A pair of specific primers was designed based on the reported Bm86 gene of Boophilus microplus,the Bm86 gene was cloned by PCR using the plasmid pMD18-T-Bm86 as templates, and subcloned into the prokaryotic plasmid pGEX-4T-1. The recombined plasmid was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by different concentration of IPTG for different time. SDS-PAGE showed that the recombinant plasmid pGEX-4T-1/Bm86 expressed a fusion protein Bm86-GST (Mr 94 000) after being induced with IPTG. High level expression of Bm86-GST was found at 1 mmol/L IPTG condition a fter incubation for 8 h at 37 degree C, and the expression level of the recombinant Bm86-GST reached up to 29% of total E coli proteins Western-blotting analysis showed that the recombinant Bm86-GST was recognized by the rabbit anti-B. microplus positive serum.
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