| Objective:Ticks feed on blood, which seriously hazards the health of human and domestic animal directly or indirectly.In this paper, we mainly focus on the research of Haemaphysalis flava to explore the full-length sequence of 4D8 gene, and examine the mRNA expression level of 4D8 gene at different stages and in different tissues of H. flava, for providing a theoretical basis about a universal vaccine development.Methods:1. The morphology characteristics of these ticks were observed by stereo-microscope. The COX1 and ITS1 genes of ticks were amplified, cloned and sequenced 2. Primes based on the sequence alignment of 4D8 among Haemaphysalis were designed for the PCR amplification of 4D8 Open Reading Frame (ORF) of H. flava; RACE Primes based on 4D8 ORF sequence were designed for the PCR amplification of the full-length sequence of 4D8.3. The standard curve of objective gene and house-keeping genes were established by using diluted template DNA samples for 1,5,25,125,625 times. Under the optimal conditions, the 4D8 gene expression levels at different stages and in different tissues of H. flava were detected by using relative quantitative RT-PCR. Results:The results of morphology and molecular biology show that the ticks are Haemaphysalis flava,and the distribution and the range of host of the tick is much broader than has been recorded.The full-length sequence of 4D8 is 1318bp, and contains an ORF of 540bp.The ORF sequences is relatively conservative between different species. The expression levels of 4D8 gene were detected in different stages. The results showed that the expression levels of the gene in the female ticks increased with their growth phase, and the expression levels of Subolesin was higher in engorgement female adult tick than in engorgement male adult tick. The results of 4D8 gene expression levels in different tissues showed that the expression levels of Subolesin was higher in blood feeding adult tick than in engorgement adult tick. |
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