The Study On The Construction Of Multi-functional Fusion Peptides And Their Curative Effect For T2DM And Thrombosis Model Mice

Diabetes mellitus is triggered due to the deficiency of insulin secreted absolutely or relatively and the low sensibility of target tissue to insulin, which produces the confusion of metabolism, and its mainly clinical symptoms are hyperglycemia, hyperlipemia, hyperviscosity syndrome and so on. With the improvement of modern life, there are more and more diabetes patients. So it is important for scientists to find a better drug, better route of administration and therapeutic method. Today, Diabetes mellitus has become a "source of sickness", it tends to bring a series of complications, and these complications make the patients worse and worse. Studies have shown that the highest mortality for patients with diabetes was cardiovascular disease, and it accounts for about80%, the incidence of diabetes suffering from cerebral thrombosis was12times than the non-diabetic people. Therefore, it is of great significance for us to find a new drug which is effective to Diabetes mellitus and its complications such as cardiovascular diseases and so on.Glucagon-like peptide-1(GLP-1) is a30amino acid peptide hormone. It is released and synthesized from the proximal small intestine L-cells. GLP-1is widely recognized as a physiological incretin hormone. It can significantly reduce blood glucose levels and improved abnormal metabolism due to high blood glucose. Thus GLP-1has a good therapeutic effect on type2diabetes. Gastric inhibitory polypeptide (GIP) is a physiological gut peptide secreted from the small intestine K-cells in response to feeding. It is a42amino acid peptide. GIP is synthesized in intestinal K-cells also and its secretion is regulated largely by the ingestion of carbohydrate. GIP can potently stimulate insulin release in a glucose-dependent manner. Also, GIP exerts other effects on pancreatic beta cell, including the stimulation of proinsulin gene transcription and translation, the enhancement of beta cell growth, differentiation, proliferation and survival. Hirudin is a small acidic protein isolated from salivary glands of Hirudomedieinalis, which is the best inhibitor of thromin we have found today. It has very significant effect on the prevention and treatment of human cardiovascular disease, and it is the first choice for patient with cardiovascular disease.In this study, we used site-directed mutagenesis construct an anti-DPP-IV GLP-1analog, recombinant orally long-acting GLP-1(rolGLP-1), which has a Ser substitute for Ala at the eighth amino acid position to resist the cleavage by DPP-IV, and one Glu26plus one Asp34substitutes for two Lys residues (lys26, lys34) respectively to inhibit peptide degradation by trypsin. Moreover, we used molecular cloning technique constructed a recombinant GIP (rGIP) by replacing Ala with Gly at the second amino acid position of GIP analog resist the cleavage by DPP-IV and constructed a recombinant HV(rHV). We ligated muti-copy rolGLP-1gene with muti-copy rGIP gene and a rHV gene to obtain4rolGG(4rolGLP-4GIP)、5rolGLP-4GIP and5rolGLP-HV fusion gene. The pET-22b(+)-rolGG pET-22b(+)-4rolGG、pET-22b(+)-5rolGLP-4GIP and pET-22b(+)-5rolGLP-HV was obtained by cloning rolGG,4rolGG, rolGLP-4GIP and5rolGLP-HV into pET22b(+) vector, and then transformed into BL21(DE3). The transformed engineering strains after induced with IPTG and then analysised with SDS-PAGE electrophoresis. The results showed that the fusion peptides achieved efficiency and soluble expression. We optimized the incucible expression condition, the results showed that, the best expression condition of rolGG is under the0.6mmol/L final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG), incubated at25℃for10h; the best expression condition of4rolGG is under the0.2mmol/L final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG), incubated at37℃for5h; the best expression condition of5rolGLP-4GIP is under the0.4mmol/L final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG), incubated at30℃for10h; the best expression condition of5rolGLP-HV is under the0.4mmol/L final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG), incubated at37℃for5h; The cell pellets resuspended into lysis buffer and disrupted by sonication. The supernatant was load to Ni-NTA resin, and then we obtained the higher purity peptides of rolGG,4rolGG,5rolGLP-4GIP and5rolGLP-HV, laid a better foundation to testify the function of these fusion peptides. The digestion assay of rolGG in vitro demonstrated that the rolGG peptide can be cleaved by modified trypsin led to production of two fragments, rolGLP-1and rGIP, and the efficiency reached to about70%.STZ-induced diabetic mice were used to determine the biological activity of rolGG,4rolGG,5rolGLP-4GIP and5rolGLP-HV fusion peptide in vivo. The results showed that, compared with0.9%NaCl processed negative control mice, rolGG,4rolGG,5rolGLP-4GIP and5rolGLP-HV fusion peptide led to significant decrease the levels of plasma glucose and cholesterol, improve the levels of oral glucose tolerance, glycosylated hemoglobin and the secretion of insulin, and alleviation of emaciation, polydipsia, and polyphagia symptoms.We used thrombin titration assay to test the anticoagulant activity of fusion peptide, the results showed that its anticoagulant activity from HV was105±1.7ATU/ml.Our study used Thrombosis model to verify the antithrombotic activity of5rolGLP-HV fusion peptide. We administered the5rolGLP-HV fusion peptide at the time of subcutaneous injection of carrageenan (50mg/kg) in KM mice to judge the time of thrombosis occurring and the relative length of thrombus in KM mice tail. Finally, the mice fed the5rolGLP-HV fusion peptide occurred thrombosis at about32h, and the relative length of thrombus in tail had reduced from37.5%to25%, so we knew that the5rolGLP-HV fusion peptide paly a great role in antithrombosis.This study provides an exciting evidence for the research of orally peptide drugs the functional-complementary dual insulinotropic peptide rolGG and muti-copy functional-complementary dual insulinotropic peptide4rolGG and5rolGLP-4GIP, establishes the foundation for the peptide drugs by oral application in the treatment of diabetes mellitus and offers a possibility in the treatment of type2diabetes by using the orally recombinant fusion peptide to produce the immunological tolerance. Otherwise, our study also indicated that the fusion peptide5rolGLP-HV has dual-function, and it is expected to be developed into an oral drug for the treatment and prevention of diabetes and thrombosis. Our study laid a solid foundation for the further pre-clinical study of5rolGLP-HV.