Cloning Of And Expression Of Bm91 Gene Of Boophilus Microplus In Pichia Pastoris

The total RNA was extracted from the salivary glands and the intestine of partially engorged adult female Boophilus microplus and double cDNA was synthesized using M-MLV RTase cDNA Synthesis Kit.According to the nucleotide sequences of Bm91 published in GenBank,a pair of primers was designed and the Bm91 gene about 1908 bp was amplified successfully by PCR.The target gene was subcloned into pGEM-T Easy vector and tested by PCR,enzyme digestion and sequencing.The sequencing result was showed that the cloned Bm91 gene was shared 97.1% homology with the date published in GenBank and this fragment was contained the complete open reading frame of Bm91 gene.Then the gene was digested from pGEM-T Easy vector by EcoRⅠand subcloned into pPIC9K secretory expression vector of Pichia pastoris.This recombinant eukaryotic expression vector of designated pPIC9K-Bm91 was constructed successfully.The recombinant plasmid was linearized with SacⅠand electroporated into competent cell GS115 of P.pastoris and then cultivated in MD medium plate at 28℃for 2～4 days.The single Multiinsertion transformant was picked and confirmed the target Bm91Gene was integrated within the genome of the Pichia by PCR.The positive multiinsertion transformants were screened in medium YPD containing different concentrations of antibiotic G418 and the multicopy recombinant plasmid was named GS115/pPIC9K-Bm91 His+Mut+.Then the multicopy recombinant was induced by 0.5%methanol for 96h.The expressed Bm91 protein was analyzed by the SDS-PAGE and Western-blot,the results indicated that the Bm91 protein was cotained in the expressed product and its molecular weight is approximately 83 kDa.At the same time the protein could be specifically recognized by the postive serum of rabbit which was infested by B. microplus.All those would be a good foundation for the development of anti- Boophilus microplus vaccine.