Construction Of CDNA Expression Library Of Salivary Gland From Female Hyalomma Anatolicum Anatolicum And Cloning And Prokaryotic Expression Of 4D8 Gene

Hyalomma anatolicum anatolicum is an ectoparasite which is distributed mainly in Uygur autonomous region. It can transmit many species of haemoprotozoa parasites and can cause the decrease in the productivity by biting and sucking of animal blood. It inhibits the development of animal husbandry and threatens the inhabitant health.The cDNA expression library of salivary gland from female Hyalomma anatolicum anatolicum was constructed. In order to clone and study functional genes of Hyalomma anatolicum anatolicum. Total RNA were extracted by salivary gland of Hyalomma anatolicum anatolicum and mRNA were further purified. A library of oligo(dT)-primed cDNA with directional EcoRⅠ/ HindⅢlinkers was synthesized and purified by Mini Column Fractionation Kit, then ligated with theλscreen vector contain EcoRⅠ/ HindⅢarms. After package in vitro, the cDNA library of salivary gland of Hyalomma anatolicum anatolicum were constructed and amplified, The cDNA primary library titer and amplification library are 4.0×105 pfu/mL and 8.8×109 pfu/mL,respectively.Specific primers were designed according to the gene 4D8 sequence of Dermacentor marginatum in GenBank. The gene 4D8 was amplified by PCR from cDNA expression library of salivary gland from female Hyalomma anatolicum anatolicum and cloned into pMD-18-T vector. Sequencing result showed that the nucleotide sequence and amino acid sequence of the cloned 4D8 gene shared 89.2% and 96.0% homology with the data published in GenBank respectively. A recombinant expression plasmid pGEX-4T-1-Hyaa4D8 was constructed by sub-cloning 4D8 fragment digested from plasmid pMD-18-Hyaa4D8 into linearized pGEX-4T-1 vector. The plasmid pGEX-4T-1-Hyaa4D8 was expressed in E. coli induced by IPTG. SDS- PAGE showed that a fusion protein with a molecular weight of 45ku was expressed. The concentration of expressed 4D8 protein was 1.24mg/mL. The gene 4D8 expressed level could reach up to 33.0% of total E. coli proteins.The recombinant fusion protein could be detected by rabbit anti-Hyalomma anatolicum anatolicum positive serum with the Western-blotting.