Study On Expression And Purification Of Recombinant Human Coagulation Factor Ⅶ In Mammalian Cell Line

Human coagulation factor VII (hFVII) is a vitamin K dependent glyxoprotein which is synthesized by liver cells and secreted into blood. The activated human coagulation factor VII (hFVIIa) belongs to serine protease family, playing a key role in the extrinsic pathway of blood coagulation, and it can also regulate the intrinsic pathway.The zymogen hFVII is a single-chain molecule consisting of 406 amino acid residues. The hFVIIa consists of a light chain of 152 amino acid residues and a heavy chain of 254 amino acid residues, which were held together by a single dissulfide bond.According to research reports, the hFVIIa was used in many clinic fields in recent years, such as alternative treatment of hemophilia patients, hFVII deficiency patients, especially in hemostasis of surgical operation and first aid of the wound.As hFVII is only present in small amounts in human plasma, it's difficult and expensive to purify it from plasma. So it is a good way using mammalian expression system to produce recombinant coagulation factor VII (rhFVII).In this study, we described the process that the rhFVII was expressed and purified.1. To clone the hFVII cDNA gene and achieve the expression of rhFVII in mammalian cell lines.The hFVII cDNA was amplified from total RNA of human embtyonic liver using RT-PCR, then cloned the gene into pGEM-T vectors. Sequence analysis showed that we have obtained the 1335bp cDNA gene fragment. Then the expression vector was constructed by subcloning the gene into the pcDNA5/FRT/TO plasmid. After identification by DNA sequencing and obtaining a correct recombinant vector, the recombinant expression vector pTO-FVII and assistant plasmid pOG44 were cotransfected into Flp-293TR cell line by means of lipofectin procedure. Then, the resultant transfected cells were selected by Hygromycin. The resistant cell clones were selected and cloned by limited dilution method, the induced culture supernatants were collected using the tetracycline media for identification by SDS-PAGE, Western blot and bioassays. The result showed that we have obtained the recombinant coagulation VII which have bioactive function. This will help us to do further research.2 To prepare monoclonal antibodies against human coagulation factor VII (hFVII) and characterize their properties.Bal b/c mice were immunized with the recombinant activated human coagulation factor VH. The splenocytes of the immunized mice were fused with SP2/0 myeloma cells by hybridoma technique. Three hybridoma cell lines were obtained using enzyme-link immnoadsorption assay method to screen and limited dilution method to clone. The McAb 3B8 belongs to IgG2a subclass, 3D2 and 1C5 belong to IgG1 subclass. The titers of hybridoma cell induced ascites were 1:1×10~7,1:1×10~6 and 1:1×10~6 respectively. We prepared ascites using the 3B8 cell line inducement method, then the monoclonal antibody was purified by protein A affinity chromatography, which will facilitate establishing purification method of rhFVII.3 To purify and characterize the recombinant human coagulation factor VII (rhFVII) from the cells expression supernatant fluid.The monoclonal antibody was coupled with the CNBr-Sepharose 6B Fast Flow gel which becomes the McAb-Sepharose 6B Fast Flow gel, and prepare a monoclonal antibody immunoaffinity chromatogrophy column. Then we validate it using the standard recombinant human coagulation factor VII. The result shows McAb-Sepharose 6B Fast Flow gel can bind the standard recombinant human coagulation factor VII, and can be eluted by 1mol/L NaCl elution buffer.Next, the rhFVII was adsorbed by DEAE-Sephadex A-50 gel from the cells expression supernatant fluid, then remove salts using Sephadex G-25 gel column and immunoaffinity chromtogrophy using McAb-Sepharose 6B Fast Flow gel, the rhFVII were eluted by elution buffer. The immunoaffinity chromtogrophy assay shows the rhFVII can be purfied through DEAE-Sephadex A-50 gel adsorption, remove salts and McAb-Sepharose 6B Fast Flow gel immunoaffinity chromtogrophy. We have established the immunoaffinity chromtogrophy method. It will be useful in the future research.In summary, we have successfully cloned hFVII cDNA and expressed the rhFVII in mammalian cell line. We also prepared the monoclonal antibody and established the immunoaffinity chromtogrophy method. These are very important to manufacture the rhFVII.