Click here for additional data file

Click here for additional data file.(23K, zip) Author Contributions Conceptualization, G.P., Y.W. (IEM), including NbSWP30(SWP1), NbSWP25(SWP2), NbSWP32(SWP3), NbSWP5, NbSWP7, NbSWP9, NbSWP11, and NbSWP12 [36,37,38,39,40,41]. NbSWP26 participates in host cell adhesion and infection through its interaction with the host protein Bmtutl-519 [42,43,44]. The AlocSWP2 in has high levels of amino acid sequence identity with its homologue protein in and specimens (weight 8 to 15 g) were collected from commercial shrimp ponds in Fujian, China. The purification of EHP spores was performed according to a previously described method, with some modifications [49]. Briefly, the infected hepatopancreas was dissected and homogenized with an electric tissue grinder. Tissue debris were removed by filtering the product twice through a syringe containing 3 layers of absorbent cotton, followed by two rounds of sucrose density gradient centrifugation (30%, 45%, 60%) (protein database deposited in Uniprot (https://www.uniprot.org/; accessed on 15 June 2020) with the following parameters: trypsin with a maximum of two missed cleavages, carbamidomethylation, methionine oxidation, reverse decoy mass tolerance 20 ppm, error tolerance 0.1 Da, and a mascot score of 20 or greater. Peptides were excluded for further analysis when the matching 0.05. A protein was considered when at least two different peptides were identified. Proteins that were unambiguously identified as nuclear and cytoplasmic ones were filtered out from raw proteins data by expert annotation information. 2.3. Bioinformatics Analysis Proteins were classified via gene ontology (GO) annotation based on three categories: cellular component (CC), biological process (BP), and molecular function (MF). COG and KEGG were analyzed using WebMGA (http://weizhong-lab.ucsd.edu/webMGA/server/cog/; Destruxin B accessed on 5 August 2020) and KAAS (https://www.genome.jp/tools/kaas/; accessed on 5 August 2020), respectively. To investigate the biological significance of annotated proteins, Omicsbean (http://www.omicsbean.cn; accessed on 5 Rabbit Polyclonal to Cyclin A1 August 2020) was used to analyze the functional features based on enriched Destruxin B GO terms and KEGG pathways. Pathway enrichment was analyzed using Fishers exact test and visualized using ggplot2 v3.3.0. Uncharacterized proteins or SWPs with a unique peptide count 5 and coverage 10% were considered to be high-abundance surface proteins (HASPs). Conserved domains were predicted with PFAM (http://pfam.janelia.org/; accessed on 5 August 2020). Signal peptide sequences and transmembrane domains were predicted using SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/; accessed on 5 August 2020) and TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/; accessed on 5 August 2020), respectively. Putative N- and O-glycosylation sites were analyzed with NETNGLYC (http://www. cbs.dtu.dk/services/NetNGlyc/; accessed on 5 August 2020) and NETOGLYC (http://www.cbs.dtu.dk/services/NetOGlyc/; accessed on 5 August 2020), respectively. The heparin-binding motifs (HBMs) were predicted with PatScanUI (https://patscan.secondarymetabolites.org/; accessed on 5 August 2020). Sequence similarity searches were performed by BLASTP against the NCBI database with an E-value cutoff of 1 1.0 103. Finally, 2D topology was predicted by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2; accessed on 5 August 2020). 2.4. Gene Cloning and Recombinant Protein Expression of EhSWP3 PCR primers with restriction enzyme sites Destruxin B were designed based on the genome DNA sequence of (GenBank No. “type”:”entrez-protein”,”attrs”:”text”:”OQS55745″,”term_id”:”1174015875″,”term_text”:”OQS55745″OQS55745) using Primer 6.0. The forward primer Roseta (DE3). A single transformed colony was grown in 5 mL LB medium containing 50 g/mL Kanamycin overnight at 37 C with shaking at 180 rpm. Then, IPTG was added to a final concentration of 0.5 mmol/L to induce protein expression at 37 C for 12 h. Cells were obtained via centrifugation at 12,000 and 4 C for 10 min, followed by ultrasonic treatment in ice for 10 min. The supernatant and precipitation were collected separately and analyzed by 12% SDS-PAGE electrophoresis. The extracted proteins were then purified with Ni-IDA-Sepharose Cl-6B affinity chromatography (Novagen, Madison, WI, USA) for later use. 2.5. Polyclonal Antibodies Preparation and Western Blotting Analysis The preparation of polyclonal antibodies against EhSWP3 (PAbs anti-EhSWP3) was performed as follows: purified recombinant EhSWP3 was intradermally injected into New Zealand white female rabbits (2C2.5 kg) at a dose of 0.4 mg protein per rabbit, and 0.01 M PBS (pH 7.4) buffer was used as the control. One week after three booster injections, rabbit blood was collected, and serum containing PAbs anti-EhSWP3 was obtained via centrifugation and purified to a concentration of 1 1 mg/uL, and stored at ?20 C for later use. For Western blot analysis, the purified recombinant rEhSWP3.

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