Bombyx Mori Apolipophorin-Ⅲ Recognition Function Of Preliminary Study

Insect Apolipophorin-â…¢(ApoLp-â…¢) is a member of apolipoprotein family, it has been recently found that ApoLp-â…¢is a munifunctional protein and plays a role as one of the pathogen recognition receptors during insect immune responses. To futher verify this, an ApoLp-â…¢was cloned from Bombyx mori P50 and fused with GFP. The recombinant protein was expressed in E. coli, purified using a Ni-NTA column and used for binding assays the cells/spores of different microorganisms.The ApoLp-â…¢mRNA sequence of B. mori (Genbank accession number: U59244) is 623 bp long, encoding a protein with 186 amino acids (aa). Signal peptide prediction and the documented report indicated that it had two different cleavge sites for signal peptides, between 17 and 18 and between 22 and 23, respectively. Thus, two different recombinant proteins by C-terminal fusion with GFP were expressed and verified by SDS-PAGE and western blotting verified. For binding assays, the recombinant proteins were incubated with the spores/cells of Aspergillus nidulans, Metarhizium anisopliae, Beauveria bassiana, Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae, respectively. The observations under the fluorescence microscope demonstrated that the ApoLp-â…¢could bind the spores of A. nidulans, M. anisopliae and Beauveria bassiana, but not the cells of E. coli, B. subtilis and yeast. The protein also failed to bind the blastospores of B. bassiana and M. anisopliae. To further prove that ApoLp-â…¢plays a role in insect immunity,the spores of M. anisopliae were incubated with the recombinant protein, demonstrated with antibacterial activity by comparison with the insect blood sample injected only with fungal spores. In general, the data of this study showed that the ApoLp-â…¢of B. mori functions in fungal cell recogenition to activate the production of downdstream antimicrobial peptides.