中国畜牧兽医2017，Vol.44Issue(6)：1645-1651,7.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
Correction of the Mutant SLA-1-632-TPK Gene from ToPigs Pig and Construction of SLA-1-TPK/pMD19-T Recombinant Plasmid
摘要
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.关键词
托佩克猪/SLA-1基因/基因矫正/剪切重叠延伸PCR(SOE-PCR)Key words
ToPigs pig/SLA-1 gene/gene correction/SOE-PCR分类
生物科学引用本文复制引用
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):1645-1651,7.基金项目
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) （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) （31672525）
辽宁省教育厅重点实验室项目(LZ2015003) （LZ2015003）
