-HB01/pEGFP N3was successfully constructed and expressed in PK15cells as fusion protein SLA-2-HB01-EGFP,which lays a base for further studying thepresentation of peptide epitopes for SLA-2-HB01.

关 键 词：荷包猪 PK15细胞 SLA-2 真核表达载体 

分 类 号：Q78[生物学—分子生物学] S828[农业科学—畜牧学]