Molecular cloning and expression analysis of a hemolin-like molecule from Antheraea pernyi

• A hemolin-like gene was identified from A. pernyi.
• The hemolin gene was highly expressed in the hemolymph and fat body.
• The expression of hemolin-like was significantly induced by microorganisms.
• This protein could enhance insect innate immunity by agglutinating microorganisms.

Hemolin belongs to the immunoglobulin superfamily and plays an important role in innate immune response of insects. In this study, a hemolin-like cDNA of 1418 bp was obtained from Antheraea pernyi (Ap-hemolin-like). Sequence analysis revealed Ap-hemolin-like was homologous to those hemolins from other insect species. Recombinant Ap-hemolin-like protein was expressed in Escherichia coli cells, and polyclonal antibodies were produced against the recombinant proteins. Real-time PCR and western blot analysis showed that the Ap-hemolin-like was expressed in hemolymph, Malpighian tubules, midgut, epidermis and fat body, with the highest expression level in hemolymph. To investigate its role in the immune response against microorganisms, fifth instar larvae were challenged by injecting nucleopolyhedrovirus (NPV), E. coli, or Beauveria bassiana. The results showed that the expression of Ap-hemolin-like in hemolymph and fat body was obviously induced by microorganisms. In addition, the recombinant Ap-hemolin-like protein promoted the agglutination of E. coli in the presence of calcium, which was confirmed by agglutination assay. These results suggested that the Ap-hemolin-like protein was involved in innate immune response of A. pernyi against pathogens.

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