Gene Cloning, Expression And RNA Interference Of Two Immunosuppressant Proteins From The Salivary Glands Of Ticks

Ticks are obligate hematophagous organisms and the most important ectoparasites of domestic animals,which can transmit lots of pathogens.Concernning the health of human and livestock, tick control make great sense. Salivary gland , one of the most important organ in ticks, secrets many functional moleculars critical to ticks'blood-feeding and pathogen-transmitting. To study the molecular interactions at the arthropod–host-pathogen interface, and also to find immunizing against targets in ticks'salivary gland,we selected two expressed sequence tags (ESTs) from the libraries of differentially expressed genes in the salivary glands of the unfed and partially engorged ticks. By the methods of gene cloning,we get the full-length sequences of two genes which have great identity to immunosupressant protein p36 in ticks. One named RH36 is from Rhipicephalus haemaphysaloides ,another named HA36 from Hyalomma asiaticum.The full-length sequence of the gene for RH36 is 844 base pairs.Open Reading Frame is from 81bp to 734bp with a coding capacity of 24 ku ,and its deduced amino acid sequence has 218 amino acids. By analysis of signal pepitide, its deduced amino acid has a signal pepitide composed by 21 amino acids. In coding region ,there is five N-glycosylation sites,ten phosphorylation sites and three N-myristoylation sites. According to the result of BlastX, the sequence show great homology with the sequences of the immunosupressant protein p36 in other ticks. The amplified PCR product was digested, and ligated into the expression vector, and sub-cloned into Ecoli. BL21(DE3), the expression bacteria. Transformed E. coli was induced by IPTG,and expressed the desired recombinant product. We do RNA interference by microinjecting dsRNA into ticks. Analyzing the effect of RNAi by indirect immunofluorescence and Real-time PCR, we discovered that messenger RNA levels for RH36 is down-regulated.Concerning the control ticks, the sticking percentage is 22.86% lower in 48 hours, and almost all of the engorged female ticks fail to lay eggs,the percentage of which is about 60% lower. The weight of engorgement tick is a bit lower ,and the time RNAi ticks need to bite on the host is a bit longer. It indicated that RH36 has something to do with blood-feeding and breeding.The full-length sequence of the gene for HA36 is 924 base pairs.Open Reading Frame is from 44bp to 752bp with a coding capacity of 27 ku ,and its deduced amino acid sequence has 235 amino acids. By analysis of signal pepitide, its deduced amino acid has a signal pepitide composed by 19 amino acids. In coding region ,there is six N-glycosylation sites,seven phosphorylation sites and one N-myristoylation site. According to the result of BlastX, the sequence show great homology with the sequences of the immunosupressant protein p36 in other ticks. In RT-PCR, HA36 mRNA transcripts were detected in the salivary gland of partially engorged adult ticks (6 days after the start of feeding), not in non-fed adult ticks,indicating that the expressiong of HA36 is induced in blood-feeding. The amplified PCR product was digested, and ligated into the expression vector, and sub-cloned into Ecoli. BL21(DE3), the expression bacteria. Transformed E. coli was induced by IPTG,and expressed the desired recombinant product. In conclusion, we cloned a gene of the immunosupressant protein p36 from Hyalomma asiaticum and Rhipicephalus haemaphysaloides for the first time,and get the recombinat protein expressed in Ecoli. BL21(DE3);we also investigated the function in a pilot study, this will be the foundation to further study.