| 奶牛骨钙素基因的克隆与原核表达 |
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| 引用本文： | 祝海宝,郭定宗,李家奎,杨世锦,周东海,杨雪.奶牛骨钙素基因的克隆与原核表达[J].中国奶牛,2007(9):2-5. |
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| 作者姓名： | 祝海宝 郭定宗 李家奎 杨世锦 周东海 杨雪 |
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| 作者单位： | 华中农业大学动物营养代谢病与畜产品品质调控研究中心,武汉,430070 |
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| 摘 要： | 以奶牛骨组织提取的RNA为模板，利用RT—PCR技术扩增出奶牛骨钙素全长cDNA，然后将扩增产物重组到PMD-18T载体中，测定了全基因的核苷酸序列。序列分析表明，奶牛骨钙素全长cDNA为303bp，编码100个氨基酸，与GenBank中的X53699的序列完全相同。通过加端PCR技术连接单链DNA片段人工定点同义突变，将奶牛骨钙素成熟蛋白基因中的大肠杆菌稀有密码子同义突变为大肠杆菌常用密码子并亚克隆至PET-32a表达载体．转化到宿主菌BL21（DE3）中，经IPTG诱导后，成功表达出奶牛骨钙素融合蛋白。
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| 关 键 词： | 奶牛 骨钙素 克隆 突变 表达 |
| 文章编号： | 1004-4264（2007）09-0002-03 |
| 收稿时间： | 2007-03-08 |
| 修稿时间： | 2007年3月8日 |
Cloning and Prokaryotic Expression of Bovine Osteocalcin Gene |
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| Zhu Haibao,Guo Dingzong,Li Jiakui,Yang Shijin,Zhou Donghai,Yang Xue.Cloning and Prokaryotic Expression of Bovine Osteocalcin Gene[J].China Dairy Cattle,2007(9):2-5. |
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| Authors: | Zhu Haibao Guo Dingzong Li Jiakui Yang Shijin Zhou Donghai Yang Xue |
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| Institution: | The Center of Animal Nutrition Metabolic Disease and Livestock Products Quality Regulation, Huazhong Agricultural University,Wuhan 430070 |
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| Abstract: | Bovine osteocalcin cDNA gene was amplified from bone of bovine by RT-PCR.PCR product was cloned into PMD-18T plasmid,Sequencing analysis showed that bovine osteocalcin cDNA gene was 303 bp and encoded 100 amino acids,completely matched with X53699 in GenBank.through connecting three artificial single strands DNA which contain fixed-point mutated sites by add-on PCR,we changed some rare codons to the codons that Escherichia coli frequently used in mature bovine osteocalcin gene,then recombined the connected production into PET-32a plasmid and transformed into the competent expressive cells of E.coli BL21(DE3).Recombinant E.coli BL21 was induced by IPTG and osteocalcin fusion protein was successfully expressed. |
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| Keywords: | Cow Osteocalcin Clone Mutation Express |
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