The Biological Functions Identification Of Subolesin And Cysteine Protease Genes From Hyalomma Delritum

The hard tick Hyalomma detritum is an obligate ectoparasite that can transmit a remarkable variety of pathogens, especially Theileria annulata, which caused massive economic losses. Recently, the main method of controlling ticks and tick-borne diseases is chemical acaricides. However, this approach has serious disadvantages. With the occurence of environment pollution and acaricide resistance problems,the immunological method was paid more and more attention. Recently, to apply this method, it is critical to search for appropriate functional genes in ticks to be potential protective antigen. The study to the biological characteristics of functional genes offers theory foundation to the vaccines in controlling ticks and tick-borne disease. At present, according to the research of reported functional genes in other ticks, it shows that subolesin gene and cysteine protease gene had been detected in several organisms.Evidence of the conservation of subolesin throughout evolution was provided by the high homology of amino acid sequences in higher eukaryotes. And cysteine protease is involved in host blood digesting in ticks and regulating reproduction and development of ticks.According to the study, the complete open reading frames(ORFs) of subolesin gene of Hyalomma detritum, Hyalomma asiaticum were firstly cloned using the special primers. Through the sequence analysis to the subolesin gene, the results indicated the ORF was 492 bp, coding 163 amino acids. The analysis results of biological information soft-wares proved that the amino acid sequences were highly conserved among ticks, but also had specificity in different species.The complete open reading frame(ORF) of subolesin gene of H. detritum was cloned into pGEX-4T-1vector, and the the recombinant plasmid was transformed into E. coil BL21(DE3) cells. The recombinant protein was expressed successfully, and was approximately 45 ku. Besides, the inclusion-body protein was detected by SDS-PAGE. The result of Western blotting demonstrated the purified recombinant protein emerged strong immunogenicity with rabbit anti-H. detritum adult and rabbit anti-H. detritum salivary gland and ovary serum. What’s more, the analysis of qPCR shows that subolesin gene was detected in all developmental stages and tissues, but had different expression level.The relative expression level of subolesin gene is comparative high in tick mid gut and salivary gland.And the relative expression level increased with feeding. However, in adult ticks the level was up-regulated in the early stage feeding and down-regulated in the latter stage of feeding. All the results indicated the subolesin gene may be involved in reproduction, development and feeding of ticks.Besides, the mid gut and salivary gland of ticks played critical role in pathogen transmission,propagation and parasitic. Herein, the result suggested that subolesin gene may be involved in pathogen transmission. The effects of immunization and challenge infestation and RNAi showed the the feeding rate, duration of feeding, mortality rates, engorgement weights, and egg laying rate were affected by the gene. Consequently, we predicted that subolesin gene may be involved in reproduction, development and feeding of ticks.The conserved CP sequences was cloned from H. detrium by primers cys500. And then a 1192 bpc DNA(507 bp conserved region) encoding cysteine protease was obtained by 5’ and 3’ RACE. The complete open reading frame(ORF) extending from position 77 to 1075 bp was 999 bp coding 332 amino acids. Besides, the analysis results of biological information showed that the amino acid sequences were different among ticks, but also had conserved sites in different species. The complete open reading frame(ORF) of CP gene of H. detritum was ligated into pET30 a vector, and the recombinant plasmid was expressed successfully with approximately 45 ku in size. The purified inclusion-body protein had strong immunogenicity with rabbit anti- H. detritum adult ticks. According to the result of qPCR provided evidence that CP gene was expressed selectively. And the expression level was high only in mid gut and could be ignored in other tissues. The high relative expression level was obtained in the development stage-eggs. Meanwhile, the relative expression level increased with feeding. So, the results indicated the cysteine protease gene may be involved in eggs development,feeding and blood digestion in ticks. Herein, the effects of immunization and challenge infestation and RNAi showed the the feeding rate, mortality rates, and egg laying rate were affected by the gene.Consequently, we predicted that CP gene may be involved in eggs development and feeding of ticks.