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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Experimental Parasitology 130 (2012) 189–194 Contents lists available at SciVerse ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr Cloning and characterization of a surface antigen CiSA-32.6 from Cryptocaryon irritans ⇑ Xiaohong Huang , Zhiyu Sun, Guowei Guo, Changfeng Zheng, Yang Xu, Liping Yuan, Cheng Liu Fujian Key Laboratory of Developmental and Neuro Biology, College of Life Science, Fujian Normal University, Fuzhou 350108, Fujian, PR China article info abstract Article history: Cryptocaryon irritans is a ciliated parasite causing cryptocaryosis in marine fish. To isolate functional Received 17 September 2011 genes, a cDNA library of C. irritans trophonts was constructed and a gene designated CiSA-32.6 (GenBank Received in revised form 17 January 2012 ID: JF812643) was cloned and characterized. The full-length cDNA (1158 bp) encoded a deduced polypep- Accepted 18 January 2012 tide of 330 amino-acid (aa) with a signal peptide of 22 aa. To express the ciliate gene, a truncated open Available online 1 February 2012 reading frame (CiSA-32.6t) was synthesized to remove fragments encoding the signal peptide and hydro- phobic C-terminal and to modify non-universal genetic codes. CiSA-32.6t was subcloned into Escherichia Keywords: coli DH5a strain using the pGEX-4T-1 vector and then expressed as a glutathione S transferase fusion pro- Cryptocaryon irritans tein (rCiSA-32.6t). Western blotting analysis showed that sera from mice immunized with rCiSA-32.6t Surface protein Molecular characterization reacted specifically with a native protein (32.6 kDa) in parasite lysates. Moreover, rCiSA-32.6t reacted Prokaryotic expression specifically with sera from mice immunized with a C. irritans trophont lysate. Expression of the Antigenicity CiSA-32.6 gene in C. irritans was detected at all developmental stages by reverse transcriptase PCR and Western blotting analysis. This study provides the basis of further investigations into the pathogenic biol- ogy of C. irritans and the control of cryptocaryosis. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction 2007; Misumi et al., 2011, 2012). This has promoted the develop- ment of vaccines capable of inducing a protective response in the Cryptocaryon irritans is a parasitic ciliate, belonging to Class host. In previous studies, live or killed parasites have been used Prostomatea, Order Prorodontida, Family Cryptocaryonidae, Genus for immunization (Luo et al., 2007; Misumi et al., 2011, 2012). Cryptocaryon (Wrighta and Colorni, 2002). Parasites penetrate the However, techniques for in vitro propagation of C. irritans are skin, gills and eyes of marine fishes, causing cryptocaryosis, more insufficiently developed rendering collection of the parasites for commonly known as white spot disease, which is a devastating vaccine production extremely laborious. Production of genetically problem in commercial marine aquaculture in tropical and sub- engineered vaccines has therefore become a focus for research. A tropical regions (Colorni and Burgess, 1997). The increased inci- full-length cDNA library of C. irritans was constructed and screened dence of cryptocaryosis outbreaks in southeast of China has led for vaccine candidates, from which a large number of novel func- to severe economic losses in aquaculture. For example, hundreds tional genes were isolated. In this study, one of these genes encod- of millions of dollars were lost during one cryptocaryosis outbreak ing a C. irritans surface antigen (GenBank ID: JF812643) was cloned in Xiapu county, Fujian province in 2009. To date, no effective and expressed in bacteria for molecular characterization. methods have been developed to control this disease although re- cently, immunoprophylaxis has been considered as a potential 2. Materials and methods control measure (Colorni, 1985; Burgess and Matthews, 1995; Yoshinaga and Nakazoe, 1997; Bryant et al., 1999; Luo et al., 2.1. Parasites and experimental animals C. irritans trophonts were collected from gills of infected Pseu- Abbreviations: CiSA-32.6 or CiSA-32.6, the gene or protein of surface antigen from Cryptocaryon irritans with a molecular mass of 32.6 kDa; rCiSA-32.6t, dosciaena crocea cultured in netcages in the coastal area of Xiapu recombinant truncated CiSA-32.6; ESTs, expression sequence tags; ORF, open county, Fujian province, China on July 2nd, 2009, during a crypto- reading frame; IPTG, isopropylthio-b-D-galactoside; GST, glutathione S transferase; caryosis outbreak in the area. The collection was carried out by RT-PCR, reverse transcriptase PCR; aa, amino-acid. soaking gills with visible white spots in