| Ticks are obligate ectoparasites that infest domestic animals and fowls . They are found in many regions of the world and they are the significant vectors transmitting a great number of pathogens of human and animals, such as arboviruses, rickettsiae, spirochetes and parasitic protozoa. At present, about 100 kinds of tick were found in china, Rhipicephalus haemaphysaloides tick is a predominance kind of ticks in south of china, which transmit Babesia bovis , B.equi and B.gibsoni. Current tick control methods depend heavily on the use of chemical acaricides. However, the accompanying problems of resistance to the acaricides and the presence of chemical residues in meat and milk show the need for alternative control methods. Immunological protection of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method comparing to the use of acaricides. On the other hand, cysteine proteinases enzymes has been demonstrated that they are involved in the mediation of key physiological functions such as embryo development and nutrition of parasite, thus they can be considered as good target antigens for a tick vaccine. In this study ,we aimed to clone , express and characterize of cysteine proteinases gene from a hard tick--- Rhipicephalus haemaphysaloides tick, respecting to the search of tick molecules for vaccine candidates.Firstaly, gene special primers which were used in 3'-RACE amplification were designed based on the consensus amino acid motifs flanking present in all papain-like cysteine proteinases and tick codon usage frequency table, GSP=5'-ACCAGGGCCAGTGCGGCTCCTGCTG-3', the PCR products of 3'-RACE were purified, linkaged to pGEM-T easy, transformed DH5α and sequenced. Gene special primers which were used in 5'-RACE amplification were designed based on the sequence of 3'-end, cysA: GSP1=5'-AGTGTCCAGCGTCAATGGCTA-3', GSP2=5'-GATGTCTACAAAGCCGGTGTC, nested GSP=5'-ACCAGGGCCAGTGCGGCTCCTGCTG-3' ; cysB: GSP1=5'-TAGACACCTTCCGAGTAGAAC-3', GSP2=5'-CGTGGCTGGCGTCGATGGCAACTGAGAC-3', nested GSP=5'- ACCAGGGCCAGTGCGGCTCCTGCTG-3', the PCR products of 5'-RACE were purified, linkaged, transformed and sequenced. Gene special primers which were used to amply full length of genes were designed based on the sequence of 5'-end, cysA: GSP3=5'-GAGCTCTCCACCAGGTGCACC-3'; cysB: GSP3=5'-AGGTCACTTGGGGTCTCCGGC-3', the products of PCR were finally sequenced and identified, then the full length of the two genes were learned. The full length of cysA is 1168bp, encoding a 332 amino acid residue polypeptide with 36.33kDa predicted molecular weight; the full length of cysB is 1153bp, encoding a 335 amino acid residue polypeptide with 37.56kDa predicted molecular weight. The consensus amino acid motifs flanking presence in both deduced amino acid sequences. And both genes of Rhipicephalus haemaphysaloides tick show high sequence homology to other tick cathepsin L-like cysteine proteinase, so they should be identified as members of the cysteine proteinase gene family. Secondly, the natural Expression of cysA and cysB in ticks were analysised by the reverse transcription polymerase chain reaction method (RT-PCR). The total RNA of eggs, larva, nymphae, unfed adult, parcially fed adult and shell , salivary gland , gut were extracted using the TRIZOL reagent respectively. The same concentration total RNA were used in RT-PCR analysis respectively, the results revealed that both cysA and cysB were expressed in all periods of tick development and in all tick organs except gut.Thirdly, the recombinant protein of Trx-cysA and Trx-cysB were expressed in E.coli with high level. Recombinant proteins were purified by Nickel affinity chromatography, then analysised by western blot. The results reveal that anti-serum against tick salivary gland can recognize recombinant proteins of Trx-cysA and Trx-cysB, in addition, anti-serum against Trx-cysA and anti-serum against Trx-cysB both can recognize native protein from crude extraction protein of full tick. These results sug...
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