Construction Of The Suppression Subtractive Hybridization CDNA Libirary Of Partial-engorged Male Haemaphysalis Longicornis And Prokaryotic Expression Of Macrophage Migration Inhibitor Factor

Haemaphysalis longicornis is one of the extremely widespread and important suck-blooding ectoparasites,which mainly infest cattle,sheep and other vertebrates. It is a major vector of many important pathogens to animals,such as virus,rickettsia, leptospira,bacteria,protozoa,mycoplasma and chlamydia.H.longicornis and the diseases which they transmitted caused severe economic lose to animal husbandry in China.For a long time,the application of acaricides is the principal method to the control of ticks.This approach is,however,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 ticks.The emergence of the disadvantages above has necessitated the search for alternative methods for tick control.Up to now,immunological control is one of the most promising methods for the tick control.To construct the suppression subtractive hybridization(SSH) cDNA library of male H.longicornis and screen the differentially expressed gene in the partial engorged condition.The cDNA library of partial engorged male H.longicornis was constructed in the vector of pGEM-T-easy by the way of SSH.The sequencing and bioinformatics analysis were performed and 18 ESTs were obtained from the SSH cDNA library.Blastx analysis revealed that the putative proteins encoded by these ESTs had similarity to protein reported before,which functioned in signal transduction,tissue morphogenesis,transcriptional regulation,carbohydrate metabolism and so on.These proteins may be valuable to the ticks to adapt the changes of physiology after feeding.With the total RNA of partial engorged male H. longicornis as the templete,the quality of the ESTs was evaluated by RT-PCR.The results revealed that 3 of 5 ESTs chosen from the library were differentially expressed in the partial engorged condition.Specific primers was designed according to the H.longicornis macrophage migration inhibitor factor(HLMIF) gene sequence published in GenBank(Accession No.AB255601.1).The HLMIF DNA fragment of 368 bp was amplified by PCR and cloned into pET-30a-(+) and transformed into E.coli BL21(DE3).The recombination colony were identified by restriction endonuclease digestion and sequencing.The recombination colony was induced by IPTG and analyzed by SDS-PAGE.The expression condition was optimized.The results indicated that the cloned HLMIF DNA fragment had a 99.7%identity to HLMIF sequence in GenBank and an 18.5 ku HLMIF fusion protein was expressed in E.coli mainly in the form of soluble protein. The optimum expression of HLMIF fusion protein was obtained with 1.0 mmol/L IPTG at 37℃for 7 h.The construction of H.longicornis SSH cDNA library and the prokaryotic expression of HLMIF,would lay the foundation for the cloning of functional genes and the development of anti- H.longicornis vaccine.