Identification and molecular characterization of a novel antigen of Eimeria acervulina

Molecular & Biochemical Parasitology 186 (2012) 21–28

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Molecular & Biochemical Parasitology

Identification and molecular characterization of a novel antigen of Eimeria acervulina Huili Zhu a,b , Ruofeng Yan a , Song Wang a , Xiaokai Song a , Lixin Xu a , Xiangrui Li a,∗ a b

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, PR China Animal Disease Prevention and Control Center, Pingdingshan City 467000, Henan, PR China

a r t i c l e

i n f o

Article history: Received 29 January 2012 Received in revised form 29 August 2012 Accepted 3 September 2012 Available online 17 September 2012 Keywords: cSZ-JN1 E. acervulina Antigen cDNA expression library immunization

a b s t r a c t Eimeria acervulina (E. acervulina) is one of the seven species of Eimeria infected in chicken. Until now, only a few antigen genes of E. acervulina have been reported. In this study, a cDNA expression library of E. acervulina sporozoites was constructed in a eukaryotic expression vector, pVAX1.0. Subsequently, the library was divided into pools and inoculated into chickens to observe the ability of the antigens to induce humoral immune response and cell-mediated immune response. The positive pools that stimulated significant immune responses were fractionated sequentially until a single positive clone was screened. After three rounds of screening, a clone, named as cSZ-JN1, with the ability to stimulate chicken immune response was obtained. The sequence analysis showed that the opening reading fragment (ORF) of cSZJN1 was 615 bp in size and encoded a predicted protein of 204 amino acids with 21.8 kDa. BLASTN and other sequence databases searches revealed that the identity of the amino acid sequence of cSZ-JN1 to the complete sequence of Eimeria tenella annotated protein (ETH 00022005.1.pep) was 31.37% and to Toxoplasma gondii ME49 hypothetical protein (gb|EEB00972.1|) 24%, and had no significant homology with the known genes of E. acervulina and other parasites. Immunofluorescence analysis using antibody against recombinant cSZ-JN1 indicated that this protein was expressed in sporozoite and merozoite development stages. Animal challenge experiments demonstrated that the recombinant protein of cSZ-JN1 and DNA vaccine carrying cSZ-JN1 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented anti-coccidial index more than 160. All the above results suggested that the cSZ-JN1 was a novel E. acervulina antigen and could be an effective candidate for the development of new vaccine against E. acervulina infection. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Avian coccidiosis, caused by intestinal infection with Eimeria spp., occurs worldwide and is economically the most important parasitic disease of the poultry industry [1,2]. At present, the controls of Eimeria infection are still based mainly on anti-coccidial drugs and live vaccines. However, the drawbacks of these measures, which include the emergence of drug resistance and high production expenses, have driven the development of new control strategies [3,4]. Recent studies have demonstrated various levels of protection by vaccination with recombinant antigen or DNA vaccines against coccidiosis [5–8]. It has also been found that DNA vaccines or recombinant antigen can provoke both humoral and cellmediated immune responses [8–12], and co-delivery of cytokines

∗ Corresponding author. Tel.: +86 25 84399000; fax: +86 25 84399000. E-mail address: [email protected] (X. Li). 0166-6851/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molbiopara.2012.09.002

as adjuvants could enhance the potential for DNA vaccines or recombinant antigen to induce broad and long-lasting humoral and cellular immunity [13–16]. Expression library immunization (ELI) is a contemporary approach to vaccine production that has the potential to identify novel vaccine antigens. To date this in vivo screening of expression libraries has been utilized to identify protective antigens against a variety of organisms, including bacteria, fungi and parasites [17–23]. E. acervulina is one of the most prevalent Eimeria species in chicken. Until now, few genes of this coccidian have been reported and tested for their immunogenicity [24,25]. The search for new antigens and testing their protective ability against challenge with E. acervulina will facilitate the development of a new generation of vaccines against this parasite. In the current study, a noval cDNA clone encoding for E. acervulina antigen gene was identified and the protective efficacies of the recombinant protein and DNA vaccine encoding this antigen were evaluated using chicken challenge experiments.

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2. Materials and methods

2.4. cDNA library screening

2.1. Animals and parasites

The plasmids in each pool were prepared using a Qiagen endotoxin-free plasmid purification system (Qiagen, USA), according to the manufacturer’s instructions. According to the permission for inoculating the chickens with the DNA vaccine issued by the Animal Care and Ethics Committee of Nanjing Agricultural University (Approval No. 201009022). Two-week-old chickens, 10 each group, were inoculated with 100 ␮g plasmids of each library pool by intramuscular injection in the leg each animal, and 100 ␮g of pVAX1.0 plasmid alone was given to chickens as the plasmid control. Chickens in the negative control group were injected with sterile PBS buffer at the same injection site. A booster immunization was given 1 week later with the same amount of components as the first immunization. Ten days after the booster immunization, all chickens from each group were killed by cardiac puncture following protocols approved by the Animal Care and Ethics Committee of Nanjing Agricultural University (Approval No. 200709005) to collect blood serum for determination of cytokine and antibody levels. The blood was allowed to clot for 1 h at room temperature (RT) and then overnight at 4 ◦ C. The serum was collected by centrifugation (800 × g, 10 min), aliquoted, and stored at −20 ◦ C until used. The positive pools that stimulated significant immune responses were fractionated sequentially and screened as above until a single positive clone was isolated.

