重组七鳃鳗PHB2蛋白的原核表达及纯化

Objective: We aimed to construct the soluble prokaryotic expression vector of PHB2, obtain adequate amount of purified rLm-PHB2 protein for further study of its biological activity in vivo. Methods: The PHB2 gene was amplified from the pMD18-T-Lm-PHB2 plasmid template which was previously constructed. The PCR products were subjected to HindⅢ and EcoRI digestion and then linked to the soluble expression vector pET-32a. The identified recombinant plasmid pET-32a-Lm-PHB2 was transformed into Rosetta Blue, and the IPTG induced expression of rLm-PHB2 was confirmed by SDS-PAGE and Western blotting assay. The rLm-PHB2 fusion protein was purified with His affinity chromatography purification system, then the highly purified protein rLm-PHB2 was obtained. Results: The correct construction of recombinant plasmid pET-32a-Lm-PHB2 was confirmed by PCR, restriction enzyme digestion and gene sequencing identification. Expression products were verified to present in the supernatant of bacteria lysis, declaring the successful soluble expression of rLm-PHB2. SDS-PAGE and Western blotting results showed that the molecular weight of rLm-PHB2 protein was about 47KD, corresponding with anticipant size. Conclusion: The pET-32a-Lm-PHB2 prokaryotic expression vector was constructed correctly, and the soluble expression of PHB2 protein was succeeded. Ultimately, the rLm-PHB2 protein with high purity was obtained after purification.