| Microsporidia, which are obligate intracellular parasites of eukaryotes, have a worldwide distribution and can infect a broad spectrum of hosts from protists to mammals, including humans. As an important infectious disease in sericulture, pebrine disease caused huge economic losses to sericulture every year. With the development of genomics, invasion mechanism of N. bombycis and the construction of detection and control system against this pathogen, studies on N. bombycis have advanced rapidly in the past decade. However, to reveal the invasion mechanism of N. bombycis and overcome the obstinate illness- pebrine of silkworm are always the emphasis and difficulty of the sericultural research in world.The microsporidian spore wall is composed of proteins and chitin, which is located in the outermost layer of the mature spore. The spore wall proteins are major component of the spore wall. Studies on the sproe wall protein of N. bombycis, researchers have identified some important proteins which related to infecting host, stabilizing spore wall structure and proliferation activities. So far, the researchers have discovered and reported several spore wall proteins of N. bombycis, which are SWP5, SWP11, SWP12, SWP16, SWP25, SWP26, SWP30 and SWP32. Among them, SWP5 interacts with the polar tube proteins and involves in phagocytosis. SWP26 involves in host cell adherence resulting in decreased host cell infection. HBM(heparin binding motif) of SWP26, plays an important role in infecting directly or adhere to the host cell. Spore wall protein, SWP25, of the microsporidian N. bombycis is localized to the endospore and possesses a signal peptide with a 20-amino acid. Remarkably, SWP25 also has a HBM, which may be involved in adhesion. Through this study, we want to screen the interactive silkworm proteins with SWP25, and explore the function of this spore wall protein during the invasion of N. bombycis.In this study, we used Bac-to-Bac baculovirus expression system to insert the SWP25 gene and EGFP gene into the baculovirus transfer vector p FastBac1. Then, the transfer vector was transformed into Escherichia coli DHl0 Bac to construct recombinant vBmEGFP-SWP25. The v BmEGFP-SWP25 was then used to transfect BmN cells to obtain the recombinant baculovirus. Successfully transfected cells could be observed significant green fluorescence signal, and western blot detected a protein band of about 55 k Da in BmN cells infected by the recombinant virus. These results indicated that the EGFP-SWP25 fusion protein had been successfully expressed in BmN ceIIs. This study has laid the foundation for screening the interactive proteins of Bombyx mori with the SWP25 of N. bombycis at the cellular level. In order to further explore the biological function of the SWP25, we screened the interactive silkworm proteins with this spore wall protein at cellular level using co-immunoprecipitation method. Combined with bioinformatics analysis by mass spectrometry, the eight candidates silkworm proteins were initially screened: Bombyx mori thioredoxin peroxidase(BmTpx), Bombyx mori receptor for activated protein kinase C RACK1(BmRACK1), Bombyx mori ras-related protein Rab-11(BmRab11), Bombyx mori ras-related protein Rab-18(BmRab18), Bombyx mori ubiquitin conjugating enzyme 4(BmUbcd4), Bombyx mori Heat shock proteins(BmHSP40/BmDnaJ), Bombyx mori BTB/POZ and MATH domain-containing protein, Bombyx mori TBC1 domain family member 15(BmTBC1D15). And these proteins were analyzed and discussed in this study.In addition, we cloned the complete cDNA sequence of the alpha-tubulin gene from Endoreticulatus sp. Zhenjiang using rapid ampli?cation of cDNA ends(RACE). The full-length alpha-tubulin cDNA sequence of Endoreticulatus sp. Zhenjiang was 1382 bp. The opening reading frame(ORF) of alpha-tubulin cDNA was 1320 bp, encoding a polypeptide of 439 amino acids with a calculated molecular weight of about 48.6 kDa, an isoelectric point of 5.1 and without signal peptide. Online BLAST homology analysis and multiple sequence alignments showed that alpha-tubulin gene sequence of Endoreticulatus sp. Zhenjiang had obvious homology with Endoreticulatus sp. CHW-2004 Taiwan with 99.4% nucleotide similarity and 99.5% amino acid sequence similarity. Based on the cDNA sequence, we designed specific primers to differentiate Endoreticulatus sp. Zhenjiang from other microsporidia, Nosema species(N. bombycis, N. philosamiae, N. hemerophila, N. phyllobrotica, N. antheraeae). The results showed that the expected fragment was amplified only from the genomic DNA of Endoreticulatus sp. Zhenjiang. Therefore, the result showed that the specific identification of Endoreticulatus sp. Zhenjiang among the other microsporidia can be a more reliable detection method to improve the epidemiology of the disease. |
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