Studies On The Expression And Localization Of Ricin B-lectin NBRTB From Microsporidia Nosema Bombycis

Microsporidia are obligate intracellular parasites, unicellular eukaryotes, which generally exsite in the natural world. They can infect almost all vertebrates and invertebrates, and they are the common pathogens for economic insects, sericulture, fishery and shrimp farms. Until now, more than 1400 microsporidia species belonging to 150 genera have been reported. Nosema bombycis, a model species of microsporidia, is known as a pathogen of silkworm Pebrine, which casuses great economic losses and must be examined in sericulture industry. So how to overcome pebrine disease is very important and difficult to sericulture and microsporidia researchers.To crack the pebrine, we sequenced the genome of Nosema bombycis, from which we identified some ricin-B-lectin genes that have not been reported.Searching by homologous alignment, we found the homologs in the genome of E. cuniculi,A. locustae, N. ceranae and E. binenus. So the ricin B-lectin genes are generally encoded in microsporidia. Considering the agglutination function of ricin-B-lectin and the infection characteri of N. bombycis, we presume that these ricin B-lectins play an important role during the infection of N. bombycis. We carried out this study to identify the microsporadia ricin B-lectin, and study their expression and locations. Furthermore, this study will provide reference to the research of microsporida infection and identification.In this study, we identified the ricin B-lectin from N. bombycis genomic data, and then the characters, sequence alignments, phylogenetic relationships of those 19 N. bombycis ricin B-lectin were analyzed. According the results, we choose one of the genes NBO₆g0049 for further resaerch. In order to easy to handwriting and remremembering, we write nbrtb649 for short and NBRTb649 is used as the name of protein it encodes. Secondly, RT-PCR, gene cloning, prokaryotic expression were employed, and polyclonal antibodies were produced using the recombination protein NBRTb649. Subsequently, indirect immunoflourescence test and immunoelectron microscopy analysis were employed for localization. The main results are as follows: 1. Bioinformatics analysis of ricin B-lectin genes in the N. bombycis genomeWe obtained 19 ricin B-lectin genes from our N. bombycis genome database by retrieve its annotations, and they mainly distribute two scaffolds (scaffold0006 and scaffold463) in the form of tandem duplication. We use the common bioinformatics tools to analysis these ricin B-lectins from N. bombycis, except for NBO₆g0050,NBO₆g018,NBO₉81g0001 and NBO₁214g0002, other 15 sequences were predicted to contain N-signal peptides and indicate that the N.bombycis has adapted these genes to fulfill an extracellular function. Besides,17 ricin B-lectins were predicted to have N-or O-glycosylation sites. By cluster and phylogenic evolution analysis, two ricin B-lectins from different scoffolds would gather as a small branch, this illustrates that those ricin-B-lectin gene from N.bombycis maybe the products of amplification. Although the amino acid sequence of ricin B-lectin from N. bombycis has low homology to the other identified ricin B-lectin, the conserved (QxW) motives were found in these sequences. Besides, ricin B-lectin genes also exist in some other sequenced microsporidia, i.e., E. cuniculi, A. locustae, N. ceranae and E. binenus. Interestingly, the form of distribution of those genes in genomes of E. cuniculi and E. binenus are very similar with which in N. bombycis. The phylogenetic tree of microsporidia ricin B-lectin indicated that the the ricin B-lectin from N. bombycis maintained its independent phylogenetic relationship.and other microsporidia also maintained their independent phylogenetic relationship, which suggest selection pressures are different due to adapt to differential host immune-response challenges.2. Expression profile of the nbrtb649 gene from N. bombycisIn this study, reverse transcription PCR was used to analysis the transcription information during different infect time of the nbrtb649, which was chosen from the genomic database of N. bombycis according to the analytic results above. Using the RNA of midguts of silkwormat different infected stages as the models, and the results showed that the transcription of nbrtb649 beginning with 24h post-infected and continued through to day 10 post-infected. This suggests that the ricin B-lectin gene may play important role during infection process.3. Prokaryotic expression of the nbrtb649 gene from N. bombycis and preparation polyclonal antibodyThe nbrtb649 was cloned and the recombinant expression plasmid pGEX4T-1-nbrtb649 was constructed. Then, the recombinant expression plasmid was transformed into Escherichia coli BL21. and expression of recombinant NBRTB649 was induced by IPTG. the fusion protein was purified by electro-eluting and dialysis. Meanwhile, the polyclonal antibody against NBRTB649 was generated by immunizing mice with the purified fusion proteins. A specific band of approximately 27kDa was detected from the total protein of N. bombycis by anti-NBRTB649 serum.4. Ultrastructural localization of NBRTB649In order to determine the cellular location of NBRTB649, the specific mouse polyclonal antibody anti-NBRTB649 was used for IFAT and immunoelectron microscopy (IEM) to examine mature spores. Both the mature spores and the spores treated with K2CO3 were fixed and permeabilized and used to IFAT analysis. The fluorescence signals only can be detected within mature spores, neither in the spore wall of mature spores nor sprout spores, which suggests that NBRTB649 is not a spore wall protein. IEM of ultrathin sections of mature N. bombycis demonstrated that the labelled gold particles are present in sporoplasm of spores, which is in agreement with the IFAT data. It indicated NBRTB649 is expressed in mature spores with no obvious location features.