Cloning And Expression Of Bm86 Gene Of Boophilus Microplus In E.coli

A pair of primers was designed based on the published Bm86 gene sequences,and the gene was amplified by RT-PCR from unfed larvae of Boophilus microplus and cloned into pGEM-T Easy vector to construct the recombinant plasmid PGEM-Teasy-Bm86. The result showed that the nucleotide and amino acid sequence of the cloned Bm86 gene shared 97% and 95.6% homologies with the data published in GenBank respectively. Recombinant expression pGEX-4T-1-Bm86 plasmid was constructed by sub-cloning Bm86 fragment with cohesive end digested from pGEM-T easy-Bm86 plasmid into linearized pGEX-4T-1 vector. The recombinant plasmid was then transformed to BL21 and induced by IPTG for expression. The analysis of SDS-PAGE showed that a fusion protein with a molecular weight of 88Ku was expressed, and the concentration was 1.08mg/ml, Which accounts for about 39% of the total proteins. The result of Western Blot showed that the recombinant protein could be recognised by rabbit anti-B. microplus positive serum .