Rhipicephalus Tick Salivary Glands Metalloproteinase Gene Cloning And Biological Function Analysis

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. At present, about 100 kinds of tick were found in china. 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. On the other hand, Metalloproteinase, existing widely in the parasites, blongs to a family of proteinase whose functions depending on the metal ion. It is believed that metalloproteinase play a role in many important functions. Some Metalloproteinases have strong immunogenicity , so they are believed to be a kind of improtmant molecule of anti-parasite vaccin and for medical research .Based on the cDNA library of Rhipicephalus haemaphysaloides partily fed female adult which was built by Weiyi Zhang , in this study , we aimed to clone , express and characterize of Metalloproteinase gene from a hard tick--- Rhipicephalus haemaphysaloides tick--- it is a predominance kind of ticks in south of china , respecting to the search of tick molecules for vaccine candidates.Firstly gene special primers which were used in 5'-RACE amplification were designed based on the EST sequence , GSP1:5'-CCTGAAGCCCGTGCT-3';GSP2:5'-GCTGTCGGCCGGGTTTCTCCTC-3';nestedGSP:5'-GCCTTCGACTTCCACGTT GTCGT-3' . The PCR products of 5'-RACE were purified, linkaged to pGEM-T-easy, transformed DH5 α and sequenced . Gene special primers which were us ed to amply full length of genes were designed based on the sequence of 5'-e nd , MP-F-5': 5' -GCTCTATATGTATCGCATGTACAAC-3' MP-F-3': 5' -CTTCCAAAG CAGCAACTCCCAC-3. The products of PCR were finally sequenced and identifie d, then the full length of the MP genes were learned .Secondly, the natural Expression of MP in ticks were analysised by the reverse transcription polymerase chain reaction method (RT-PCR). The total RNAof different phase and organ were extracted using the TRIZOL reagent respect...