| 巴什拜羊SPLUNC1基因的克隆与真核表达 |
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| 引用本文： | 王继雪,沈文,孙延鸣.巴什拜羊SPLUNC1基因的克隆与真核表达[J].中国畜牧兽医,2018,45(3):628-634. |
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| 作者姓名： | 王继雪 沈文 孙延鸣 |
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| 作者单位： | 石河子大学动物科技学院, 石河子 832003 |
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| 基金项目： | 国家自然科学基金项目（31460686） |
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| 摘 要： | 为了克隆巴什拜羊SPLUNC1基因并表达该蛋白，本试验从巴什拜羊肺脏组织中提取总RNA，利用RT-PCR方法扩增出SPLUNC1基因开放阅读框序列，将该基因片段插入到真核表达质粒pPIC9K中构建pPIC9K-SPLUNC1重组质粒，然后将pPIC9K-SPLUNC1重组质粒线性化并电击转化入毕赤酵母GS115中，经甲醇诱导蛋白表达后用SDS-PAGE和Western blotting分析鉴定目的蛋白。结果表明，经RT-PCR扩增成功获得大小为748 bp的SPLUNC1基因；构建的pPIC9K-SPLUNC1重组质粒经PCR、酶切及测序鉴定与预期结果一致；SDS-PAGE及Western blotting检测鉴定结果表明获得大小为25.53 ku的SPLUNC1蛋白。本试验结果为进一步研究巴什拜羊SPLUNC1蛋白的生物学活性奠定基础。
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| 关 键 词： | 巴什拜羊 SPLUNC1基因 克隆 真核表达 |
| 收稿时间： | 2017-09-18 |
Cloning and Eukaryotic Expression of SPLUNC1 Gene of Bashibay Sheep |
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| WANG Jixue,SHEN Wen,SUN Yanming.Cloning and Eukaryotic Expression of SPLUNC1 Gene of Bashibay Sheep[J].China Animal Husbandry & Veterinary Medicine,2018,45(3):628-634. |
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| Authors: | WANG Jixue SHEN Wen SUN Yanming |
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| Institution: | College of Animal Science and Technology, Shihezi University, Shihezi 832003, China |
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| Abstract: | In order to clone Bashibay sheep SPLUNC1 gene and express its protein,total RNA was extracted from lung of Bashibay sheep,RT-PCR was used to amplify SPLUNC1 gene open reading frame sequence,the gene fragment was inserted into eukaryotic expression plasmid pPIC9K to construct plasmid pPIC9K-SPLUNC1,then the plasmid pPIC9K-SPLUNC1 was linearized and electroporated into Pichia pastoris GS115,the protein was induced by methanol,and the target protein was identified by SDS-PAGE and Western blotting.The results showed that the 748 bp SPLUNC1 gene was successfully amplified by RT-PCR;The plasmid pPIC9K-SPLUNC1 was consistent with expected results after PCR,enzyme digestion and sequencing identification;Detection results of SDS-PAGE and Western blotting showed that the SPLUNC1 protein was successfully expressed with 25.53 ku.This study laid a foundation for further research of SPLUNC1 protein biological activity of Bashibay sheep. |
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| Keywords: | Bashibay sheep SPLUNC1 gene cloning eukaryotic expression |
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