The Study In Interaction Of Coagulation Factors With Recombinant Protein Hq012 From Salivary Glands Of HaemapHysalis Qinghaiensis

Objective To express recombinant protein Hq012 from the salivary gland of the Haemap Hysalis qinghaiensis in Escherichia coli,purify the recombinant protein and study the interaction between recombinant protein Hq012 and coagulation factors.Methods A gene fragment of a novel gene Hq012 of H.qinghaiensis was PCR amplified and inserted into a p GEM-T easy vector and transformed into DH5α competent cells.Positive clones were selected by blue-white screening.Plasmids were extracted from positive clones and digested by restriction enzymes Nde I and Xho I.The obtained Hq012 gene fragment was ligated into a prokaryotic expression vector p ET30 a excised with Nde I or Xho I in frame with the 6X histidine tag at the C-terminus.The constructed recombinant expression plasmid was transformed into E.coli Rosetta(DE3)and recombinant protein Hq012 was induced by IPTG.The expressed r Hq012 was analyzed by 12% SDS-PAGE.The recombinant protein in inclusion bodies was purified by either under native or denatured conditions.Native purification means the solubilized protein was refolded before passing through a Ni-NTA affinity chromatography followed by renaturation by dialysis,while the strategy of purification under denatured condition means the denatured protein was first purified by a Ni-NTA affinity chromatography column and then refolded by a series of urea dilution dialysis.The purified r Hq012 was further evaluated for its effect on APTT,PT and TT assays.The potential effects of r Hq012 on the coagulation process was analyzed by incubation of the protein with various serine proteases and their corresponding chromogenic substrates.Results The prokaryotic expression vector p ET-30a-Hq012 was successfully constructed.Recombinant protein Hq012 was successfully expressed in E.coli as a form of inclusion bodies.A higher amount of purified r Hq012 was harvested under a native condition compared to under a denatured condition.Except for a enhanced effect on the amidolytic activity of FXIa towards its substrate,r Hq012 showed no significant effect on the amidolytic activity of other serine proteases towards their corresponding substrates.And r Hq012 could not affect the APTT,PT and TT tests significantly.Conclusion r Hq012,a novel tick-derived protein with no anticoagulation function at present was obtained.