The Cloning,Expression And Identification Of Haemalin Of Haemaphysalis Flava

Ticks are a kind of vampire living parasites that had serious public health implications,which spread a variety of zoonotic infectious diseases.to date,there was not an effective alternative to control of tick infestations.In current study,we was based on the study of salivary gland transcriptase group to screened Haemalin gene fragment.The full length was obtained by cloning.The prokaryotic expression and in vitro activity showed that the protein had anticoagulant function and had the potential of new drug development.The 3 'end sequence of contig38438 gene fragment was successfully amplificated by RACE and the full sequence of Haemalin was obtained by splicing.The sequence analysis showed that the length of Haemalin(KX094561.1)607bp encoded by a gene with ORF of 423 bp that was translated into a deduced 141 aa with a 16aa-putative signal peptide at N-terminal and protein molecular weight was 14 kDa,inclueding two Kunitz domains.the ORF fragment was cloned into pGEX-4T-1 vector,and the recombinant plasmid was transfected into BL21(DE3)cells.The recombinant protein was expressed by SDS-PAGE.SDS-PAGE showed that the protein had good water solubility.Western Blot proved that the protein as the recombinant protein,the protein molecular weight of 40KDa(GST tag protein molecular weight of 26KDa).The result showed that Heamalin fusion protein could inhibit PT and APTT,suggesting that Heamalin could prevent blood coagulation by exogenous and endogenous coagulation factors.