The Study In Interaction With Coagulation Factors Of Recombinant Protein Hq002 In Salivary Glands Of Haemap Hysalis Qinghaiensis

Objective The salivary gland recombinant protein hq002 of Haemap Hysalis qinghaiensis is expressed and purified in E.coli BL21 to study the interaction between recombinant protein Hq002 and coagulation factors.Methods The gene sequence of hq002 is digested by Nde I and Xh O I and then inserted into the prokaryotic expression p ET-30a vector to obtain the recombinant expression plasmid pet30a-Hq002.The recombinant expression plasmid is transformed into E.coli BL21（DE3）and induced by IPTG.The expression of r Hq002 is identified by 12%SDS-PAGE and Western blot and rhq002 is purified by Ni²⁺ion metal chelation chromatograp Hy,and r Hq002 is obtained by dialysis and ultrafiltration.Analyze the influence of r Hq002 on APTT,PT and TT tests.React r Hq002 with a variety of serine proteases and their chromogenic substrates to analyze the possible role of recombinant protein in the process of coagulation.Results r Hq002 has two Kunitz domiant and it can significantly enhance the amide decomposition activity of thrombin,FXa and TF-FVIIa complexes on chromogenic substrates.It prolongs APTT and has no effect on the amide decomposition activity for FIXa,FXIa,FXIIa,α-Trypsin,plasmin,kallikrein,u PA and other active molecules involved in coagulation.Conclusion r Hq002 enhances the amide decomposition activity of thrombin,FXa and TF-FVIIa complexes on chromogenic substrates and prolongs APTT.Recombinant protein Hq002 is a novel tick-derived protein with anticoagulant potential.It provides a new idea to develop serine protease inhibitor anticoagulant drugs.It provides a new idea for the research and development of tick-borne vaccine.