Cloning And Eukaryotic Expression Of Bm86 Gene And Construction Of CDNA Library Of Boophilus Microplus

In order to clone and expression Bm86 of Boophilus microplus in Pichia pastoris system.Bm86 gene was cloned from the larvae of B.microplus by RT-PCR. The results show that the nucleotide sequence of the Bm86 gene sequenced shares 97.4% homology with that published in GenBank data and this fragment contained the complete open reading frame (ORF) of the Bm86 gene. The interested Bm86 gene was inserted into the secretory expression vector pPIC9K, and pPIC9K-Bm86 was constructed successfully. The recombinant was linearized with Sac Ⅰand electroporated into the yeast cell GS115. It was confirmed that interested Bm86 gene was intergreted within the genome of the P.pastoris by PCR, and the multicopy recombinant was named GS115/pPIC9K-Bm86 His+Mut+. After being induce by 0.5% methanol for 96h in shaking flask to express Bm86 and took time points, the expressed Bm86 protein was analyzed by SDS-PAGE and Western blot. The results show that Bm86 protein expressed successfully at the 48th hour in P.pastoris. The concentration of expressed Bm86 protein was 0.36mg/mL and its molecular weight is approximately 68KD.The Bm86 expressed level could reach up to 32% of total proteins in culture supernatant and could be specifically recognized by the postive serum of rabbit which infected by B.microplus. At the same time, in order to study functional genes of B.microplus, total RNA extracted by B.microplus and mRNA were further purified. A library of oligo(dT)-primed cDNA with directional EcoRⅠ/HindⅢlinkers was synthesized and purified by Mini Column Fractionation Kit, then ligated with the λscreen vector containing EcoRⅠ/HindⅢarms. After package in vitro, the cDNA library of B.microplus was constructed and amplified. The cDNA primary library titer and amplification library are 4.5×105pfu/ml and 7.2×1010pfu/ml, respectively.