Studies On The Salivary Gland Of Phipicephalus Haemphysaloides Haemphysaloides And Its Expressed Sequcence Tags

Rhipicephalus haemaphysaloides haemaphysaloides is one of the most important ticks in South China and can cause many diseases such as bovine babesiasis. Its salivary gland is an important organ during biting and secretes a number of functional molecules which are potential candidates for vaccine development. The objectives of the present study were to examine the salivary gland's compositions and to look for molecules which could be used vaccine candidates for controlling tick and tick-borne diseases.At 25℃ and relative humidity of 92%, R. haemaphysaloides haemaphysaloides was raised by biting rabbits and propagating. The salivary gland was obtained by dissecting the R. haemaphysaloides haemaphysaloides. Salivary gland extract (SGE) was extracted by freezing and thawing the salivary gland in liquid nitrogen several times, followed by homogenization by ultrasound and centrifugation.Ten micrograms of SGE from non-biting Rhaemaphysaloides haemaphysaloides and from those bitted for 5 days were analysed by SDS-PAGE, and the results revealed that their protein expressions were obviously different. The SGE changed after biting and produced some new molecules in the range of 45-100kDa. The proteins were transferred to PVDF and analysed by Western-blotting using the serum from rabbit which was bitted by R. haemaphysaloides haemaphysaloides thress times. The results indicated that the protein of 35 kDa could be a very important one and the protein of 100kDa is a newly expressed protein after biting. The dynamics of antibody responses in rabbit infected with the tick was examined using indirect ELISA. The antibody titer increased after biting and the more biting the higher titer.Using the 'Smart technique' and LD-PCR, a cDNA library of the salivary gland of R. haemaphysaloides haemaphysaloides was constructed with a capacity of 2.75x106pfu/mL and a recombinant rate of 95%. The inserted fragments were in the range of 0.5-2.0kb. The constructed library provided the resources for cloning valuable new genes. 100 ESTs from this library with an average length of 745bp were obtained by random sequencing. 27% of the ESTs represent new unknown genes. The others included two complete genes, some inhibition factors, cements and other valuable ESTs. The results of the present studies have important implications for further studies of the salivary gland and for the development of molecular vaccines for controlling tick and tick-borne diseases.