The Screening, Cloning And Expression Of Functional Gene Of Haemaphysalis Longicornis And Studies On Their Immune Function

Haemaphysalis longicornis are blood sucking ectoparasites and served as one of the most important vectors transmitting bacteria, viruses, parasites and rickettsia. It has been posing a serious threat to the animal husbandry in China. At present, although the principal tick control measure is chemical acaricides method, the development of alternative methods for tick control is important and urgent due to the appearance of acaricide-resistant ticks, environmental and food chain contamination. The use of tick vaccines has been shown to be the most promising alternative tick control method. The success of this method depends on the identification, cloning and in vitro expression of tick molecules which play a key physiological role in the survival of ticks and spread of disease.In this study, a cDNA expression library of H. longicornis unfed female tick was screened with anti-H. longicornis unfed female tick serum, anti-H. longicornis larva serum and anti-H. longicornis egg serum produced from rabbit, respectively. Eleven genes encoding immunodominant antigens were obtained, and named for HL01 to HL11. The nucleotide and deduced amino acid sequence analysis showed that HL07 shared 99% similarity with H. longicornis vitellogenin, and the others including were novel gene of H. longicornis, which had significant homology with the H. qinghaiensis Hq05 gene, H. qinghaiensis myogenic protein T, H. qinghaiensis myosin alkali light chain molecules, Boophylus microplus paramyosin, Ixodes scapulars surface protein and mitochondria of a variety of tick, respectively. Five genes were submitted to GenBank, accession numbers were GU932666, GU932667, GU932668, GU932669, GU932670, respectively. Eight genes including HL02, HL04, HL05, HL06, HL07, HL09, HL10, HL11 were expressed in the Escherichia coli BL21(DE3)pLysE. All of them except HL10 were expressed successfully. The expression products of HL02, HL04, HL05 and HL06 gene revealed good reactogenicity by Western-blot with anti-H. longicornis unfed female tick serum and rabbit anti-H. longicornis larva serum. These will provide candidates for further screening of protective antigen genes of H. longicornis.The HL05 gene was amplified by 5'rapid amplification of cDNA ends (5'RACE), and the full-length cDNA was obtained by splicing. The full-length of HL05 gene was 1053bp and contained an open reading frame (ORF) of 540 bp that codes for 179 amino acid residues with a coding capacity of 20.01ku. Bioinformatics analysis indicated that HL05 protein has a glycosylation site, it was secreted, no transmembrane domain and more potential antigenic determinants. The full-length sequence was submitted to GenBank and accession number was GQ499841. The recombinant plasmid pSCREEN/HL05 was expressed in E. coli BL21 (DE3), and expression products were 55 ku. Affinity chromatography was used to purify the recombinant protein, and the protein HL05 had good immunogenicity by indirect ELISA. Native HL05 was detected in ticks larvae, nymphs and adult except egg successfully, and it may have two isoforms which were 21ku, 23 ku. Vaccination of rabbit with rHL05 resulted in reduction of engorged weight of female ticks compared to the controls, and it conferred that rHL05 is a significant protective immunity. These results revealed that rHL05 could be a candidate vaccine molecule for the control of H. longicornis.