| 水牛MyD88 cDNA的克隆与原核表达 |
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| 引用本文： | 张晓茹,匡文华,成鹰,杜丽,张冬琳,郝永昌,雷明,焦寒伟,刘涛,祁超,王凤阳.水牛MyD88 cDNA的克隆与原核表达[J].广西农业生物科学,2011,30(2):190-196. |
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| 作者姓名： | 张晓茹 匡文华 成鹰 杜丽 张冬琳 郝永昌 雷明 焦寒伟 刘涛 祁超 王凤阳 |
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| 作者单位： | 1. 华中师范大学生命科学学院,武汉,430079
2. 海南大学农学院海南省热带动物繁育与疫病研究重点实验室（筹）,海口市动物基因工程重点实验室,海口570228 |
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| 基金项目： | 国家转基因生物新品种培育重大专项 |
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| 摘 要： | 采用RT-PCR方法从水牛外周血白细胞总RNA中扩增出髓样分化因子88（mydoid differentiation factor88,MyD88）cDNA序列,PCR产物分离纯化后,与pMD20-T载体连接,重组质粒经PCR、酶切鉴定后测序,并进行生物信息学分析;构建pET28a-MyD88表达载体,并将其转化至E.coli BL21（DE3）,经IPTG诱导表达后,进行SDS-PAGE、镍柱亲和层析纯化和Western blotting分析。结果显示,克隆到的水牛MyD88cDNA全长为1189bp,含有1个891bp的开放阅读框,编码296个氨基酸,理论等电点为5.65。经IPTG诱导表达后,得到一个带His·Tag的约39kD的重组融合蛋白。用抗His单克隆抗体进行Western blotting,得到1条约39kD特异性抗体结合带,表明水牛MyD88原核表达载体成功构建并表达。本研究为进一步开展水牛MyD88的结构功能分析奠定了基础。
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| 关 键 词： | MyD88 水牛 基因克隆 原核表达 |
Gene Cloning and Prokaryotic Expression of MyD88 cDNA from Buffalo |
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| Zhang Xiaoru,Kuang Wenhua,Cheng Ying Du Li Zhang Donglin Hao Yongchang Lei Ming Jiao Hanwei Liu Tao Qi Chao Wang Fengyang.Gene Cloning and Prokaryotic Expression of MyD88 cDNA from Buffalo[J].Journal of Guangxi Agricultural and Biological Science,2011,30(2):190-196. |
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| Authors: | Zhang Xiaoru Kuang Wenhua Cheng Ying Du Li Zhang Donglin Hao Yongchang Lei Ming Jiao Hanwei Liu Tao Qi Chao Wang Fengyang |
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| Institution: | College of Life Sciences,Huazhong Normal University,Wuhan,00; College of Agriculture,Hainan University,Hainan Key Lab of Tropical Animal Reproduction ＆ Breeding and Epidemic Disease Research (Construction Period),Animal Genetic Engineering Key Lab of Haikou,Haikou,570228 |
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| Abstract: | Mydoid differentiation factor 88 （MyD88） cDNA was amplified from total RNA of peripheral blood leukocyte in buffalo by RT-PCR,and cloned into pMD20-T vector after purified.The recombinant plasmid was confirmed by PCR,endonuclease digestion,sequencing and |
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| Keywords: | MyD88 Buffalo Cloning Prokaryotic expression |
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