| Ticks are haematophagous ectoparasites that infest domestic animals and fowls. As the media ,they could be disseminated by a variety of viruses, bacteria, spirochetes, rickettsia and protozoan diseases such as blood, not only to the livestock industry has resulted in serious economic losses, but also a great human public health hazards.Nowadays,there are about 100 kinds of tick were found in china, Hyalomma asiaticum is a predominance kind of ticks in china, which transmit T.annulata, Babesia and hemorrhagic fever of Xinjiang. Immunization protection is deemed as the most potential control strategies in the world.However, the effective target antigen may be the base for immunization protection. Histamine binding protein is an significant kind of protein in ticks,and mainly existing in haemolymph and salivary which are able to bind host Histamine that entering the haemolymph of ticks, thus avoiding the damage to the their internal organs. HBPs are female specific espression which is essential for them get a successful meal.tick's HBP can be used as the target nolecules for anti-tick and anti-tick vaccine.This study cloned and expressed a HBP gene of Hyalomma asiaticum for the first time and analyzed its function,which will be for the tick and tick-borne diseases and their immunization of work to do some exploring.Firstly,The gene special primers 3'-GSP and 3'-Nested-GSP which were based on the EST data of the libraries of differentially expressed genes in the salivary glands of the unfed and partially engorged Hyalomma asiaticum, the PCR products of 3'end Amplified was purified, cloned to pGEM-T easy, transformed DH5αand sequenced. Gene special primers 5'-GSP1,5'-GSP2 and 5'-Nested-GSP which were used for 5'end Amplified were designed based on the sequences of EST and 3'end sequences of target gene, the PCR products of 5'end was purified, cloned, transformed and sequenced. Gene special primers F-Primer and R-Primer which were used to amply full length of genes were designed based on the sequence of the full length of target gene , the PCR products of the full length of target gene was sequenced and identified, then the full length of the genes was learned. The full-length sequence of Hyalomma asiaticum Histamine Binding protein (HaHBP) was obtained by rapid amplification of cDNA ends protocol.The HaHBP's cDNA is composed of 902 nucleotides and encodes 227 amino-acid resides ,the deduced mass is 25.9kDa . And the gene of Hyalomma asiaticum tick show high sequence homology to other tick's HBP whose were repoted in the NCBI, so it should be identified as the HBP gene,and named HaHBP. The sequence of HaHBP has the typical characteristics of secondary structure of HBP, what is the structure contains aβbarrel, which is histamine binding site, and the secondary structure was consisted of two disulfide bonds and only four or five tryptophan residues (W) part of the decision. HaHBP encoded amino acid sequences have been identified with the histamine-binding protein in a very similar position or have the same or similar cysteine (C) and tryptophan residues (W), which is the structure of histamine-binding protein characteristics coincide. Comprehensive homology, structural features, molecular weight, signal peptide, gene expression characteristics of the results of bioinformatics analysis, the initial forecast for a coding HaHBP Hyalomma Asia histamine binding protein of a new gene.Secondly, Expression of HaHBP in Hyalomma asiaticum were analysised by RT-PCR. The total RNA of eggs, larva, nymphae, unfed adult, parcially fed adult and shell , salivary gland , gut.Those total RNA were used in RT-PCR analysis respectively, the results revealed that the expression of HaHBP gene had female specific characteristic, and the HaHBP gene only was expressed in parcially fed female adult,which include the fed female adult's salivary, shell and gut, but not expressed in eggs, larva, nymphae and unfed adult.Thirdly, The recombinant proteins of GST-HaHBP was expressed by pGEX-4T-1 vector in E.coli by induction of IPTG and the fused proteins was purified by GST Sepharose. The result indicated that it can be expressed in high level in E.coli and have a better immunogenicity by Western-blot analysis .At last, To the fusion protein purified from GST-HaHBP (GST protein as negative control) as the coating antigen in ELISA ELISA plates 4℃overnight, after washing, by adding Histamine-HRP enzyme conjugate 37℃reaction of 2h, after the full washing, by adding K -Blue color substrate after 10min by adding the termination of liquid 5min, microplate reader OD values measured.It was proved that HaHBP can combined with histamine by ELISA-Histamine for the first time.All in all, we successfully cloned a new HBP from Hyalomma asiaticum .and the HaHBP could be expressed in Bl21 with high level. The recombinant protein has strong immunogenity and immunization.It was proved that HBP can combined with histamine by ELISA at the first time.The study establishes an important base for further research on the function of HaHBP.
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