黄瓜过敏性诱导反应蛋白基因(CsHIR1)原核表达及其多克隆抗体制备  

Prokaryotic Expression and Polyclonal Antibody Preparation of Cucumber CsHIR1 Gene

作　　者：毛双双[1] 李玉红[1] 周旋[1] 李万青[1] 陈菲帆 程智慧[1] 陈鹏[2] 

机构地区：[1]西北农林科技大学园艺学院,陕西杨陵712100 [2]西北农林科技大学生命科学学院,陕西杨陵712100

出　　处：《西北植物学报》2014年第5期884-889,共6页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31071791);高校基本科研业务费专项资金(YQ2013003);西北农林科技大学国际科技合作基金

摘　　要：该试验用黄瓜霜霉菌侵染黄瓜幼苗,并通过PCR方法克隆其过敏性诱导反应蛋白(HIR)的基因CsHIR1,构建原核表达载体pET28a-CsHIR1,实现在大肠杆菌(E.coli)BL21(DE3)中的高效表达;对诱导表达的时间和IPTG的浓度进行了优化;利用钴离子螯合层析纯化了重组蛋白并制备高效价多克隆抗血清。结果表明:该黄瓜过敏性反应诱导蛋白以包涵体的形式表达,最佳诱导时间和IPTG浓度分别为4h和0.5mmol·L-1;经纯化,得到高纯度的分子量为34kD重组蛋白CsHIR1。Western blotting显示CsHIR1的抗体具有较好的特异性。原核表达体系的建立和多克隆抗体的制备为进一步研究CsHIR1基因在黄瓜中的功能奠定了基础。In this study,the cucumber seedlings were infected by Pseudoperonospora cubensis,and the gene of hypersensitive induced reaction proteins （CsHIR1） was cloned.The prokaryotic expression vector of pET28a-CsHIR1 was constructed and efficiently expressed in E.coli BL21（DE3）.The induced expression duration and the concentration of IPTG were optimized.The recombinant protein was purified by cobalt chelating chromatography to prepare the high titer polyclonal antiserum.The results showed that CsHIR1 were expressed in the form of inclusion body,and the optimal induction duration and IPTG concentration were 4 h and 0.5 mmol·L-1,respectively.High purity recombinant protein CsHIR1 with molecular weight 34 kD was obtained.Western blotting results showed that the antibody of CsHIR1 has good specificity.The prokaryotic expression system establishment and polyclonal antibodies preparation lay the foundation for further investigating the function of the CsHIR1 gene in cucumber.

关 键 词：黄瓜 过敏性反应诱导蛋白基因 原核表达 多克隆抗体 

分 类 号：Q785[生物学—分子生物学] Q789