Analysis On The Excrement Of Haemaphysalis Flava And Expression And Identification Of HafAV422

Tick is a parasite have great public health significance, but so far there is no environmental friendly, safe and efficient method of tick controlling. This study was based on fecal protein component analysis of Haemaphysalis flava, reference salivary transcriptome date screening out research target. Through cloning, expression and in vitro experiments to evaluate the value of HafAN422 as anti-tick vaccine.LC/MS/MS analysis H.flava fecal protein components, selected AV422 as research target protein; obtained AV422 protein-coding gene fragment (Contig2959) by search into the salivary transcriptome libraries established and midgut transcriptome library; cloning HafAV422 missing 5' and 3' with RACE, acquire HafAV422 full length sequence;clone ORF insert into pET28a vector, conversion with BL21 to obtain HafAV422 recombinant protein;Measure the effects of different concentrations of HafAV422 purified protein work on PT, APTT, TT and other indicators of coagulation.In this study, H.flava facal samples were detected 841 peptides,66 unique peptides, thereby identifying 18 proteins. Among specific peptides CLTDIQAGLEK, HAPCLGQTLPDQKK, APCLGQTLPDQK and ELIGFVAEGSQELFK were functional annotations for AV422 proteome. Contig2959 is a partial sequence in salivary transcriptome library of H.flava, missing 5'-end and 3'-end sequence. Cloning results showed HafAV422 of H.flava gene full length of 1152bp, the size of the open reading frame of 696bp, and AV422 similarities were more than 92.6%. With pET28a system successfully expressed HafAV422, the protein can significantly extend the TT, PT and APTT, therefore inhibit coagulation factor and plasminogen.There are variety of protein components in ticks feces, some of them are secretions residue in the salivary glands and midgut, it's highly conserved, play an important role in anti-coagulation, is a potential anti-tick vaccine.