| 破伤风毒素C片段基因的克隆及在大肠杆菌中的高效表达 |
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| 引用本文： | 程宁宁,单艳菊,潘志明,刘婷,耿士忠,黄金林,焦新安.破伤风毒素C片段基因的克隆及在大肠杆菌中的高效表达[J].中国预防兽医学报,2007,29(6):439-441,447. |
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| 作者姓名： | 程宁宁 单艳菊 潘志明 刘婷 耿士忠 黄金林 焦新安 |
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| 作者单位： | 扬州大学,江苏省人兽共患病学重点实验室,江苏,扬州,225009 |
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| 基金项目： | 国家自然科学基金;教育部全国优秀博士学位论文作者专项基金 |
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| 摘 要： | 本研究对破伤风毒素C片段进行了基因克隆、重组表达、蛋白纯化和免疫原性分析。应用PCR技术直接从破伤风梭菌64008菌株基因组中扩增出大小为1356 bp的破伤风毒素C片段(TetC)基因,经DNA序列测定分析,扩增出的基因与GenBank上登录的序列AF154828的同源性达到99.2%。将此基因克隆至大肠杆菌融合表达载体pGEX-6P-1,构建成重组表达质粒pGEX-6P-1-TetC,并在大肠杆菌BL21中表达,重组蛋白的表达量占菌体总蛋白的21%。经SDS-PAGE蛋白电泳鉴定,表达产物为76 Ku左右的重组蛋白,经免疫印迹试验证实该重组蛋白是破伤风毒素C片段抗原。
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| 关 键 词： | 破伤风毒素 大肠杆菌 基因克隆 表达 蛋白纯化 |
| 文章编号： | 1008-0589（2007）06-0439-04 |
| 修稿时间： | 2006-09-28 |
Cloning and high-level expression of tetanus toxin fragment C in E.coli |
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| CHENG Ning-ning,SHAN Yan-ju,PAN Zhi-ming,LIU Ting,GENG Shi-zhong,HUANG Jin-lin,JIAO Xin-an.Cloning and high-level expression of tetanus toxin fragment C in E.coli[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(6):439-441,447. |
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| Authors: | CHENG Ning-ning SHAN Yan-ju PAN Zhi-ming LIU Ting GENG Shi-zhong HUANG Jin-lin JIAO Xin-an |
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| Institution: | Key Laboratory of Zoonosis of Jiangsu Province, Yangzhou University, Yangzhou 225009, China |
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| Abstract: | Gene expression and immunogenicity of the freagment C of tetanus toxin was investigated.The gene of tetanus toxin fragment C was amplified by PCR from the genomic DNA of Clostridium tetani strain 64008.The PCR product was inserted into the pMD19-T vector for sequencing and subcloned into the high-expression vector pGEX-6P-1 and expressed in E.coli BL21. The results showed that the fragment C shared 99.2% sequence homology with AF154828 deposited in GenBank.The expressed protein was purified by Redipack GST purification kit.SDS-PAGE revealed that the recombinant protein has a molecular weight of 76 Ku and accounted for 21% of the total bacterial protein.Western blot analysis further indicated that this protein possessed full immunogenicity of tetanus toxin fragment C. |
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| Keywords: | tetanus toxin fragment C E coli gene cloning expression protein purification |
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