托佩克猪SLA-1-632-TPK基因的矫正及重组质粒SLA-1-TPK/pMD19-T的构建

doi:10.16431/j.cnki.1671-7236.2017.06.010

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (6): 1645-1651.doi: 10.16431/j.cnki.1671-7236.2017.06.010

托佩克猪SLA-1-632-TPK基因的矫正及重组质粒SLA-1-TPK/pMD19-T的构建

翟晓鑫, 高花, 韩勇, 高凤山

大连大学生命科学与技术学院, 基因和蛋白质工程实验室, 大连 116622

收稿日期:2016-12-06 出版日期:2017-06-20 发布日期:2017-06-28
通讯作者: 高凤山 E-mail:gfsh0626@126.com
作者简介:翟晓鑫(1993-),男,山西晋中人,硕士,研究方向:动物分子免疫学,E-mail:xiaoxinzhai@qq.com
基金资助:
国家自然科学基金项目：猪源病毒CTL多肽表位与SLA-Ⅰ结晶研究（31172304）；国家自然科学基金项目：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）

Correction of the Mutant SLA-1-632-TPK Gene from ToPigs Pig and Construction of SLA-1-TPK/pMD19-T Recombinant Plasmid

ZHAI Xiao-xin, GAO Hua, HAN Yong, GAO Feng-shan

Gene and Protein Engineering Laboratory, College of Life Science and Technology, Dalian University, Dalian 116622, China

Received:2016-12-06 Online:2017-06-20 Published:2017-06-28

摘要/Abstract

摘要：

猪白细胞抗原（swine leukocyte antigen，SLA）在猪免疫系统中起递呈抗原作用，对其展开研究可为猪相关传染病的预防提供依据。研究发现，托佩克猪SLA-1-632-TPK基因编码序列发生碱基缺失（距离SLA-1-632-TPK基因5'端632 bp处丢失1个碱基"C"），导致移码突变。为矫正SLA-1-632-TPK基因，设计2对矫正引物，以原重组质粒SLA-1-632-TPK/pMD18-T为模板，利用剪切重叠延伸PCR（splicing overlap extention PCR，SOE-PCR）技术分别扩增SLA-1-632-TPK 5'和3'端，之后进行拼接，最后扩增全序列从而矫正目的基因，并进一步连接pMD19-T Simple载体，通过单菌落PCR筛选阳性克隆并测序，并通过GENETYX version 9.0软件对所测序列进行分析。结果显示，SOE-PCR成功扩增得到5'和3'端片段，大小约为650和590 bp，与理论设计值大小（648和585 bp）接近，经过拼接以后，得到全长约1 200 bp，与理论设计值1 223 bp接近。菌落PCR结果显示，矫正基因成功克隆入pMD19-T Simple载体。序列分析结果显示，托佩克猪SLA-1-632-TPK基因距离5'端632 bp处丢失的碱基"C"得到矫正并正确编码。本研究成功矫正了SLA-1-632-TPK基因，并构建其重组质粒SLA-1-TPK/pMD19-T，为下一步研究SLA-1-TPK蛋白表达和相关功能奠定基础。

关键词: 托佩克猪; SLA-1基因; 基因矫正; 剪切重叠延伸PCR（SOE-PCR）

Abstract:

The swine leukocyte antigen (SLA) genes in pigs were the important immune gene group in antigen presentation, and studing on SLA could provide the references for the prevention of some infectious diseases. Earlier studies found that SLA-1-632-TPK gene in ToPigs pig had a deleted base in its coding sequence (a single base "C" was lost in 632 bp from the 5' end of the SLA-1-632-TPK gene), which lead to frameshift mutation. In order to correct the SLA-1-632-TPK gene, two pairs of gene-correction's primers were designed to correct the gene by the splicing overlap extension PCR (SOE-PCR) in template of recombinant plasmid of SLA-1-632-TPK/pMD18-T. Firstly,the 5'and 3'ends of SLA-1-632-TPK gene were amplified, respectively, then both of them were spliced and amplified to form an intact SLA-1-632-TPK gene. After detected by agarose electrophoresis, the interest of the product was further cloned into pMD19-T Simple vector. The positive clones were screened by colony PCR and then sequenced. The result showed that the 5'and 3' ends of the SLA-1-632-TPK gene were all amplified successfully by SOE-PCR, with the products of about 650 and 590 bp, which were consistent with the theoretical value of 648 and 585 bp, respectively. After spliced, the intact sequence of SLA-1-632-TPK gene was obtained with the product of about 1 200 bp, which was close to the theoretical value of 1 223 bp. The colony PCR result showed that the corrected gene was successfully inserted into pMD19-T Simple vector . After the sequence was analyzed by GENETYX version 9.0, it was shown that the nucleotide "C" in 632 bp numbered from the 5'end of the gene was added and the SLA-1-632-TPK gene was coded correctly. In this study, the SLA-1-632-TPK was corrected successfully, and the recombinant plasmid SLA-1-TPK/pMD19-T was constructed, which would lay a foundation to further study the protein expression and associated function of SLA-1-TPK.

Key words: ToPigs pig; SLA-1 gene; gene correction; SOE-PCR

中图分类号:

翟晓鑫, 高花, 韩勇, 高凤山. 托佩克猪SLA-1-632-TPK基因的矫正及重组质粒SLA-1-TPK/pMD19-T的构建[J]. 《中国畜牧兽医》, 2017, 44(6): 1645-1651.

ZHAI Xiao-xin, GAO Hua, HAN Yong, GAO Feng-shan. Correction of the Mutant SLA-1-632-TPK Gene from ToPigs Pig and Construction of SLA-1-TPK/pMD19-T Recombinant Plasmid[J]. China Animal Husbandry & Veterinary Medicine, 2017, 44(6): 1645-1651.

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托佩克猪SLA-1-632-TPK基因的矫正及重组质粒SLA-1-TPK/pMD19-T的构建

Correction of the Mutant SLA-1-632-TPK Gene from ToPigs Pig and Construction of SLA-1-TPK/pMD19-T Recombinant Plasmid

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