Cloning of Bm91 gene of Boophilus microplus and its expression in Pichia pastoris

Total RNAs were extracted from the salivary glands and the intestine of semiengorged adult female Boophilus microplus,respectively.According to the nucleotide sequences of Bm91 gene of B.microplus available in GenBank,a pair of primers were designed and the Bm91 gene was amplified by RT-PCR from the total RNAs.Then the verified gene was sub-cloned into EcoRⅠ site of the secretory expression vector pPIC9K,and the verified recombinant expression vector pPIC9k-Bm91 was linearized with SacⅠ and electrotransformed into yeast cell GS115.The multicopy recombinant Pichia pastoris strains were screened by G418 and induced by methanol.The expressed product was analyzed by SDS-PAGE and Western-blotting.The results showed that Bm91 gene was expressed successfully and the expressed Bm91 protein of 83 ku in molecular mass in supernatant had good reactogenicity.