Preparation Of Mice Immunized With Human-Derived D-dimer To Obtain CDNA

Objective:In order to overcome the defects of monoclonal antibodies technology in the applications of D-dimer clinical diagnosis, looking for more specific D-dimer Antigenic epitopes, this project determine to prepare mice immunized with human-derived D-dimer for obtaining cDNA. It will also provide the conditions for constructing a phage display library of human-derived D-dimer and screening library in later research.Methods:Freeze-thaw method was used to extract from fresh human plasma fibrinogen. Mix the solution of human fibrinogen with fresh plasma of healthy people (for preparation of cross-linked fibrin that thrombin and clotting factorⅩⅢare in need) and add imidazole buffer (IBS); cross-linked fibrin was prepared and then dissolved by the Plasmin from human fibrin. Products get through Sephadex gel (Sephadex) G-150 gel filtration to obtain crude D-dimer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the protein purity, after we add an equal volume of complete Freund's adjuvant to get BALB / C mice immured. Then every other week with the D-dimer with an equal volume of crude Freund's adjuvant 3 times to strengthen the immune spleen, cold milling, homogenization, extraction of total spleen cell RNA, reverse transcription to cDNA. The cDNA as template designed heavy chain and light chain genes amplified VH and VL genes in mice, by VH, VL antibody library.Results:1. Through freezing and thawing method extracted from fresh human plasma fibrinogen precipitation, dissolved by the imidazole buffer with fresh human plasma at 37 Celsius degree water bathing for 10 minutes, fibrinogen completely solidified into a white gel-like. After determined by the urea dissolution experiments, it was confirmed cross-linking of fibrin gel.2. The cross-linked fibrin gel was placed into pH7.6 TBS buffer, adding fibrinolytic enzymes, after shaking at 37 degrees Celsius for 48 hours, cross-linked fibrin almost completely degraded into soluble degradation products. Solution in 78 hours by cold and dry, it completely dissolved into pale yellow liquid.3. Get the liquid through the non-irreducibility SDS-PAGE, compassed brilliant blue R250 staining, bleaching; after we observe several clear color display bands. Marker protein molecular weight in accordance with the control, you can obtain the solution that contained most of the protein is about 190KDa. D-dimer with the same molecular weight, indicate that hydrolysis conditions are right.4. The liquid gained at the rate of 10ml per hour is balanced again with the pH7.6 TBS buffer solution. Get it on sample and through gel filtration. Once every 10 minutes for liquid filtration, collecting dialysis, desalting, and then the SDS-PAGE, suggested that the reduction ofβ-mercaptoethanol protein fragments about 90,80KD should be D-dimer fragments andβ-D-Chain5. D-dimer in the crude product obtained by the filter membrane sterilization was used for immunization of mice whose spleen cells were strengthened the immune by four weeks. After the extraction of total RNA, we extracted total RNA by 1% agarose gel electrophoresis. With three clearly visible lines, it means the RNA meets the standard of reversing transcription to cDNA.Conclusion:This project from extracted from fresh human plasma fibrinogen, preparing to form cross-linked fibrin gel, getting through the proteasome degradation, gel filtration separation and purification, immunization of mice to extract total RNA, to using RT-PCR technique to obtain antibodies against D-dimer Variable region of VL, VH genes is significant. The success of this experiment is of separation and purification of the crude D-dimer, providing the antigen for mice being immunized. Successfully build a full set of foundation for construction of an anti-D-dimer antibody library, as well as separation and purification of human plasma proteins providing for the relevant experience.