黄瓜CsERECTA基因的原核表达及其多克隆抗体制备  

Prokaryotic Expression of Cucumber CsERECTA Gene and Preparation of Its Polyclonal Antibody

作　　者：王琪琦[1] 李玉红[1] 胡亮亮[1] 秦亚光[1] 赵子瑶 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》2016年第5期1031-1038,共8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项目

摘　　要：ERECTA基因编码一个富含亮氨酸重复序列结构的丝/苏氨酸类受体蛋白激酶,参与调控植物器官的形态建成,在株型控制及抗逆方面也有重要作用。该研究通过构建带有maltose binding protein(MBP)标签的pET21a-CsERECTA融合蛋白原核表达载体,实现了在大肠杆菌(E.coli)BL21(DE3)中的高效表达,并对诱导表达的温度、时间和IPTG浓度进行了优化。利用镍离子螯合层析纯化得到MBP-CsERECTA融合蛋白,再用rTEV蛋白酶对其进行酶切,得到CsERECTA蛋白并制备了该蛋白的多克隆抗体。结果表明,黄瓜CsERECTA蛋白以可溶和包涵体2种形式表达,低温有助于蛋白以可溶性形式大量存在。最佳诱导温度为23℃,诱导时间为6h,IPTG浓度为0.5mmol·L^(-1)。通过Western blot可检测到黄瓜内源的CsERECTA蛋白,说明制备的多可隆抗体具有较好的特异性。多克隆抗体的成功制备为进一步研究CsERECTA的功能奠定了基础。ERECTAgene encodes a leucine-rich repeat receptor-like Ser/Thr kinase.It participates in regulating morphogenesis of plant organs and plays an important role in the change of plant type and resistance to adversity.In the present study,pET21a-CsERECTA,aprokaryotic expression vector with maltose binding protein tags was constructed and successfully expressed in E.coli BL21（DE3）.Expression conditions were optimized in the optimal temperature,induction duration,and IPTG concentration.The fusion protein MBP-CsERECTA was purified by nickel chelating chromatography and enzyme digestion was conducted by rTEV protease.The CsERECTA protein was obtained and it was used to prepare polyclonal antibody.The results showed that CsERECTA was expressed in the form of soluble and inclusion bodies in E.coli BL21（DE3）,and a large number of proteins were soluble at low temperature.The optimal temperature,induction duration and IPTG concentration were 23 ℃,6hand 0.5mmol·L^-1,respectively.Western blot showed that the polyclonal antibody of CsERECTA has good specificity,because endogenousCsERECTA was detected.The successfully prepared polyclonal antibody can be used for further investigation,which establish the foundation for investigating the function of the CsERECTAgene in cucumber.

关 键 词：黄瓜 CsERECTA基因 原核表达 多克隆抗体 

分 类 号：Q785[生物学—分子生物学] Q786