Cloning&Expression And Application Of Human Procalcitonin Gene

Calcitonin is a protein glycosylation in the human body, because of its specificity, high sensitivity and become clinical detecting bacterial infection, sepsis, and an important indicator of sepsis and other diseases.To express the recombinant Human Procalcitonin (PCT) in sf9cells using insect baculovirus expression system and E.coli systerm. PCT gene was synthesized and connected with pFastBacTM1vector. The plasmid pFB-PCT was constructed to recombinant and identified by restriction enzyme. The pFB-PCT was transformed into the DH10Bac competent cells. To obtain the recombinant baculovirus vector, the positive clones were screened. The Bacmid-PCT plasmid was extracted and identified by PCR. Bacmid-PCT was transfected into sf9cells by liposome method, harvested recombinant baculovirus. The expression of PCT was further conformed by Western-blot. The PCT gene was connected with PET-22b vector and transformed into the BL21(DE3) competent cells.The expression conditions of recombination PCT were optimized.The Ni-affinity chromatography was used to purified PCT fusion protein which connected with His tags.The immunogenicity of PCT was characterized by ELISA. The results indicated that the recombinant baculovirus vector Bacmid-PCT was successfully constructed. Western-blot results showed that the recombinant protein specific binding with anti-mouse PCT monoclonal antibody. Recombinant baculovirus was successfully expressed human PCT protein in sf9cells. And the recombinant protein has immune activity.The fusion protein was successfully expressed in BL21(DE3) cells and purified for the suloble PCT.The successful expression of PCT by the two methods laying foundations for further study of PCT.In this paper, the experimental results for the future use of PCT as a monitoring postoperative disease such as bacterial infection provide technical basis and application foundation.