Cloning, Prokaryotic Expression Of PDGF-C And TGF-β1and Preparation Of Its Specific Polyclonal Antibody

Objective:The human platelet-derived growth factor C(PDGF-C)and transforming growth factor betal (TGF-β1) play important roles in hepatic cirrhosis caused by hepatitis B virus infection. However, the methods of detecting PDGF-C and TGF-Bl are not available at present. The aim of this thesis was to prepare their specific antibodies for establishing the method to detect them.Methods:We chose the regions encoding TGF-β1and PDGF-C, and designed the primers for amplified them using bioinformatics. The fragments which amplified by reverse transcription polymerase chain reaction (RT-PCR) were inserted into the EcoRI and Xhol sites of pGEX-5X1and pRSET-A2, respectively. The expressions of TGF-β1and PDGF-C were induced by IPTG in E.coli, BL21or BL21(DE3), respectively. The fusion proteins which were PDGF-C and TGF-β1with GST tag were purified by recovery from SDS-PAGE(12%).The purified TGF-β1fusion protein with GST and PDGF-C fusion protein with GST were used to immune the New Zealand white rabbits with Freund’s adjuvant, respectively. Using the polyclonal antibodies of PDGF-C and TGF-β1detected the PDGF-C or TGF-β1fusion protein with HIS tag from E.coli. and endogenous protein of PDGF-C and TGF-β1in eukaryotic cells with method of ELISA and western blot. Then we used these antibodies to detect the PDGF-C and TGF-β1in serum of patients with hepatic cirrhosis and healthy people serum by western blot preliminarily.Results:1.The DNA fragments encoding gene of PDGF-C and TGF-β1, respectively, were amplified by RT-PCR.2.Recombinant plasmids, pGEX-5X-1/PDGF-C, pRSET-A2/PDGF-C, pGEX-5X-1/TGF-β1and pRSET-A2/TGF-β1for expressing PDGF-C and TGF-β1fusion proteins with GST or His tag were made.3.Expressed PDGF-C or TGF-β1fusion protein with GST or His tag from E.coli. BL21.4. Purified PDGF-C or TGF-β1fusion protein with GST or His tag from E.coli. BL21.5.Generated the polyclonal antibodies of PDGF-C and TGF-Bl in New Zealand white rabbits.6. It was detected for the fusion protein of PDGF-C with HIS tag and endogenic protein of PDGF-C in eukaryotic cells by ELISA and western blot with the prepared polyclonal antibody of rabbit. It was detected for the fusion protein of TGF-β1with HIS tag and endogenic protein of TGF-β1in eukaryotic cells by ELISA and western blot with the prepared polyclonal antibody of rabbit.7.Used the polyclonal antibodies of PDGF-C and TGF-B1to detect the PDGF-C and TGF-β1in serum specimens from patients with hepatic cirrhosis and healthy people serum by western blot. The result showed that the levels of PDGF-C and TGF-B1in serum from patients with hepatic cirrhosis were significant different from serum of health people.Conclusions:The specific polyclonal antibodies for PDGF-C and TGF-β1were prepared using fragments expressed in E.coli successfully. They are applicative to PDGF-C and TGF-B1of molecular pathomechanism research in hepatic cirrhosis caused by hepatitis B. The result showed that the levels of PDGF-C and TGF-B1in serum from patients with hepatic fibrosis were significant different from serum of health people. It was not observed at home and abroad by choosing the same coding gene as our research in PDGF-C and TGF-β1for clone, prokaryotic expression and preparation of polyclonal antibody.