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Binding of Rhipicephalus (Boophilus) annulatus tropomyosin and cathepsin l like as vaccine candidate using OE-PCR
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عنوان دوره: دومین کنگره بین المللی حشره شناسی ایران
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چکیده
Introduction: The cattle tick, Rhipicephalus (Boophilus) annulatus, is one of the most abundant tick species worldwide, specifically in tropical and subtropical territories. This species is mainly found in North of Iran. Additionally, tick infestation usually provokes notable financial burden in livestock industry through blood loss, weight reduction, decreased lactation, anorexia, irritation, and toxic conditions such as paralysis and allergies. The tick control programs have frequently been relied on the utilization of anti-tick chemical compounds, i.e. acaricids. However, the emergence of acaricid resistant tick populations and persisting of chemical residuals in animal productions, justify the demand for other prevention strategies. So far many proteins have been introduced as a vaccine candidate. One of the suggested anti-tick vaccine candidates is the actin-binding Tropomyosin protein. Tropomyosin protein plays an important role in regulating actin function, immune and allergic reactions and vaccine production. Cathepsins are lysosomal proteases with several members such as cysteine, aspartic and serine proteases. The physiologic actions of cathepsins are enclosing degradation of intracellular and endocytosis-introduced extracellular proteins. Several methods are used for binding proteins. The overlap extension polymerase chain reaction (or OE-PCR) is designed to attach two separate PCR products together that have been designed to have a short overlap of complementary sequence. This overlap can be produced by the addition of bases to the ends of the internal primers for the respective PCR products. In this study we aimed to binding of two genes of Rhipicephalus annulatus tick larva as fused potent vaccine candidates, cathepsin and tropomyosin, using OE-PCR.
Material and method: Reference sequence of tropomyosin and cathepsin proteins were obtained from NCBI data base. Common fragments of each proteins sequence among ticks, differing from those of cows, were selected. The specific primers (Forward, Reverse and complementary primers) were designed for OE-PCR. Two genes were linked to each other by OE-PCR and the PCR product was run on the agarose gel and sequenced by TakapouZist Company.
Result: PCR product on the agaros gel showed a sharp band in intended spot. The result of sequencing confirmed these findings.
Conclusion: There have been introduced several anti-Boophilus vaccines so far, but they were imperfect. There is hope that in future it will become possible to use this construct as a Protein Vaccine Candidate with none of the previous vaccine defects. This requires cloning and expression of these construct and inject it into animals.
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