The Immuno-Screening Of CDNA Expression Library Of Haemaphysalis Qinghaiensis Adult Tick And Cloning And Expression Of The Gene

Haemaphysalis qinghaiensis tick and the protozoal diseases that transmitted by it are major economic burden to animal husbandry in China. Currently the principal tick control method is the application of acaricides. 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 potential for development of resistance against acaricides by ticks. These limitations have necessitated the search for alternative tick control measures. Only anti-tick vaccination among the several alternative tick control measures appears promising.Recombinant phages were plated on a host BL21(DE3)pLysE,and when plaques begain to form, the plates were overlaid with nitrocellulose filters and incubated for an additional 3.5 hours. The filters were processed by successive incubations with positive serum of rabbit against Haemaphysalis qinghaiensis larval tick, sheep anti- rabbit IgG alkaline phosphatase conjugate, and color development substrates and thus positive plaques with intense purple color were found. Pure phage stocks were obtained by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids subcloned in BM25.8 were isolated and then transformed into host strain JM109. Ten genes were obtained after analysis of plasmids abstracted from JM109 by PCR, restriction digestion and sequencing,and they are designated respectively HqLA-1, HqLA-2, HqLA-3, HqLA-4, HqLA-5, HqLA-6, HqLA-7, HqLA-8, HqLA-9, HqLA-10. The sizes of those genes were 798bp, 421bp, 637bp, 392bp, 297bp, 1473bp, 626bp, 201bp, 989bp, 551bp respectively. HqLA-1~ HqLA-9 of those genes were new ones by comparation to known gene sequences, and the new genes were submited to GenBank, accession numbers provided by them were FE674076,FE674077,FE674078,FE674084,FE674079,FE674080,FE674081,FE674082,FE674083.The 5 new genes of HqLA-2,HqLA-6,HqLA-7,HqLA-9,HqLA-10 were expressed successfully,and molecular weight of expression products were 47kDa,87kDa,48kDa,57kDa,68kDa respectively. The expressed products of the gene HqLA-7 and HqLA-9 were proved to be immunoreactive by hybridizing anti-sera of rabbit against Haemaphysalis qinghaiensis larval tick by Western blot analysis. All those would be a good foundation for the development of anti-H.qinghaiensis vaccine.The cDNA Expression Library of Haemaphysalis qinghaiensis adult Tick constructed by Gao Jinliang was screened with positive serum of rabbit against Haemaphysalis qinghaiensis larval tick. Thus the expressed products of obtained antigen genes were immunogenic and immunoreactive for Haemaphysalis qinghaiensis larval tick and Haemaphysalis qinghaiensis adult Tick in theory.