Construction Of MASP-2Different Domains With The Adenovirus Vector

As a tightly regulated network of proteolytic plasma proteins, the complement system is a fundamental part of the immune system, providing protection against invading microorganisms through the binding of various serine protease. In the lectin pathway, mannose-binding lectin (MBL) and ficolins bind to pathogens and activate MBL-associated serine protease-2(MASP-2), the key enzyme which can directly start the complement cascade. Recently, the growing list of reports with MASP-2elucidate that the SP domain can cleave the second complement component C2, but not C4, whereas the CCP domain is responsible for this phenomenon. In order to study the effective domain and to find the effects of CCP1, CCP2and SP, this experiment successfully constructed a series of adenovirus vectors.Objective:①Transfect the constructed recombinant pGEM-T-CCP1-CCP2-SP, pGEM-T-CCP2-SP and pGEM-T-SP into E.coil BL21(DE3) strain. The proteins CCP1-CCP2-SP, CCP2-SP and SP are expressed and purified for the further experiments.②Construct the adenovirus vectors of pYr-adshuttle-1-CCP1-CCP2-SP, pYr-adshuttle-CCP2-SP and pYr-adshuttle-1-SP to study the influence of inherent immune molecules MASP-2on occurrence and development of infection.Methods:①The recombinant pGEM-T-CCP1-CCP2-SP, pGEM-T-CCP2-SP and pGEM-T-SP were transferred into E.coil DH5a strain for amplification, then the correct plasmids were imported into E.coil BL21(DE3) strain for expression. Take the best conditions(37℃;3h; the IPTG final concentration of0.75mmol/L) to express proteins and use the nickel ion column to obtain the purified proteins.②The gene encoding CCP1-CCP2-SP(1185bp), CCP2-SP(996bp) and SP(735bp) were amplified from plasmid pGEM-T-CCP1-CCP2-SP using the PCR technique. The amplification products were purified and inserted into the multiple cloning sites of the pYr-adshuttle-1(4470bp) plasmid digested with BamH I and EcoR I to construct recombinant pYr-adshuttle-l-CCP1-CCP2-SP, pYr-adshuttle-CCP2-SP and pYr-adshuttle-1-SP respectively and then they were homologous recombinated with framework plasmidof adenovirus expression vector. Results:①The weight of recombinant product CCP1-CCP2-SP, CCP2-SP and SP are around44KDa,37KDa and27KDa which had been identified by SDS-PAGE.②The successful construction of adenovirus vectors pYr-adshuttle-1-CCP1-CCP2-SP, pYr-adshuttle-CCP2-SP and pYr-adshuttle-1-SP had been identified by PCR and Sequencing.Ronclusion:The successful construction of three kinds of adenovirus vectors has made a foundation for the further experiments about homologous recombination.