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

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.