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蛋鸡TRPV6基因的原核表达、纯化及抗体制备
发表时间：2012-06-15 浏览量：1518 下载量：660
| 全部作者： | 郑燕玲，侯加法 |
| 作者单位： | 南京农业大学动物医学院 |
| 摘 要： | 为制备蛋鸡TRPV6蛋白多克隆抗体，根据基因序列，设计一对特异性引物，以卵巢组织中提取的总RNA为模板，扩增蛋鸡TRPV6（transient receptor potential vanilloid receptor 6）基因1 801~2 176位置的375 bp序列，构建原核表达质粒pET- 32a(+)- TRPV6；将重组质粒转化BL21（DE3），经异丙基硫代半乳糖苷（IPTG）诱导表达TRPV6融合蛋白，通过镍离子螯合柱纯化后免疫新西兰大白兔，获得兔抗鸡TRPV6多克隆抗体，再分别通过ELISA方法和Western blotting检测抗体的效价和抗体特异性。结果表明：实验成功地构建了原核表达载体pET- 32a(+)- TRPV6，十二烷基硫酸钠- 聚丙烯酰胺凝胶电泳（SDS- PAGE）蛋白电泳检测发现目的蛋白约35 ku；Western bloting分析显示：表达的TRPV6融合蛋白具有良好的免疫源性，其抗体可与大肠杆菌表达的产物以及天然TRPV6蛋白特异性结合；ELISA显示其抗体效价达1∶100 000. 因此，获得纯化的融合蛋白和多克隆抗体对研究TRPV6钙离子通道在蛋鸡髓质骨形成过程中的作用机制起到了重要作用。 |
| 关 键 词： | 临床兽医学；多克隆抗体；原核表达；纯化；TRPV6；蛋鸡 |
| Title： | Prokaryotic expression, purification and polyclonal antibody preparation of TRPV6 gene in laying hen |
| Author： | ZHENG Yanling, HOU Jiafa |
| Organization： | College of Veterinary Medicine, Nanjing Agricultural University |
| Abstract： | To prepare the polyclonal antibody of transient receptor potential vanilloid receptor 6 (TRPV6) protein in laying hen, a pair of primers was designed and synthesized to clone the 1 801 to 2 176 position of TRPV6 gene about 375 bp by RT- PCR from the total RNA, which was extracted from ovarium. The amplified segment was inserted to the expression plasmid pET- 32a(+), then the prokaryotic expression vector pET- 32a(+)- TRPV6 was constructed and transformed into BL21(DE3) and induced by IPTG. The recombination proteins were purified by using Ni2+- NTA chelating column. The purified proteins were inoculated into New Zealand adult rabbits to develop polyclonal antibody of rabbit against chicken TRPV6. ELISA and Western blotting were then performed to evaluate the specificity of the prepared antibody. The results showed that the prokaryotic expression vector pET- 32a(+)- TRPV6 was successfully constructed in this experiment and the clone recombinant protein relative molecular weight was 35 ku detected with polyacrylamide gel electrophoresis. Western blotting indicated that the TRPV6 fusion protein displayed great immunobiological activity and anti- TRPV6 polyclonal antibody, which was purified by immunizing rabbit could bind with TRPV6 protein specially expressed in E. coli and natural TRPV6 protein extracted from long bone. The titer of antiserum generated was 1∶100 000 by ELISA. Therefore, the TRPV6 fused protein and polyclonal antibody obtained could play an important role in studying the function and mechanism of TRPV6 calcium ion channel in the formation of medullary bone in laying hen. |
| Key words： | clinical veterinary medicine; polyclonal antibody; prokaryotic expression; purification; TRPV6; laying hen |
| 发表期数： | 2012年6月第11期 |
| 引用格式： | 郑燕玲，侯加法. 蛋鸡TRPV6基因的原核表达、纯化及抗体制备[J]. 中国科技论文在线精品论文，2012，5（11）：1087-1093. |
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