Cloning And Expression Of Human Parathyroid Hormone (1-34)

Human parathyroid hormone (hPTH) is a polypeptide hormone whichis secreted from the parathyroid glands and composed of 84 aminoacids. It regulates the concentration of blood calcium and phosphateby binding to specific receptors in the renal and osseous tissues,in which process the adenylate cyclase system is stimulated and somerelated enzymes are activated. There are some conserved domains inhPTH which are responsible for its function. The (1-34) amino acidsequence of N-terminal region of hPTH is the shortest fragment whichkeeps the full activity of PTH. The biologically active hPTHfragment has been reported to be effective in the treatment ofdisorders in muscle and bone. It also has effects on cardiovascularsystem. Because it is difficult to get plentiful PTH by extract andchemosynthesis, so we attach great importance to obtain rhPTH and itsanalogues by the genetic engineering techniques in the recent years.In this report,the recombinant human parathyroid hormone(1-34) gene was PCR amplified by using synthesis oligonuleotidesPTH(1-34) as the template, the Xa recognition site was added to theDNA gene, then cloned into pET-32a vetor. The recombinant plasmid wasidenified by DNA aequencing. The Trx-fusion and His-tagged proteinwas produced in E. coli BL21(DE3) after IPTG induction, itconstituted about 55ï¼…of total bacterial protein, and exited insupernatant after sonication. The fusion protein was purified byHis-Bind column and cleaved by Factor Xa to release PTH(1-34). AfterFactor Xa cleavage and repurification by His-Bind column, the electronic purified PTH(1-34) was obtained. Cardiovascular and cerebrovascular diseases have become the biggest killer to people' s health nowadays, and acute myocardial infarction is . the most serious disease in some western developed countries.As thrombosis is the most important inducement of this disease, thrombolytic therapeutics become the main way in clinics. In the process of the thrombolytic therapeutics, the combination of both thrombolytic and anticoagulant drugs are adopted to prevent the occurrence of rethrombosis.Reteplase(r-PA) is the recombinant type of novel tissue plasminogen activator (t-PA) variant , the domains of kringle I,Finger and EGF were deleted from the original t-PA, and kringle II and protease domains are maintained in the structure of r-PA which containing 355 amino acid residues with molecular we ight of 39 kDa . Reteplase is the third generation thrombolytic drug. Because the advantages of long half-life , low hemorrhaging in clinics , reteplase is a promising thrombolytics in clinics. Hirudin, as a potent and specific inhibitor of thrombin, can be used to protect from and to treat clinically thrombosis. It contains 9 pairs of disulfide bonds, 65 amino acid residues with molecular weight of 7000 D. Its C-terminus rich in acidic amino acid residues possess hydrophilicity, free on the molecular surface, and can bind with fibrin recognition site of hirudin. The minimal segment of 12 C-terminal acidic residues keeps the activity of anti-thrombosis.In this paper, we want to combine the fibrinolytic with anticoagulant activities for therapy for thrombotic disease, a fusion protein made of 12 peptides of hirudin and reteplase was firstly constructed and expressed.(1)12 peptides of hirudin located in the C- terminal of fusion proteinWe amplify the objective gene from rPA gene with PCR, the fusion gene (r-PA-the (Gly)₃ link and 12 peptides of hirudin )is obtained. Then clone it into pET-21a vector, the sequence is identical with which we predicted .The recombinant gene transformed in E.coli BL21(DE3),BL21(DE3)pLysS,HMS174(DE3),0rigamai(DE3), but the objective fusion protein didn't express after IPTG induction. Then we change another pBAD expressional system, the fusion gene was ligated with pBAD/g III A vetor, then transformed in E. coli T0P10F' ,but got the failing result as before. We deduce the failure of the protein expression is relevant with it spacial structure.(2)12 peptides of hirudin located in the N-terminal of fusion proteinWe amplify the objective gene from rPA gene with two cycles PCR, the fusion gene(12 peptides of hirudin-the (Gly)₃ link-r-PA)is obtained. Then clone it into pET-21a vector, the sequence is identical with which we predicted .The recombinant protein was produced in E. coli BL21(DE3) after IPTG induction , it constituted about 12% of total bacterial protein, and exited in inclusion body after sonication. After renaturation by molecular-exclusion chromatography in vitro,the fusion protein has showed effective biofuntional activities, So the fusion protein is a biofunctional molecular having good prospect to develop into a new effective therapeutic agent for thromobotic disease.