| 烟台黑猪SLA-2-YTH原核表达载体的构建及表达 |
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| 引用本文： | 姜巍,董宋鹏,李子彬,姜龙,冯磊,高凤山.烟台黑猪SLA-2-YTH原核表达载体的构建及表达[J].中国畜牧兽医,2014,41(4):85-89. |
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| 作者姓名： | 姜巍 董宋鹏 李子彬 姜龙 冯磊 高凤山 |
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| 作者单位： | （1.大连大学生命科学与技术学院，辽宁大连 116622；2.吉林农业大学生命科学学院，吉林长春 130118；3.河北省涿鹿县医院，河北涿鹿 075600） |
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| 基金项目： | 国家自然科学基金项目（31172304）；地方高校国家级大学生创新创业训练计划（201211258006）。 |
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| 摘 要： | 为构建烟台黑猪SLA-2-YTH基因原核表达载体，本研究设计引物PCR扩增SLA-2-YTH胞外区，将其克隆至pMD 19-T Simple 载体，筛选阳性克隆。阳性克隆经酶切后，进一步与表达载体pET-28a(+)连接，转化BL21（Rosseta）感受态细胞并进行诱导表达，SDS-PAGE检测蛋白表达情况。结果显示，SLA-2-YTH胞外区亚克隆大小为834 bp，酶切鉴定证实其成功插入pET-28a（+）表达载体。SDS-PAGE结果显示，SLA-2-YTH基因导入宿主菌后成功表达，蛋白大小约31.0 ku，与预期结果相符，优化后蛋白相对表达量达25%以上。本研究成功构建了烟台黑猪SLA-2-YTH原核表达载体，获得了表达蛋白，为今后进一步的结构和功能研究奠定基础。
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| 关 键 词： | 烟台黑猪 猪白细胞抗原 原核表达 载体 |
| 收稿时间： | 2013-11-14 |
Construction and Expression of Prokaryotic Expression Vector of SLA-2-YTH Derived from Yantai Black Pig |
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| JIANG Wei,DONG Song-peng,LI Zi-bin,JIANG Long,FENG Lei,GAO Feng-shan.Construction and Expression of Prokaryotic Expression Vector of SLA-2-YTH Derived from Yantai Black Pig[J].China Animal Husbandry & Veterinary Medicine,2014,41(4):85-89. |
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| Authors: | JIANG Wei DONG Song-peng LI Zi-bin JIANG Long FENG Lei GAO Feng-shan |
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| Affiliation: | (1. College of Life Science and Technology, Dalian University, Dalian 116622, China; 2. College of Life Science, Jilin Agricultural University, Changchun 130118, China; 3. Zhuolu County Hospital in Hebei Province, Zhuolu 075600, China） |
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| Abstract: | In order to construct the SLA-2-YTH gene prokaryotic expression vector of Yantai Black pig, a pair of primers for amplifying the extracellular domain of SLA-2-YTH by PCR was designed, followed by cloning the gene into pMD 19-T Simple vector, and then the positive clones could be analyzed directly. After cleavage with NdeⅠand XhoⅠ, the positive clone was successfully inserted into pET-28a(+) and then the recombinant plasmids were transformed into competent cell BL21 (Rosseta).After induction, the protein expression could be detected by SDS-PAGE. The results showed that sub-clone of the extracellular domain of SLA-2-YTH was about 834 bp, and it was successfully cloned into pMD 19-T Simple vector showed by the enzyme analysis. By SDS-PAGE, SLA-2-YTH gene was successfully expressed in Escherichia coli BL21 (Rosseta) and the target protein was about 31.0 ku, which was consistent with prediction. After optimization, the relative expressed content of recombinant SLA-2 protein reached more than 25%. Through the research, we successfully constructed the SLA-2-YTH gene prokaryotic expression vector of Yantai Black pig, and then gained the expressed protein, which would lay the foundation for the future study of structure and function of SLA-2. |
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| Keywords: | Yantai Black pig swine leukocyte antigen prokaryotic expression vector |
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