Human Protein C Development And Correlated Study

Human protein C, a vitamin K-dependent serine protease, is a plasma glycoprotein with relative molecular mass(Mr) of 62x103. Activated protein C potently inhibits coagulation and inflammation by inactivating factors V and VIII and facilitates fibrinolysis in vivo. In recent years, activated protein C attracted more attention in the treatment of infectious diseases, especially in the treatment and prevention of pyaemia, endotoxin blood poisoning and disseminated intravascular coagulation in endotoxemia.With the extensive application of human protein C products (protein C purified from human plasma and recombinant protein C) in the treatment of varied diseases, it is urgent to establish a simple, fast, sensitive and specific measure for protein C detection. At present, abroad protein C products (protein C purified from human plasma and recombinant protein C) and diagnostic kits are very expensive, which is impossible for extensive clinical application.There are four parts in this experiment:1 Purifying protein C by physical-chemical methods: The major steps are as follows: After collecting elution products from DEAE-Sephadex A-50 absorbed, DEAE-Sepharose Fast Flow and Heparin-Sepharose CL 6B chromatography, protein C was confirmed by APTT, SDS-PAGE and Western blotting. The purity of protein C was estimated to be more than 95% by SDS-PAGE.2 Expression of human protein C cDNA in CHO cells: By using lipofectamine-mediated gene transfer technique, recombinant expression vector containing human protein C cDNA gene and dhfr gene was transfected into CHO cells.Six higher expression cells were obtained by MIX (6.4umol/L) selecting. Protein C was confirmed by APTT, SDS-PAGE and Western blotting.3 Preparation and identification of monoclonal antibodies to human protein C: Five monoclonal antibodies designated as PC1 to PC5 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C and the titer, subclass, specificity and affinity were detected.4 Application of monoclonal antibodies to human protein C.1) Method of human protein C detection through ELISA (Enzyme-linked immunoadsordent assay): The ELISA method was established through two monoclonal antibodies (McAb) identification with different antigenic sites of human protein C which enabled the determination of protein C in concentrations range of 3.12~ 200ng/mL in less than 4 hours with a single antigen-antibody reaction. Within-run and between-run coefficients of variation were less than 7%. The reference range of protein C in plasma of 102 normal samples was 4.02?.295ug/mL.2) Immunoaffinity purification of protein C by monoclonal antibody to human protein C: Protein C was isolated from DEAE-Sephadex A-50 adsorbed fresh plasma by immunoaffinity chromatography on a column of Sepharose 4B coupled with monoclonal antibody to protein C. This immunopurification resulted in a 16700-fold purification of fully functional zymogen from plasma. The purity of human protein C was estimated to be more than 99% by SDS-PAGE.The research established purification methods (physical-chemical method and affinity chromatography) for human protein C, which is hopeful for large scale protein C production; High expression of human protein C cDNA in CHO-dhfr" cells laid a foundation for the research of protein C; In addition, we developed Five hybridoma cell lines of monoclonal antibodies to human protein C and established ELISA to detect human plasma protein C.