Identification, Expression And Functional Analysis Of Lectin Gene From The Chinese Oak Silkworm (Antheraea Pernyi)

Innate immunity is an important way for insects to resist microbial infection. It includes humoral and cellular reactions.Lectins are important pattern recognition receptors (PRR), widely identified in plants, animals and insects.They play an important role in the defense of pathogen.A lectin gene (ApL3) of C-type lectin supper-family was cloned from Antheraea pernyi using reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE-PCR).Sequence analysis revealed that the sequence length of the lectin gene is 1017 bp,the open reading frame is 927 bp and codes for 308 amino acids with a signal peptide,located between 1～15 amino acid.The calculated molecular mass of the mature peptide is about 35 kD and the isoelectric point is 4.99.Bioinformatics analysis revealed that this lectin has two conservative polysaccharide recognition domains(CRD),which belong to C-type lectin supper-family.We further investigated the tissue distribution of ApL3 by RT-PCR and found that the expression of ApL3 mRNA was very high in fatbody,and also can be found in other tissue. Real-time fluorescence quantitative methods were used to analysis the expression difference of ApL3 after infection by different pathogenic microorganisms and ApL3 expression was up-regulated after bacteria infection.The ORF were amplified by PCR and ligated to pET-28a vector for protein expression. The recombinant proteins were induced by different IPTG concentrations and detected by SDS-PAGE.The recombinant protein was purified by a Ni-NTA nickel affinity column.Western blot analysis of recombinant protein showed that a protein band about 35kD was detected using anti-His antibody, while there was none in the control group.The purified ApL3 protein was renatured and agglutination of microorganism by ApL3 was tested,then was used to immunize rabbits to generate polyclonal antisera. The titer of the antiserum was more than 1:240000 determined by ELISA. Western blotting revealed that the prepared antibody could specifically recognize the recombinant and endogenous ApL3 protein.Results showed the successful expression of the recombinant lectin protein in E.coli Rosetta (DE3) cells.Distribution of ApL3 is not tissue-specific and has function of agglutination. These will provide the foundation for further study on eukaryotic expression and function of ApL3 gene.