Cloning And Expression Of C-reactive Protein(CRP) Gene

C-reactive protein(CRP) involves in human inflammatory response, it is an important clinical diagnosis detection index. Bombyx mori baculovirus expression system has high expression and post-translational modification function. It is the ideal of exogenous recombinant protein expression system and can be produced on a large scale. This study attempts to express the recombinant human CRP by the system.CRP is amplified using human liver tissue c DNA as a template by designing specific primers, and then CRP was connected to the p ColdTMII carrier, recombinant expression vectors p ColdTMII-CRP was obtained. p ColdTMII-CRP was transformed into E.coli BL21(DE3) and induced to get inclusion protein. After inclusion bodies dissolved, Ni-NTA column purification, refolding, recombinant human protein CRP is obtained by prokaryotic expression. After SDS-PAGE, Western Blot, as well as mass spectrometry, it determined that the protein is expressed successfully.Recombinant human CRP obtained by prokaryotic expression immunizes New Zealand rabbits. First time, freund’s complete adjuvant immunizes rabbits, after a week, incomplete freund’s adjuvant should be used to strengthen immunity, and immuned once a week for four consecutive weeks. Before the final immunization, The titer of the antiserum should be determined by ELISA. If the titer of the antiserum attain purpose titer, a week later, whole blood should be taken from the carotid arteries of rabbits. Antiserum against CRP was obtained by centrifugation and its titer was determined by ELISA. Antigen concentration is 10 μg/m L,we can get antiserum titer reached 1:6, 4000. Finally, the CRP polyclonal antibody was acquried. Western Blot was performed for testing strip. It can be used for future research.After restriction enzyme digestion, CRP was connected to the p Fast BacTMHTB transfer vector. Using the Bac-to-Bac system, p Fast BacTMHTB-CRP was transferred into E.coli Bm DH10 Bac, and then recombinant Bacmind-CRP was screened. Bacmind-CRP transfected Bm N cells, recombinant baculovirus v Bm His-CRP was obtained about 5 d. v Bm His-CRP was seeded onto Bm N cells and 5 instars larva, the target protein was detected in Bm N cells and silkworm fifth instar silkworm larvaes. To get cheap recombinant product, we choose to express it in silkworm. SDS-PAGE analysis of protein expression and Western Blot detection of protein distribution, found that human recombinant protein CRP exists in precipitation. The recombinant virus which introduced with the signal peptide aslo expressed protein in an inclusion body form. So we purify recombinant protein CRP with the method of the inclusion body protein purification. But express output is not high and have complex proteins. Western Blot detected target band using homemade CRP polyclonal antibody, and mass spectrometry determined that the protein is recombinant human CRP protein. It laid a foundation for the silkworm bioreactor large-scale preparation of recombinant CRP protein, and the standard antigen source problem is sloved in the clinical CRP related inflammatory response rapid diagnostic kits.