1.Preparation Of Human Parathyroid Hormone 1-34 2.Preparation Of Thymosin α1 And The Fusion Expression Of Thymosin α1 And IFNα-1b

Part One: Preparation of human parathyroid hormone 1-34Chapter one: Solid-phase synthesis of N-terminal 1-34 peptide of human parathyroid hormone Purpose The human parathyroid hormone 1-34 peptide was synthesized by Fmoc/tBu method out on wang resin. Methodes In coupling step, Fmoc-amino acid was activated with TBTU/HoBt.After the completion of peptide elongation, and the crud peptide product was cleaved out from wang resin by TFA treatment. Then purified with SP-Sepharose-FF and semi-preparative HPLC. Results the purity of final product confirmed by HPLC and capillary zone electrophoresis to be more than 92%. Ectrospray mass spectrometry analysis showed the right molecular weight of the synthetic peptide. The total yield was 8%. Conclusion the technology is samples and can be enlarged easily.Chanpter two: Fusion expression of hPTH(1-34) in E.coliAbstract An engineering E.coli strain, BL21 (DE3)/pGEX-4T-human Parathyroid Hormone (hPTH) (1-34), was constructed by oligonucleotide annealing and PCR amplifying the target gene, then ligating it with pGEX-4T-3 vector and transferring into BL21 host. The purity of soluble fusion protein of GST-hPTH(1-34) expressed from BL21(DE3)/pGEX-4T-hPTH(1-34) is over 90% after high-density, high-expression culture and purification by affinity chromatography. The product was harvested following the enzyme digestion and affinity purification. The product is checked by HPLC,MS and N-terminus sequence analysis. The purified recombinant hPTH(1-34) stimulated adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic human parathyroid hormone standards, indicating that the recombinant product has full biological activity.Part Two: Preparation of thymosin α1 and the fusion expression of thymosin α1 and IFNα-1bChapter one: SOLID PHASE SYNTHESIS OF THYMOSINα1An solid phase synthesis of thymosinα1 is described. The Fmoc-amino acid and wang resin were used. TBTU-HoBt was used as coupling reagent.Then cleaved with TFA.The general average yield is no less than 13%.The technoiogyis sample and can be enlarged easily.Chanpter two: Fusion expression of human thymosinα1 in E.coli Engineering E.coli strain, BL21 (DE3)/pGEX-4T-human Thymosinα1, was constructed by oligonucleotide annealing and PCR amplifying the target gene, then ligating it with pGEX-4T-3 vector and transferring into BL21 host. The yield of soluble fusion protein of GST-Thymosinα1 expressed from BL21(DE3)/ pGEX-4T-thymosinα1 is about 35-40% of total protein after fermentation. Following the simple cut of thrombin or CNBr, about 0.2g/L thymosinα1 can be harvested. The product is checked by MS and activity test, which indicates that the recombinant product has full biological activity of native thymosinα1.Chanpter three: fusion expression of thymosin α1 and IFNα-1b An fusion gene, thymosinα1~IFNα-1b, was constracted by by oligonucleotide annealing and PCR amplifying method. The fusion protein was existed as an inclusion body. After refolding,purification of Q-Sepharose column and Hiload 26/60 Sephadex75 column, the purity of the fusion protein is about 95% by HPLC analysis. By activaty test, the fusion protein has the same activaty of thymosinα1 and IFNα-1b inspectively...