Expression And Pharmacodynamic Study Of The Recombinant Trypsin Inhibitor

Partâ… The Eukaryotic Expression and Characterization of the Recombinant Trypsin InhibitorObjective:To construct the eukaryofic expression system to produce the recombinant trypsin inhibitor;To purify and characterize recombinant peptide and explore its large-scale production techniques.Methods:The primers were designed according on the cDNA sequence of the trypsin inhibitor.The gene encoding the trypsin inhibitor was amplified from the spider venom cDNAs.The expressing vector of the trypsin inhibitor was constructed by linking the gene with the plasmid pVT102U,and then transfected into the yeast Saccharomyces cerevisiae S-78.After cultured YPD,the yeasts were centrifuged and the supernatant was collected.The recombinant peptide was purified with a combined use of ion exchange chromatography and RP-HPLC.The expressed product was characterized by Edmam degradation,SDS-PAGE and RP-HPLC, and MALDI-TOF.Results: 1.The expression vector pVT-HW11 was constructed successfully.2.The trypsin inhibitor could be produced with high yield by using the yeasts S-78 expression system,and the expression yield is about 13 mg/L.3.The recombinant peptide could be purified by a combined use of centrifugation,filtration,ion exchange chromatography and RP-HPLC.The purity of the recombinant peptide was over 98%.4.The recombinant peptide has the same molecular weight and amino acid sequence as natural one.Conclusion:The eukaryotic expression system of yeast Saccharomyces cerevisiae S-78 for recombinant trypsin inhibitor was successfully constructed and a simple and effective purification method was established.The recombinant peptide has the same molecule weight and amino acid sequence with its natural one.By using the method established in this study,the trypsin inhibitor would be produced with large-scale.Therefore,the present research may be innovative and has the value of practical application. Partâ…¡The Pharmacodynamic Study of the Recombinant Trypsin InhibitorObjective:To inestigate the pharmacodynamic activities of the recombinant trypsin inhibitor and explore its prospect of clinical applicationMethods:Firstly,the inhibition constants of the recombinant peptide and BPTI against serine proteinases such as trypsin,chymotrypsin,kallikrein and thrombin,were assayed by analyzing the reaction of the enzyme with the relative substrate with spectrophotometer.As the inhibition of recombinant peptide against trypsin was very potent,spectrophotometric method could hardly be used to evaluate its inhibition dynmics.Therefore Isothermal Titration Calorimetry(ITC) was applied,in which the binding dynamics of recombinant peptide and trypsin was studied by thermodynamic changes.To construct a L-Arg-induced acute necrosis pancreatitis in mice model in which therapeutic effect of recombinant peptide upon acute necrosis pancreatitis could be studied from biochemical indicators and pathological sections.The inhibition of the recombinant peptide against voltage-gated potassium channels was studied by whole cell clamp recordings on rat dorsal root ganglion(DRG) neurons,in which the inhibition effects were examined under variant voltage and different concentration of recombinant peptide.Results:1.Both recombinant trypsin inhibitor and BPTI have potent inhibition effect on the activity of trypsin.Though recombinant peptide had lower inhibition effect upon the activities of chymotrypsin,kallikrein and thrombin than BPTI,it has 30 times stronger inhibition effect on the activity of trypsin than BPTI and could be one of the most potent inhibitors against trypsin indentified so far.2.With the inhibition against trypsin,the recombinant peptide could potently suppress the activity of amylase and lipase in the plasma of the mice with acute necrosis pancreatitis.Earlier medication could markedly reduce high secrete of pancreas and protect the pancreas cell from digestion injuries with the result of good protection for the acute necrosis pancreatitis in mice and great improvement of pathological symptom.3.The blocking function on the voltage-gated potassium channels of the recombinant peptide was concentration-dependant.With the concentration of 10^-6M,potassium current could not be further reduced and were saturated,which means the strongest inhibitation was achieved.The concentration of 10^-12M was the one which recombinant peptide completely inhibitated the activity of trypsin, which indicated that it had weaker inhibition effect on voltage-gated potassium channels.And when the inhibitation against trypsin happened,the side effects in relation to the pharmacology of potassium channels were very low.Conclusion:The recombinant trypsin inhibitor is the most potent peptide inhibitor against trypsin identified so far.It has unique biological function that inhibits trypsin.It has potent protection from acute necrosis pancreatitis in mice and markedly improves its pathological symptoms. The recombinant trypsin inhibitor has promising clinical application for the better treatment of acute necrosis pancreatitis and trypsin-related diseases.