4 Sorts Of Transcript Isoforms And Protein Structureanlynisis Of Aquaporins(AQPs) In Haemaphysalis Qinghaiensis

Tick is obligate hematophagous ectoparasite in vertebrates,it is the second largest biological vector for many pathogens.Blood sucking and transmission ofdiseases have harmed livestock industry and human health.The prevention andcontrol of tick have become the focus of attention and at home and abroad.Thecontrol strategies for ticks mainly depend on the application of chemicals.However,the use of chemicals could result in resistance and environmental contamination.The alternative control strategy is the development of the new vaccines to anti-tick infections.Liquid transport during transmission of pathogens and life cycle of ticks are essential.Aquaporins(AQPs)are importantfunctional proteins and widely present in cell membrane of organisms.At present,only few studies investigated AQPs in ticks,the transportation mechanism remains unknown.A pair of degenerate primers was initially designed based on the AQPs protein sequences of 6 tick species deposised on Gen Bank.Firstly,total RNA was extracted from salivary glands of famele adult Haemaphysalis qinghaiensis.Followed by RT-PCR,PCR,partial sequence of AQPs in Haemaphysalis qinghaiensis was obtained.Based on the partial sequence,5’ and 3’ RACE primers were designed and the 5’ and 3’ terminal sequences were amplified by applying the method of RT-PCR and RACE amplification,full-length sequence of AQPs in Haemaphysalis qinghaiensis was obtained.According to bioinformation analysis,the AQPs protein sequences of H.qinghaiensis were only differed by 3’-end of amino acid sequence.There are six transmembrane areas,no signal peptide sequenceand a strong hydrophobicity.Aphylogenetic tree was constructed based on all of AQPs amino acid sequences deposited on Gen Bank,the AQPs sequence of H.qinhaiensis were clustered with Rhipicephalus appendiculatus and Rhipicephalus sanguineus(82 %)on the same clade.The conserved region of AQPs gene was amplified using designed primers basedon the AQPs sequences and then cloned into the p ET-28a(+)vector,identified by PCR,double restriction enzyme digestion and sequencing analysis,the recombinant protein was expressed by acellulare pression system and purified.In addition,3 peptides of outer membrane region of AQPs protein,as well as polyclonal antibodies were genreated.