| 奶牛β-防御素5基因的克隆与原核表达 |
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| 引用本文： | 周雷,张勇,王建锋,赵兴绪.奶牛β-防御素5基因的克隆与原核表达[J].甘肃农业大学学报,2009,44(1). |
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| 作者姓名： | 周雷 张勇 王建锋 赵兴绪 |
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| 作者单位： | 甘肃农业大学动物医学院,甘肃,兰州,730070 |
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| 基金项目： | 国际科技合作计划项目 |
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| 摘 要： | 根据已发表的β-防御素5基因序列设计引物,采用RT-PCR技术扩增奶牛白细胞β-防御素5基因片段,构建原核表达载体并进行诱导表达.结果表明:将PCR产物插入pGEM-T easy载体后,经琼脂糖凝胶电泳、PCR、酶切及DNA测序证明构建的重组克隆载体完全正确,以该重组质粒为模板扩增目的基因的成熟肽片段,产物用EcoR I+NotⅠ双酶切并与原核表达载体PET28a连接,获得了预期的重组表达质粒.将重组表达质粒转化到BL21(DE3)宿主菌中,用异丙基--βD-硫代半乳糖苷诱导表达,经尿素-SDS-PAGE检测表明表达产物为6.4 kda的融合蛋白.
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| 关 键 词： | 奶牛 β-防御素5 基因克隆 原核表达 |
Cloning and prokaryotic expression of beta-defensin 5 gene in dairy cattle |
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| ZHOU Lei,ZHANG Yong,WANG Jian-feng,ZHAO Xing-xu.Cloning and prokaryotic expression of beta-defensin 5 gene in dairy cattle[J].Journal of Gansu Agricultural University,2009,44(1). |
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| Authors: | ZHOU Lei ZHANG Yong WANG Jian-feng ZHAO Xing-xu |
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| Institution: | ZHOU Lei,ZHANG Yong,WANG Jian-feng,ZHAO Xing-xu(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China) |
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| Abstract: | A pair of primers were designed and synthesized based on the published β-defensin 5 gene sequence of dairy cattle,and then β-defensin 5 gene was amplified using RT-PCR technique.Then,the PCR product was inserted into pGEM-T easy vector to construct the recombinant plasmid that was correct identified by agarose gel electrophoresis,PCR,enzymatic digestion and DNA sequencing.The mature peptide fragment of the target gene was amplified according to the recombinant plasmid template.After digested with EcoR I and Not I,the amplified product was ligated with PET28a expressive vector to form recombinant expressive plasmid.Sequence analysis showed that the inserted target gene was completely correct.Then,the recombinant plasmid was transformed into BL21(DE3) and induced with IPTG to express the fusion proteins with relative molecular mass of 6.4 kda. |
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| Keywords: | dairy cattle β-defensin 5 gene clone prokaryotic expression |
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