Construction and identification of eukaryotic expression plasmid of hpaA of H.pylori / 第三军医大学学报

ABSTRACT

Objective To clone hpaA gene of H.pylori and construct its eukaryotic expression plasmid. Methods Gene hpaA amplificated from genome of H.pylori 11637 strain by PCR was subcloned into pMD18-T vector. Then hpaA cut down from the vector with restriction enzyme was inserted to pTCAE, and the product confirmed by restriction enzyme digestion and sequence was transformed to E.coli DH5?. pT-hpaA was transfected into CHO cell by electroporation, and HpaA protein was detected by Western blot. Results hpaA gene was inserted into pTCAE and the immunological reaction band with anti-HpaA serum at MW 30 000 was detected by Western blot. Conclusion Eukaryotic expression plasmid of hpaA of H.pylori is successfully constructed. Western blot analysis demonstrates that the expression of HpaA protein can be detected in culture supernatants of transfeced CHO cells, which lays the foundation for the further study.