Identification Of Haemaphysalis Longicornis By Scanning Electron Microscope And P27/30 Gene Clone, Prokaryotic Expression

The hard tick Haemaphysalis longicornis Neumann are is widely distributed in 17provinces,It considered to be most important ticks as pathogen vectors in china.it is bad topublic safety and economy of animal husbandry.Morphology of adults,nymphs,larvaes andeggs of likely Haemaphysalis longicornis of sicuan were observed by scanning electronmicroscope(SEM) . In adult stage:dentition formula 4|4 or 4—5|5—4,there were 30—35pores on pores area, distance of the two pores areas was double than the long path of poresarea,spiracular plate liked word "D",there were five pairs of anal setas.In nymphalstage:dentition formula 2—3|3—2,the dorsal aspect of the palpal segment 3 had nospine ,had no pores area and no genital opening,Spiracular plate was ellipse and had nomacula,There were three pairs of anal setas .In larval stage :dentition formula 2|2,had nospiracular plate,there were a pairs of anal setas and had no anus sulcus posterior. The Haller'sorgan of all developmental stages of H. Longicornis were alike,but in adult stage there wereseven sensofy stetas in the anterior pit, in nymphal and larval stage only had six sensofy stetasin the anterior pit. The surface of Eggs was smooth and glossy. The result of observation bySEM connect with bionomics,it is sure that this tick is parthenogenetic Haemaphysalislongicornis. In Adult stage of Haemaphysalis longicornis,there were 7or 8 pores in anterior ofventral of spiracular plate, and many spinule and several pores below the anal valve.In this study ,we aimed to research tick molecules for vaccine candidates. 1 pair of genespecial primers were designed based on the published amino acid motifs of the P27/30 gene ofJapan.Haemaphysalis longicornis .total RNA isolated from adult hunger tick, was used astemplate to generate complementary DNA by reverse transcription .the 670bP DNA fragmentwas amplified by polymerase chain reaction,and clones it into PMD 18-T vector.DNA sequencing confirmed the inserted fragment was P27/30 gene,The complete ORF is603bP,encoding a 201 amino acid residue polypeptide with 23.38kDa predicted molecularweight.By blasting the homologous sequence in GeneBank databases, the sequence of P27/30gene of China Haemaphysalis longicornis is 99.85% percent identity to Japan Haemaphysalislongicornis.By the technology of recombination,the P27/30 gene was cloned into expressionplasmid PET-32a(+),and was transformed to Ecoli BL21 .in SDS-PAGE with molecular massof about 45KDa,which is consisted of about 24 KDa protein of the P27/30 gene andPET-32a(+)(20.4KDa).use the same concentration of IPTG,we find it is almost no effect toamount of proteinum,but with the time go on the amount of proteinum more and more.