Identification And Function Of Intestinal Immunity- Related Molecules From Silkworm, Bombyx Mori

Insects possess an evolutionarily conserved host innate immune system to recognize and eliminate invading microorganisms. Gut epithelium acts as the first line of defense in the innate immunity. To prevent infection of pathogenic microorganisms, the intestines of insects form a complete set of the immune response against them. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics and binding with the pathogen approach,the mechanism and the host factors that participate in intestinal immunity response remain largely unknown. Therefore, this study has identified the unknown immune related molecules in the intestine through a new method and does a further study into their mechanism of participating in the immune response. 1. Construction of a T7 phage display cDNA library of the silkworm midgut and panning of ligand-binding peptides from T7 phage display libraryWe have successfully constructed T7 phage display library of midgut in Bombyx mori. The insert fragments were 300 bp-2000 bp. The original titer of library was 1×105 pfu/mL, and 8×1010 pfu/mL after amplification. Several immune related proteins were selected including translationally controlled tumor protein(TCTP), Chitin-binding protein, Aminopeptidase-N, Fungi protease inhibitor F and β-1, 3-glucan recognition proteins using chitin, LPS, B.bombyseptieus and S.marcescens as ligands. Additionally, TCTP was selected by all these four ligands, which suggested that TCTP might be involved in the recognition of these ligands. 2. The function identification of TCTP at cellular levelThe immunohistochemical and western blotting showed that TCTP was synthesized from intestinal cells and secreted into the enteric cavity, and lower concentration of TCTP was detected in the haemolymph. The haemocytes phagocytosis results showed that TCTP could bind with microorganisms and promote the haemocyte phagocytosis. We propose BmTCTP to be a novel opsonic molecule for the phagocytosis of microorganisms. In addition, TCTP could stimulate the production of antimicrobial peptides in BmNs cells. The inhibitor of ERK, U0126 and PD098059, could inhibit the production of antimicrobial peptides and it was in a dose-manner. Western blotting result showed that TCTP could cause the change of ERK phosphorylation, and the strongest signal was detected in 5 min to 10 min, decreasing gradually after 10 min and to the initial level in 30 min. 3.Verification of immunologic function of TCTP at individual levelConstructing the transgenic vector and generation of the transgenic silkworm, and the stable strains was obtained. The result of qRT-PCR and western blotting showed that the RNAi resulted in significant decrease both in transcription and translation level. Down-regulation of BmTCTP by RNAi in the midgut may cause some defects in production of anti-microbial peptide under immune challenges. We found that in the midgut of wild-type silkworm, phosphorylation kinetics of ERK demonstrated a persistent increase, with the maximum level reached at 6 h post infection followed by a gradual decrease. In contrast, the level of ERK phosphorylation in transgenic silkworm was much lower and showed a tendency of declining throughout the experiment. The result suggested that dynamics of ERK phosphorylation from transgenic silkworm midgut was disrupted, leading to less production of anti-microbial peptides as a result.