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Cloning and Expression of Gene Encoding the Anticoagulant Protein of Rhipicephalus Haemaphysaloides Tick and the Activity Analysis of Its Expressed Products

Author: ShiLei
Tutor: ZhouJinLin；YueCheng
School: Xinjiang Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Rhipicephalus haemaphysaloides C18 gene C201 gene cloning expression activity analysis
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 63
Quote: 0
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Abstract


Tick is a special category of the blood-sucking arthropods, not only an important foreign animal parasites, but the second media of mosquito and animal diseases. Compared to other blood-sucking animals , ticks spend more time to vampire, usually need 10-14 days to reach a state of full blood. Of the blood-sucking, the host produced coagulation reaction, such as microvascular reaction、platelet aggregation and blood coagulation. Tick salivary gland secretion the anticoagulant to overcome the host coagulation response for vampirring successful.Researching of Tick anticoagulant protein, not only helps to understand the blood-sucking principle and the spread of pathogenic mechanisms, but open up tick and tick-borne disease prevention and control of new technology. In addition, tick anticoagulant peptide have great value for developing the antithrombotic drugs.Rhipicephalus haemaphysaloides is the edge species in southern of China. This study was based on the EST data of the libraries of differentially expressed genes in the salivary glands of the unfed and partially engorged Rhipicephalus haemaphysaloides. Cloning of a gene encoding anticoagulant gene C18.The C18 cDNA is composed of 580 nucleotides and encodes 164 amino-acid resides, the deduced mass is 18.04kDa. In the 5 ’end of this protein there is a 18 amino acids constitute the signal peptide sequence and contains a typical Kunitz domain anticoagulant. By homology comparison of the gene and tissue inhibitor gene sequence is highly homologous. Using RT-PCR analysis of the C18 in Rhipicephalus the expression of blood before and after shows, C18 for the induction of expression after blood-sucking ticks; The recombinant proteins of GST-C18 were expressed by pGEX-4T-1 vector in E.coli and the fused protein were purified by GST Sepharose. The result indicated that it can be expressed in high level in E. coli.; through RT and APTT test of the recombinant protein proved that the recombinant protein at the same time endogenous and exogenous significant inhibition of blood coagulation.At the same time, the full-length sequence of C201 protein was obtained by rapid amplification of cDNA ends protocol.The C201’s cDNA is composed of 693 nucleotides and encodes 195 amino-acid resides, the deduced mass is 22.62kDa. In the 5 ’end of this protein there is a 20 amino acids constitute the signal peptide sequence and contains a typical Kunitz-BPTI domain anticoagulant. By homology comparison of the gene and tissue inhibitorⅡgene sequence is highly homologous .In conclusion, the study cloned two anticoagulant gene C201 and C18, expressed C18 and researched the anticoagulant activity, this will be the foundation to further study.

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