毛果杨漆酶基因PtrLAC-2的原核表达与多克隆抗体制备研究

Prokaryotic Expression and Polyclonal Antibody Preparation of PtrLAC-2 Gene of Populus trichocarpa

目的:克隆毛果杨漆酶基因PtrLAC-2,并对其进行原核表达,同时制备高特异性、高效价的PtrLAC-2多克隆抗体,为研究漆酶基因蛋白在体外结构与功能奠定基础。方法:利用拟南芥中的漆酶基因序列对毛果杨基因组数据库进行同源检索,PCR同源克隆并扩增得到毛果杨PtrLAC-2基因序列,将其亚克隆到pET30a(+)载体得到PtrLAC-2-pET30a(+)原核表达载体,转化到大肠杆菌BL21(DE3)中诱导表达并纯化得到PtrLAC-2重组蛋白。采用皮下免疫法将纯化的PtrLAC-2蛋白免疫兔子制备多克隆抗体,用ELISA和Western blot检测血清多克隆抗体的效价和特异性。结果:克隆获得了毛果杨漆酶基因的序列(命名PtrLAC-2,基因登录号XP_002308164),成功构建了PtrLAC-2-pET30a(+)原核表达载体,诱导表达获得了62 KDa的重组蛋白,ELISA检测显示抗体效价达1:102,400,经Western blot鉴定,制备的多克隆抗体能特异性检测毛果杨中的PtrLAC-2蛋白。结论:毛果杨漆酶PtrLAC-2基因可以在体外成功进行原核表达,同时获得了高特异性、高效价的兔抗PtrLAC-2多克隆抗体,为后续研究该酶生化功能提供了一定的理论基础。

Objective: To clone and express the Populus trichocarpa laccase PtrLAC-2 gene. This study prepared polyclonal antibody with high affinity and specificity to improve the study of structure and function of laccase. Methods: According to the principle of homologous cloning, laccase gene from Arabidopsis thaliana was used to blast the database JGI of Populus trichocarpa. The Populus trichocarpa laccase PtrLAC-2 gene was cloned by PCR before being ligated with pET-30a(+) to construct prokaryotic expression vector PtrLAC-2-pET30a(+). PtrLAC-2-pET30a(+) was then transformed into E. coli BL21(DE3) competent cells for induction expression. Recombinant protein was purified as antigen to immune rabbit to prepare polyclonal antibody. Titer of the polyclonal antibody and specificity were analyzed using ELISA and Western bolt at last. Results: Populus trichocarpa laccase gene was isolated (renamed PtrLAC-2, Genebank: XP_002308164). Prokaryotic expression vector PtrLAC-2-pET30a(+) was constructed successfully. The recombinant protein with the length of 62 KDa was obtained. ELISA analysis showed that the titer of the obtained antibody was 1:102,400. Western blot showed that the antibody could specifically combine with PtrLAC-2 protein in Populus trichocarpa. Conclusion: The PtrLAC-2 genes successfully expressed in E. coli and the PtrLAC-2 polyclonal antibody with high affinity and specificity was generated. This study will supply theoretical foundation in the following enzyme biochemical function.