Studies On The Role Of HSP70-b2 And Its 14-mer Peptide From Haemaphysalis Flava

HSP70 has antigen presenting function;HSP70 of ticks does not possess TKD peptide but has anticoagulant activity.In this study,Haemaphysalis flava HSP70-b2 was used to change its TKD-like peptide sequence into TKD-like peptide sequence by mutating the coding gene,and then their anticoagulant activity and immunogenicity changes were observed to provide a scientific basis for the use of protein in the future.⑴Contig41014 is a Unigene in the H.flava midgut transcriptome library,which encodes a protein that is a member of the tick HSP70 family,but it lacks the 3’end sequence.In this study,specific primers were designed with the help of DNAman,and the deleted sequence was amplified using RACE.The full-length c DNA was obtained by cloning and splicing.The results showed that the total length of HSP70-b2 gene was 2413 bp,and the total length of ORF was 1902 bp;the ORF frame encoded 634 aa of protein,with a molecular weight of70.0 KD,with a typical HSP70 protein family tag and a terminal mapping motif DEVD.⑵Through amplification,ligation and cloning,the nucleic acid sequence encoding TKD-like peptide in HSP70-b2-ORF was modified,so that its coding peptide sequence was TKD peptide,and the mutated sequence was recorded as HSP70-b2-ORF~M.HSP70-b2-ORF and HSP70-b2-ORF~M were inserted into p ET-28a,respectively,and transformed into BL21(DE3)to obtain r HSP70-b2 and r HSP70-b2~M by induction and purification.⑶The prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT)and fibrinogen(FIB)of r HSP70-b2 and r HSP70-b2~M were measured in rat plasma.The results showed that the anticoagulant activities of r HSP70-b2 and r HSP70-b2M were similar.Rats were immunized with r HSP70-b2 and r HSP70-b2~M,and the serum antibody titers at 14 days after the first immunization,the second immunization,and the third immunization were determined by ELISA.The results showed that r HSP70-b2~M had poor immunogenicity.From the experimental results of the above two aspects,r HSP70-b2 can be a candidate factor for anti-tick vaccines;r HSP70-b2~M has the potential to be developed into antithrombotic drugs.