sterile sea water for 2 h be- ⇑ Corresponding author. Address: Fujian Key Laboratory of Developmental and Neuro Biology, College of Life Science, Fujian Normal University, Qishan Campus, fore the gills were discarded. Trophonts were collected in the sed- Fuzhou 350108, Fujian, PR China. iment and allowed to develop into tomonts, which were washed E-mail address: [email protected] (X. Huang). three times with sterile sea water. Tomonts were sampled for 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2012.01.012 Author's personal copy 190 X. Huang et al. / Experimental Parasitology 130 (2012) 189–194 extraction of genomic DNA and the remainder was incubated at and used as primers for RT-PCR detection of CiSA-32.6 expression 27 °C for development of theronts. The newly hatched theronts were at the transcriptional level. Expression of a C. irritans actin gene used for the infection and passage of Sebastiscus marmoratus main- (GenBank ID: JN399999) was used as a control and amplified using tained in an aquarium. The parasite was then defined as the same the following of primer pair: 50-ATGGCCGAAGACTAACAAGCAG-30 strain as PYH4.12 or Chiayi strain by molecular phylogeny based and 50-TCAGAAGCATTTTCTGTGTACA-30. on ribosomal DNA internal transcribed spacer sequences (Diggles and Adlard, 1997; H.Y. Sun et al., 2006; Z. Sun et al., 2011). The 2.5. Modification of the CiSA-32.6 gene sequence by artificial synthesis C. irritans life cycle was established in vitro and parasites were col- lected at every stage for further investigation. Theronts were har- The CiSA-32.6 gene open reading frame (ORF) was modified for vested by centrifugation at 1000g for 5 min at 4 °C. Trophonts, the the expression in bacteria by removal of the signal peptide, muta- parasitic stage of the ciliate, were collected from S. marmoratus 3d tion of TAA into CAA and also removal of the hydrophobic C-termi- post-infection by scraping fish body surfaces in a dish of sterile sea nal to improve the solubility of the recombinant protein. The signal water with a glass slide. Motile trophonts were harvested individu- peptide was predicted by using the online Signal P 3.0 Server (Cen- ally and immediately using a pipette. Protomonts and tomonts were ters for biological sequence analysis, Technical University of Den- collected 3 d post-infection of fish by placing dishes on the bottom of mark). The hydrophobicity of the polypeptide was predicted by the aquarium for 2 and 24 h, respectively, to allow mature trophonts DNAMAN (Lynnon Biosoft, Quebec, Canada). The truncated form lost from fish to sediment into the dishes automatically. Parasites at of CiSA-32.6 without genetic coding sequences for the signal pep- each stage were washed in sterile sea water and stored immediately tide and a 25 amino acid fragment at the C-terminal was synthe- at À80 °C for further use. All experiments were conducted in accor- sized artificially and inserted into plasmid pUC57 (Genscript Ltd. dance with the Guiding Principles for the Care and Use of Research Company in Nanjing, China). Animals outlined by Fujian Normal University. 2.6. Subcloning of the truncated ORF from recombinant pUC57 into 2.2. Construction of cDNA library pGEX-4T-1 Total RNA was extracted from trophonts using TRIzol™ reagent The truncated ORF (CiSA-32.6t) was amplified using a set of oli- (Invitrogen, Carlsbad, CA). Poly A+ RNA was then purified using oli- gonucleotide primers, 50-CGGGATCCGAAAAAACTGCAGTTGCA-30 go-dT resin (Wako, Tokyo, Japan). First strand cDNA was reverse and 50-CGGAATTCTCAAGTAGAGGTAACTTC-30 (underlining in each transcribed from poly A+ RNA using SMARTScribe™ MMLV Reverse primer indicates BamHI and EcoRI recognition sites, respectively), Transcriptase, SMART IV Oligonucleotide and CDS III/30 Primer using pUC57/CiSA-32.6 as template DNA. The PCR product was (TaKaRa, Otsu, Japan). Second strand cDNA was amplified by double-digested with BamHI and EcoRI (TaKaRa, Otsu, Japan), li- long-distance PCR using the first strand as a template. Following gated to the BamHI–EcoRI ends of pGEX-4T-1 and transformed into restriction enzyme digestion, double-stranded cDNA was size-frac- E. coli DH5a. tionated using a CHROMA SPIN-400 column. All cDNAs larger than 0.4 kbp were inserted into the k phage vector kTriplE Â 2 and pack- 2.7. Expression, extraction and purification of recombinant CiSA-32.6t aged using Gigapack III Gold Packaging Extract (Stratagene, La Jolla, protein CA). The titer of the primary cDNA library was determined and amplification was carried out. Individual phage plaques from the Bacteria containing plasmid pGEX-4T/CiSA-32.6t were cultured primary cDNA library were picked at random and subjected to at 37 °CtoOD600nm between 0.3 and 0.5.