1
目 录
Abstract1
Key words1
引言1
1 材料与方法2
1.1材料 2
1.2 方法 2
1.2.1 引物设计2
1.2.2 重组表达质粒的构建2
1.2.3重组蛋白的诱导表达3
1.2.4 SDSPAGE的步骤3
1.2.5 表达产物的Western blot检测4
1.2.6 确定蛋白表达的位置4
1.2.7 重组蛋白的纯化与复性5
1.2.8 抗HA1蛋白的多克隆抗体制备5
1.2.9 ELISA检测血清抗体效价5
2 结果与分析6
2.1 HA1基因的克隆及重组表达质粒的构建与鉴定6
2.2 重组蛋白的诱导表达及纯化6
2.3 多克隆抗体的免疫学分析7
3 讨论 7
致谢8
参考文献8
犬流感病毒HA1蛋白的表达及抗体制备
动物医学专业学生 许姝雅
指导教师 刘永杰
摘要：犬流感病毒（Canine influenza virus, CIV）是一种可感染犬的流感病毒，在犬间传播迅速，随着近年来宠物犬行业和犬养殖业快速发展，犬流感引起较大关注。本试验将分离的H3N2亚型犬流感病毒（Canine influenza virus, CIV）A/Canine/Nanjing/06/2011（H3N2）株的HA1蛋白基因PCR扩增后克隆至原核表达载体pET28a(+)中，构建重组表达质粒pETHA1，将重组质粒转化大肠杆菌BL21(DE3)感受态细胞后,经IPTG诱导表达。表达的重组蛋白经过纯化、复性后，免疫家兔获得多克隆抗体血清, 并对多克隆抗体进行间接ELISA和Western blot分析。结果显示，该重组蛋白在大肠杆菌中以包涵体的形式表达，大小约为36kD；多克隆抗体血清效价为1:20000，能够与重组蛋白特异性结合，显示抗体制备成功。该试验为深入探讨病原HA蛋白的致病作用提供了科学依据。
Prokaryotic expression of  *51今日免费论文网|www.jxszl.com +Q: #351916072# 
canine influenza virus HA1 protein and its antibody preparation
Student majoring in Veterinary Medicine Xu Shuya
Tutor Liu Yongjie
Abstract：Canine influenza virus (CIV) is a influenza virus that can infect dogs and spread rapidly among dog’s, In recent years, with the rapid development of the pet dog industry and dog breeding industry, more and more people have taken notice of canine influenza. In this experiment, the HA1 gene of H3N2 subtype CIV named A/Canine/Nanjing/11/2012(H3N2), was amplified by PCR and subcloned into pET28a(+) vector. The resulting recombinant HA1 protein was expressed in E. coli BL21(DE3) by IPTG induction. The recombinant protein after purification and renaturation was used to immune rabbit to get polyclonal antibody, and the polyclonal antibody was analysed by ELISA and Western blot. Results showed that the recombinant protein was expressed in the form of inclusion bodies in E. coli, and the size is about 35 kD; Polyclonal antibody titer was 1:20000, and the antibody could react with the recombinant protein specifically. It suggests that the antibody against HA1 was prepared successfully. This experiment lays a foundation for further study on the role of HA protein in CIV pathogenicity.
Key words: canine influenza virus; H3N2 subtype; HA1 gene; polyclonal antibody

原文链接：http://www.jxszl.com/yxlw/dwyx/559417.html