Construction And Eukaryotic Expression Of Recombined Gene Of Tick Anticoagulant Peptide And RGDS Peptide

Tick anticoagulant peptide is a 60-amino acid polypeptide originally isolated from the soft tick Ornithodoros moubatu that shows both potent and exquisitely selective inhibition of blood coagulation FactorⅩa and limited homology to Kunitz-type inhibitors. Structure/function studies of TAP led to the discovery of a double mutation that showed a 37-fold increase in FactorⅩa inhibition in vitro relative to the wild-type protein. This mutant had a tryptophan in place of tyrosine at position 1 and an arginine in place of aspartic acid at position 10.The compound of the platelet glycoprotein (Gp)Ⅱb/Ⅲa is the major integrin of platelets and the only adhesion receptor which participate in the aggregation of the plate, is also the receptors of the von Willebrand factor(vWF) and fibronectin, involved in the adhere reaction of the platelet. At present considering, GpⅡb/Ⅲa is the important adhesion molecule in haemostasis and thrombogenesis. The receptor antagonists of GpⅡb/Ⅲa possess the specificity function in the aggregation of the antiplatelet and antithrombosis. It has been found that GpⅡb/Ⅲa binds the ligand by a conformational change that allows the binding of macromolecules ligand that contain the Arg-Gly-Asp (RGD) motif, the ligand of FB and Fn and VWF have such a motif. The polypeptide with RGD motif was synthesized through gene engineering or synthesized artificially can inhibit ligand (such as fibrinogen) bind the GpⅡb/Ⅲa. therefore, the polypeptide with RGD motif have the function of anticoagulation and become the hot spot in clinical research. In this study, the RGDS (Arg-Gly-Asp-Ser) sequence was designed to inserte into top of theβ-fold's finger on the TAP in some conformational restriction by the loop grafting method, and the combined gene encoding the new peptide was expressed in yeast, and obtained a difunctionality anti-thrombus preparation having both antithrombase activity and antiplatelet aggregation activity.The synthetic gene segment encoding was designed accordance with the native TAP's amino acid sequence double mutant's TAP gene sequence which had a tryptophan in place of tyrosine at position 1 and an arginine in place of aspartic acid at position 10 and the RGDS sequence was inserted into top of theβ-fold's finger on the TAP. The total length have 64 amino acids residue's sequence and 216bp including the basic groups for the cutting site of restriction enzyme. The synthetic gene was constructed from 4 overlapping oligonucleotides and synthesized. The internal oligomers were ligated by T4-DNA ligase at 16℃over a night after been phosphorylated. The product amplified by polymerase chain reaction (PCR) was purified from an agarose gel electrophoresis, and was cloned into the plasmid PMD18-T. E.Coli DH5αwas transformed with the plasmid PMD18-T. The sequence of the correctly oriented inserted gene was confirmed by DNA sequencing and restriction enzyme analysis designed PMD18T-TAP-RGDS. The TAP-RGDS gene was amplified by polymerase chain reaction (PCR). The PCR product was purified after agarose gel electrophoresis. The double restriction enzymes (EcoRI and NotI) were used to digest the PCR product and expression vector pPIC9k for the yeast Pichia pastoris. The objective fragment was retrieved from the agarose gel electrophoresis, and was linked by the T4-DNA ligase. The plasmid was renamed pPIC9k -TAP-RGDS. The E.Coli DH5αwas transformed with the plasmid pPIC9k -TAP-RGDS. The positive clon was selected, and confirmed by restriction enzyme ( EcoRI and NotI) analysis and DNA sequencing. It was confirmed that TAP-RGDS gene was inserted correctly into the vector pPIC9K by the above mentioned techniques. The reconstructed expression plasmids pPIC9K-TAP-RGDS of tick anticoagulant peptide and RGDS peptide gene were linearized by restriction enzyme SalⅠdigest and transformed into susceptible yeast Pichia pastoris GSl15 cell by the electroporation transformation method. The reconstructed yeast engineering strains was renamed GS115 /pPIC9K-TAP. The GS115/pPIC9K-TAP were identified by MD and MM and G418 plate selecting and PCR. The induced interests proteins was expressed after methanol inducement, and analyzed by SDS-PAGE. The interests protein was purified by using precipitation, and renatured by density gradual dialysis. The prothrombin time (PT) and activated partial thromboplastin time (APTT) was used to detect the anticoagulant property of interests protein. The platelet aggregation tes(tPAgT)was used to detect antiplatelet aggregation activity of interests protein.The construction of the recombinant express plasmid pPIC9K -TAP-RGDS was proved by restriction enzyme analysis and DNA sequencing and PCR. The reconstructed expression plasmids were transformed into susceptible yeast Pichia pastoris GSl15 cell by electroporation transformation method. The reconstructed yeast engineering strains GS115/pPIC9K-TAP were identified by MD and MM and G418 plate selecting and PCR. The induced interests proteins were analyzed by SDS-PAGE. There is a protein band at standard molecular weight 8kD. The results showed that the interest genes were expressed exactly. Blood clotting experiment showed that both supernatant and sediment of the reconstructed yeast had anticoagulant property.In conclusion, The eukaryotic expression vector of rTAP-RGDS was successfully constructed with the gene synthesis; the recombination production was obtained by the inducement and expression; the crude extract of the reconstructed yeast showed an anticoagulant property.