Construction Of Subtractive CDNA Library Of Salivary Gland Of Female Boophilus Microplus And Prokaryotic Expression Of Vitellogenin Gene

Boophilus microplus is one of the universal-spread, one-host ticks, distributes in tropical and subtropical regions of the world with range expansion for some species due to changes in climatic. Infections with Boophilus microplus economically impact cattle and buffalo production by reducing weight gain and milk production, and by transmitting pathogens that cause babesiosis (Babesia bovis and B. bigemina) and anthropo zoonosis (such as rickettsia, forest encephalitis, lyme disease), severely threating human health and livestock production.Control of tick infestation has been difficult because ticks have few natural enemies. Now, the major tick control method is the application of acaricides. However, use of acaricides is often accompanied by serious drawbacks, including environmental contamination, the selection of acaricide-resistant ticks, and contamination of milk and meat products with drug residues. Furthermore, development of new acaricides is a long and expensive process. Vaccines contain Bm86 antigen is considered to be the forerunner in preventing tick and tick-borne diseases.With cDNA from salivary gland of semi-engorged adult female B. microplus as tester and cDNA from starved adult female B. microplus as driver, a subtractive cDNA library of salivary gland of female B. microplus was constructed using suppression subtractive hybridization(SSH) and T/A cloning method. The inserts were from 200 bp to 550 bp in size according to PCR identification of the 158 positive clones. Sixty white clones were sequenced and twenty valid EST sequences were obtained. Bioinformatics analysis revealed that proteins encoded by these ESTs had similarity to proteins such as vitellogenin, matrix protein, acidic proline-rich protein and OTM-3 hypothetical protein. PCR primers were designed based on the obtained ESTs, and the obtained ESTs were detected using genome DNA of B.microplus as templates. The results showed that all the ESTs belonged to B. microplus.Specific primers were designed according to vitellogenin sequence according to the subtractive cDNA library. The 627 bp amplied fragment was cloned into pGEX-4T-1 and then the recombinant plasmid was tansfered into E.coli BL21 (DE3) for expression.49.4kD inclusion body form fusion protein was obtained. The fusion protein was washed with 2M urea, dissolved with 8M urea, and then purified in electric elution method. SDS-PAGE analysis showed that the target gel is perfect. Detected the immunogenicity by Western-blot, and the reaction band is apparente.The construction of subtractive cDNA library of salivary gland of female B. microplus provides foundation to study differentially expressed genes in salivary gland of female B. microplus. The expression of vitellogenin gene and the detection of protein immunogenicity lay the foundation for the development of anti-B. microplus vaccine.