Identification Of Haemaphysalis Concinna By Hc-23 Gene Clone And Prokaryotic Expression

The hard tick Haemaphysalis concinna is widely distributed in world and 10 provinces in China,it is one of the important suck-blooding ectoparasites in Sichuan province.In humans and animals,bites result in skin rashes,red swelling,itching and infested ulcerations of bite sites.It considered to be most important ticks as pathogen vectors of diseases was recorded for a number many of which are life-threatening.It is bad to public safety and economy of animal husbandry,at present the chemical were used to control Haemaphysalis concinna.However,this approach is associated with a number of disadvantages such as chemical pollution of the food chain and the environment as well as the quick development of resistance against acaricides by tick.These limitations have necessitated the search for alternative tick control measure and vaccination is one of the best control strategies.In this study,we aimed to research tick molecules for vaccine candidates 1 pair of gene special primers were designed based on the published amino acid motifs of the P27/30 gene of China and Japan strain Haemaphysalis longicornis.Total RNA isolated from unfed adult tick,was used as template to generate complementary DNA by reverse transcription,the 631 bp DNA fragment was amplified by polymerase chain reaction. DNA sequencing confirmed the inserted fragment was Hc-23 gene.The complete ORF is 603 bp,encoding a 201 amino acid residue polypeptide.The sequence produced in this study was deposited in Genbank(Accession No.FJ425897).The molecular mass of polypeptide was 23.386 kDa,its isoelectric point was 9.77,also has the strong hydrophilicity.By blasting the homologous sequence in GenBank databases,the sequence of Hc-23 gene of Haemaphysalis Concinna is 99.83%percent identity to China strain Haemaphysalis longicornis,c-t transverion only happened in 300^th base and 99.67%percent identity to Japan strain Haemaphysalis longicornis,a-g and c-t transverion only happened in 183^th and 300^th base respectively.The sequence of Hc-23 gene is 88.06%percent identity to Rhipicephalus haemaphysaloides troponin I-like protein gene.The amplified fragment was inserted into pMD18-T vector,then the pMD18-T-Hc-23 was sub-cloned into prokaryotic vector pET-32a.The recombinant plasmid was used to transform into E.coli BL21(DE3) competent cell.A single positive colony was picked and identified by PCR and restriction analysis.The recombinant bacteria were induced to expression through the changes of IPTG The results showed that a new protein band was found in inclusion with molecular mass of about 43 kDa, which was consisted of a 23.386 kDa protein of the Hc-23 gene and pET-32a(+)(20 kDa). The amount of proteinum was to peak at 4 h after induced.There were almost no difference to amount of proteinum in different IPTG concentration,all be high.By Western-blotting,the rHc-23 was evaluated by immunoblot analysis using recombinant proteins.Sera from repeatedly inoculated rabbits with Haemaphysalis concinna also reacted with rHc-23,did not react with negative control.