Midgut Transcriptome Analysis Of Haemaphysalis Flava And Expression And Identification Of Serpin 2

Tick is a parasite which have great public health significance, but so far there is no environmental friendly, safe and efficient method of tick controlling.Bm86 antigen molecule found and Boophilus vaccine applied are greatly stimulated people to develop anti-tick or tick-borne disease vaccine initiative. In this study, proteomic data as clues, transcriptome date as the basis,we clone,express and analyse serpin 2 of Haemaphysalis flava,there sults are shown below.This result not only deepen the understanding of the biological characteristics of ticks, but also provide new ideas for tick and tick-borne diseases controlling.We utilize high-throughput sequencing to characterize the midgut transcriptome of fully engorged and partially engorged female H. flava.by using Illumina sequencing technology,all high-quality sequencewere finally assembled into 76,556 unigenes with mean length of 704 bp. Functions of the assembled unigenes were annotated by Blastx against the NCBI Nr and Swiss-Prot databases, sequences were also aligned to the KOG and KEGG databases,GO functional.Swiss-Prot databases classify unigene functional annotation of 26,652, accounting for 34.81%; gene expression levels were calculated with the RPKM value,a total of 5508 differentially expressed genes (DEG) were identified between FE and PE.Compared with the PE, the numbers of up-and down-regulated genes in FE were 3940 and 1568, respectively.Contig7475 is a unigene belongs to midgut transcriptome of female H. Flava, functional annotation is:serpin 2 encoding gene fragment.analysed with serpin 2 of Haemaphysalis longicornis suggests this unigene lacks the 5' and 3'end of the sequence, we obtained serpin 2 full-length sequence of H. Flava(1488 bp) by 5'-RACE and 3'-RACE,the open reading frame is 1197bp, which encoded 398 amino acid,signal peptide position for the first 1-17aa; their sequence similarity with H.longicornis highest at 85.14%.We inserted cloning ORF of serpin 2 into pET-28a(+) vector, the recombinant plasmid is transformed by BL21(DE3), a protein highly expressed after induction, the relative mass of 42kDa, western-blot showed that the protein is a recombinant protein. In vitro experiments showed that the purified serpin 2 fusion protein can significantly prolong the APTT, PT and TT are also extended, but the effect was not significant (not more than 3S), it indicate that serpin 2 fusion protein anticoagulant mechanism work on endogenous coagulation system.