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632bp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,SOEPCR)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590bp,与理论设计值大小(648和585bp)接近,经过拼接以后,得到全长约1 200bp,与理论设计值1 223bp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632bp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。
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-I-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 spli- cing 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 showedthat the 5rand 3r 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 ob- tained 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 founda- tion to further study the protein expression and associated function of SLA-1-TPK.
出处
《中国畜牧兽医》
CAS
北大核心
2017年第6期1645-1651,共7页
China Animal Husbandry & Veterinary Medicine
基金
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)
