| Microsporidia, a pathogen that can infect almost all animals and insects. Therefore, all kinds of microsporidian are difficult to be cured definitely. Microsporidian in silkworm, Nosema bomybics was found by Nageli in 1857, from then on negative influences sericulture in various sericulture countries continuely, moreover serious impact the economy of sericulture. Met-AP2(methionine aminopeptidase 2) is the target gene of fumagillin, its derivatives or analogues, which was found in treatment of intestinal microsporidian in human. Derivatives like TNP-470, ovalicin which were less toxicity and more effective, get great clinical the rapeutic effect in intestinal microsporidian in immunocompromised patients, tumor, rheumatoid arthritis, obesity and some other diseases these years.On the basis of transcriptomics of Nosema bombycis(Canton), this study confirmed that the Met-AP2 gene was related to the target genes of the drug treatment of microsporidian Nosema bombycis,. Despite of sequencing Met-AP2 of Nosema bombycis, different primers and different microsporida of insects were also sequenced and aligned in this study. K inds of bioinformatics software application make it possible to detect feature of Met-AP2, homology of Met-AP2 of different microsporidia even different species, and Phylogenetic relationship of Met-AP2 in diversity species. Digital microscope and LAMP was established to detect therapeutic effect of fumagillin. The main results of this study are as follows:(1) Verify the presence of Nosema insects Met-AP2 gene and bioinformatics analysis of Met-AP2 gene in transcriptomics of Nosema bombycis(Canton).After BLAST, the result show there is only one Met-AP2 in transcriptomics of Nosema bombycis(Canton). Information of this gene are as follows: the length of ORF is 1077 bp, the protein encodes 358 amino acids, predicted molecular weight is 45 k Da.Sequencing result of different primers shows highly homology 93%-99.9% with data in bioinformatics, proof the result of transcriptomics of Nosema bombycis(Canton) is reliable. Preliminary verified there is only one Met-AP2 gene in transcriptomics of Nosema bombycis(Canton).Met-AP2 gene is almost exist in all organisms, it exists in the cytoplasm of stability hydrophobic protein state. Met-AP2 gene do not have predicted Signal P, glycosylation sites and the across membrane structure. The structure of the protein have typical alpha helix structure components, and it is conserved within species.(2) Conducted the prokaryotic recombinant plasmid p ET-28a–metp ET-28 A plasmid and Rosetta were used in prokaryotic expression of Met-AP2 protein. SDS-PAGE and Western blot result shows the molecular weight 45 k Da as predicted. Nickel was carried out on the fusion protein agarose affinity chromatography highly purity was obtained after purification and the concentration of the protein is 0.56mg/ml. These results provide a theoretical basis For futur e immune positioning and related antibody therapy.(3) Conducted the therapeutic effect of fumagillinDigital microscope and LAMP was established to detect therapeutic effect of fumagillin. Health silkworm, silkworm feed with microsporidia, silkworm feed with fumagillin, microsporidian silkworm feed with fumagillin were applied in this experiment. 5 pairs of LAMP primers targeted Met-AP2 were designed, one pair called LM1 primer pair was chosen for later study. We get the optimum reaction condition obtained the suitable ratio of inner and outer primers and expansion temperature. Based on detection of LAMP of 4 kinds of processing of midgut RNA of silkworm, established the LAMP method of therapeutic effect. |
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