Construction And Expression Of Fusion Gene TGF- ?RII /Fc And Identification Of Its Biological Activities

Pulmonary fibrosis is a devastating disorder that is resistant to treatment. Patients with idiopathic pulmonary fibrosis have a median survival of 4 to 5 yr after onset of symptoms. First- line therapy with corticosteroids offers only a 15% to 20% response rate despite very significant side effects. More aggressive immunosuppressive therapy with cytotoxic agents has had only a modest impact on the outcome of the disease. It's very urgent to search for a new therapy. The role of TGF- P in fibrotic disease has been acknowledged. Targeting TGF- P in fibrotic disease reduces scarring and fibrosis. Attempts to block the effects of excessive TGF- P activity have involved the use of neutralizing TGF- P antibodies and natural TGF- P inhibitors, both of which inhibit TGF- P binding to its receptors, and gene therapy with the inhibitory Smads or dominant negative TGF- P receptors, both of which block TGF- P signaling. To inhibit TGF- P binding to its receptors is the purpose of this study. By constructing the fusion gene TGF- P RII /Fc, it will not only facilitate thepurification of the chimerical protein but also help to form a disulfide-linkedhomodimer. The chimerical protein with a dominant negative type II receptor can reverse lung fibrosis and inhibit scar formation. This study is made up of three parts as follows:I ? Construction of eukaryotic expression vector of fusion gene TGF- 3 RII /Fc and its expression in CHO cells.The aim is to construct the plg-TGF- 3 RII eukaryotic expression vector of the cDNA sequence encoding bioactive extracellular domain of human transforming growth factor beta type II receptor (TGF- 3 RII) to form the fusion gene TGF- 3 RII /Fc and to express the chimerical protein TGF- 3 RII /Fc in CHO cells. Total RNA was extracted from human normal lung tissue and subjected to reverse transcription, and then the human TGF- 3 RII cDNA gene was amplified by RT-PCR. The PCR product was cloned into pMD18-T vector and the sequence was confirmed by restriction enzyme digestion and sequencing. Then the specific TGF- 3 R II encoding region fragment was subcloned into pig plasmid to form the plg-TGF- 3 RII eukaryotic expression vector. The recombinant plasmid was transfected into CHO cells with liposome transfection reagent for transient expression. The expression of the chimerical protein TGF- 3 R II /Fc was identified by immunofluorescent assay and immunoprecipitation technology. Restriction enzyme digestion and DNA sequence analysis revealed that the insert fragment was identical to the published TGF- 3 R II cDNA sequence. The chimerical protein could be detected by cellular immunofluorescence and immunoprecipitation technology. The construction of the plg-TGF- 3 RII eukaryotic expression vector of the fusion gene TGF- 3 RII /Fc and its expression in CHO cells are both successful. 2. Expression of fusion gene TGF- 3 RII /Fc in Bac-to-Bac Baculovirus Expression SystemThe objective of this part is to construct the baculovirus expression system of the chimerical protein TGF- 3 RII /Fc, and make it be expressed successfully. Recombinant Bac-TGF- 3 RII /Fc baculovirus expression system is constructed by molecular cloning technology; The DNA liposome transfection technology and insect cell culture technology are employed to culture and amplify the recombinant Bac-TGF-3 RlI/Fc baculovirus particles. The chimerical protein is detected by cellular immunofluorescence technique. The chimerical protein can be detected in sf9 cells infected by the recombinant Bac-TGF- 3 RII /Fc baculovirus particles. It is concluded that the recombinant Bac-TGF- 3 RII /Fc baculovirus expression system can be used to express the chimerical protein TGF- 3 RII /Fc correctly. The baculovirus expression system can express the target protein in large scale, so it could facilitate the purification of the target protein and the other experimental study.3. Purification and identification of the chimerical protein TGF- 3 RII /FcThe first stage is to express the chimerical proteinTGF- 3 RII /Fc in l...