Anti-BmHh Preparation And Functional Analysis In Midgut Of Silkworm, Bombyx Mori

Hedgehog(Hh) signaling pathway is one of the major developmental pathways conserved from Drosophila to mammals. Hh signaling also plays important roles in the regulation of adult tissue homeostasis and repair in mammals, and its deregulation has been implicated in several types of human cancers. The hedgehog gene was first discovered in drosophila, and members of the family have since been found in most metazoa. Hh proteins are composed of two domains, an amino-terminal domain HhN,which has the biological signal activity, and a carboxy-terminal autocatalytic domain HhC, which cleaves Hh into two parts in an intramolecular reaction and adds a cholesterol moiety to HhN.The Hedgehog family of secreted signaling proteins is a master regulator of cell fate determination in metazoans, contributing to both pattern formation during embryonic development and postembryonic tissue homeostasis. In a universally used mode of action, graded distribution of Hh protein induces differential cell fate in a dose-dependent manner in cells that receive Hh. Recent studies have shown that Hh signaling pathway have been implicated in the regulation of ISC proliferation during midgut homeostasis and regeneration. However, The regulatory roles underlying regenerative proliferation in midgut of silkworm are still poorly understood.Silkworm is of great economic value and has been studied as a model of Lepidopteran insect. As the second largest organ, intestine plays an important role in tissue reconstruction, degradation, and regeneration in the process of silkworm metamorphosis development.In order to understand the possible regulatory roles that maintain midgut homeostasis and mediate regeneration of the intestinal epithelium after injury, our studies reveal that, based on bioinformatics research methods, the silkworm BmHh has been successfully identified and cloned and we analysis the gene sequence information. Furthermore, this research analyzes and studies its function in intestinal of silkworm, Bombyx mori. The main results are as follows:1. Gene sequence information analysis of Bm Hh in silkwormBased on the NCBI and silkworm genome sequence databases, we identified the homologous genes of BmHh in silkworm. Using PCR and RACE technology, we cloned and obtained its complete cDNA sequence. Further analysis showed that BmHh located on chromosome 9 and composed of five exons and four introns. The full-length was 3172 bp and the CDS length was 1149 bp. The full-length coded 383 amino acids and Prediction of protein molecular weight was 42.37 kDa. The multiple sequence alignment and reconstruction of aphylogenetic tree of homologues from different species showed that Hh were conserved from inverterbrates and verterbrates and contained two typical domain: HhN and HhC.2.The expression analysis and subcellular location of BmHh in silkwormWe detected BmHh gene expression by using qRT-PCR technology. It turned out that BmHh expressed in all tissues at day 3 of 5th instar. This result suggested that HH pathway widely exist in silkworm organizations. Furthmore, It expressed in the intestinal throughout the developmental stages, and its expression level in pre-molting period was higher than other stages. Results indicated that the gene might be involved in proliferation and differentiation in intestinal stem cell. The BmHh gene was a kind of secretory proteins with signal peptide by bioinformatics analysis. Whereafter, we successfully constructed BmHh over-expression vector,and transfected it into ovary cell line, the results showed that the gene location to the cytoplasm near the nucleus. We speculated that the BmHh protein in silkworm had the similar protein processing and modification process.3. Preparation and specific detection of BmHh polyclonal antibodyCombining with silkworm genome sequence databases and predictions of antigenic determinant of BmHh,we select an specific fragment which can encode 253 aa. Furthmore, behind designed primers and expanded with PCR, we successfully constructed recombinant prokaryotic expression plasmid by connecting BmHh to protein expression vector PET-32a(+). Next, a large number of recombinant proteins was obtained that induced under the best temperature and the most suitable IPTG concentration, then protein purification and mice injection,we got the immune serum.Result showed that we successfully achieved polyclonal antibody of BmHh after ELISA to detect the titer of 1:16000. Finally, we detected antibody specificity using western blot and immunofluorescence that the materials were collected from ovary cell line.It proved that the preparation of antibody was available.4. The BmHh function analysis in ovary cell line and midgut in silkwormAdded synthetic dsRNA interference fragment of BmHh and cyclopamine of HH pathway inhibitor were added to ovary cell line, result showed that HH pathway major gene down-regulation detected by qRT-PCR, and BrdU-positive cells also decreased significantly.The same results occurred when cyclopamine injected into silkworm body.It implied that BmHh and HH pathway may be involved in the proliferation of intestine stem cell in silkworm midgut. Feeding Bt(a Gram-positive bacteria),E.Coli(a Gram-positive bacteria)and Bleomycin to stimulate intestine of silkworm,results showed that BmHh was up-regulation obviously through the detection of qRT-PCR and western blot.In addition, BrdU-positive cells also increased significantly. Hh signaling pathway have been implicated in the regulation of ISC proliferation during midgut homeostasis and regeneration. As a consequence, we suggested that BmHh had been implicated in the regulation of ISC proliferation in response to tissue damaging induced by enteric infection and cytotoxic agents in silkworm midgut.