| 猪去乙酰化酶SIRT3的克隆及表达 |
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| 引用本文： | 王候光,马苗鹏,黄魁英,李华周,明飞平,王伟芳,夏枫耿,罗梦晓,杨军,蔡海明,施巨清,黄朝远,楚品品,董建明,朱红霞,张玲华.猪去乙酰化酶SIRT3的克隆及表达[J].中国畜牧兽医,2014,41(5):53-57. |
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| 作者姓名： | 王候光 马苗鹏 黄魁英 李华周 明飞平 王伟芳 夏枫耿 罗梦晓 杨军 蔡海明 施巨清 黄朝远 楚品品 董建明 朱红霞 张玲华 |
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| 基金项目： | 广州市科技攻关项目（201300000040）；广州市科技攻关项目（2014Z1）。 |
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| 摘 要： | 本试验旨在原核表达猪去乙酰化酶SIRT3，并初步鉴定重组蛋白的抗原性和特异性。采用RT-PCR扩增包含SIRT3的完整编码序列；通过Ω-PCR，将SIRT3完整编码序列克隆至原核表达载体pET-28a(+)中，转入大肠杆菌Rosetta（DE3）中进行诱导表达和亲和层析纯化；经免疫印迹分析初步评价SIRT3的抗原性和特异性。重组原核表达质粒pET28a-SIRT3经双酶切及测序鉴定证明构建正确，表达的重组蛋白相对分子质量约39 ku，能被相关抗体识别。结果表明，本试验成功在大肠杆菌中表达猪去乙酰化酶SIRT3，为对其进行进一步研究奠定了基础。
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Cloning and Expression of Sus scrofa Sirtuin 3 |
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| WANG Hou-guang,MA Miao-peng,HUANG Kui-ying,LI Hua-zhou,ING Fei-ping,WANG Wei-fang,XIA Feng-geng,LUO Meng-xiao,ANG Jun,CAI Hai-ming,SHI Ju-qing,HUANG Chao-yuan,CHU Pin-pin,DONG Jian-ming,ZHU Hong-xia.Cloning and Expression of Sus scrofa Sirtuin 3[J].China Animal Husbandry & Veterinary Medicine,2014,41(5):53-57. |
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| Authors: | WANG Hou-guang MA Miao-peng HUANG Kui-ying LI Hua-zhou ING Fei-ping WANG Wei-fang XIA Feng-geng LUO Meng-xiao ANG Jun CAI Hai-ming SHI Ju-qing HUANG Chao-yuan CHU Pin-pin DONG Jian-ming ZHU Hong-xia |
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| Abstract: | The study was aimed to obtain recombinant Sus scrofa sirtuin 3 (SIRT3) in prokaryotic system and preliminarily evaluate its antigenicity and specificity. The fragment which included the SIRT3 complete coding sequence was obtained by RT-PCR and cloned into pET-28a(+) vector, then the recombinant vector was transformed into E.coil Rosetta （DE3） to induce, express and purify, the antigenicity of recombinant SIRT3 was identified by Western blotting. The double enzyme analysis and sequencing proved that recombinant plasmid pET28a-SIRT3 was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 39 ku, was recognized by positive serum antibody. The result showed that the SIRT3 was successfully expressed in E.coil, which would provide a useful tool for designing an in-depth investigation of the role of SIRT3. |
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