New-hatched Chinese Yellow chickens were reared in clean brooder cages and were screened periodically for their Eimeria infection status by microscopic examination of feces. The birds were provided with coccidiostat-free feed and water ad libidum. Birds were shifted to animal containment facility prior to challenge with virulent oocysts. The study was approved by the Institutional Animal Experiment Commission in accordance with the Chinese regulations of animal experimentation. E. acervulina JS strain was maintained in the Laboratory of Veterinary Parasite Disease, Nanjing Agricultural University, China. Sporulated oocysts of E. acervulina JS strain were stored in 2.5% potassium dichromate solution at 4 ◦ C and passaged through chickens every 3 months interval. Sporozoites from E. acervulina oocysts were purified on DE-52 anion exchange columns using the protocol described previously [26]. E. acervulina merozoites were harvested from the duodenal loop of chickens 54 h and 89 h post-infection (p.i.) and purified using standard methods [27,28] before being pelleted and frozen in liquid nitrogen.

2.2. Soluble antigens of E. acervulina A count of 5 × 109 E. acervulina sporozoites were washed three times by centrifugation with 0.1 M PBS (pH 7.2) at 2000 × g for 10 min at 4 ◦ C. The pellet was dissolved respectively in 2 ml of PBS and PBS containing 0.5% Triton X-100 and was disrupted by ultrasound in ice bath (200 W, work time 5 s, interval time 10 s, 50 cycles). After high-speed centrifugation, the supernatant proteins were separated and estimated spectrophotometrically, adjusted to 1 mg/ml with PBS and stored at −20 ◦ C until to be used. The soluble antigen dissolved by PBS was used for ELISA and that dissolved by PBS containing Triton X-100 was used for western blot to analyze the native protein of the cSZJN1.

2.3. Construction of cDNA expression library According to the manufacturer’s protocol, total RNA from purified E. acervulina sporozoites was isolated with TRIZOL Reagent (Invitrogen, USA). Subsequently, the mRNA was extracted using Oligotex mRNA Kit (Qiagen, USA). cDNA synthesis was primed with oligo(dT) primers and 5-me dCTP was incorporated into both strands without extraction or precipitations between first and second strand synthesis. The cDNA was treated with T4 DNA polymerase to flush the ends and ligated with EcoR I–Not I–BamH I adaptors (TaKaRa Biotech, Dalian, PR China). Following adaptor ligation and phosphorylation with T4 polynucleotide kinase, the cDNA was digested with Xho I. The cDNA was passed through a Mini Column Fractionation Kit (Novagen, USA) to remove excess adaptors and small cDNA products (<300 bp). The fractionated cDNA bigger than 300 bp in size was ligated to the EcoR I and Xho I digested expression vector PVAX1.0 (Invitrogen, USA). The ligation mixes were transformed into Escherichia coli TOP10 and selected on solid media containing 50 ␮g/ml kanamycin. After overnight growth, the colonies of E. coli were combined into pools. To estimate the size of the inserts of the cDNA library, eight independent clones were picked randomly and the plasmids were extracted with an Axgen Plasmid miniprep kit (Axgen, USA) and digested with EcoR I and Xho I.

2.5. Determination of serum antibody level by enzyme-linked immunosorbent assay (ELISA) The IgG antibody levels against E. acervulina soluble antigen in the serum samples were determined by ELISA as described previously [15]. Briefly, flat-bottomed 96-well plates (Marxi-Sorp, Nunc, Denmark) were coated overnight at 4 ◦ C with 100 ␮l per well of the solution of soluble antigens of E. acervulina (50 ␮g/ml) in 0.05 M carbonate buffer, pH 9.6. The plates were washed with 0.01 M PBS containing 0.05% Tween-20 (PBS-T) and blocked with 5% skim milk powder (SMP) in PBS-T for 2 h at 37 ◦ C. The plates were incubated for 2 h at 37 ◦ C with 100 ␮l of the serum samples diluted 1:50 in PBS-T with 1% SMP in duplicate. After three washes, the plates were incubated for 1 h at room temperature with 100 ␮l/well of horseradish peroxidase-conjugated donkey anti-chicken IgG antibody (Sigma) diluted 1:1000 in 2% SMP in PBS-T. Color development was carried out with 3,3 ,5,5 -tetramethylbenzidine (TMB) (Sigma), and the optical density at 450 nm (OD450 ) was determined with a microplate spectrophotometer. All serum samples were investigated by ELISA at the same time under the same conditions, and the serum samples collected on each occasion were included on one plate. 2.6. Determination of serum cytokine concentration The concentration of interleukin-2 (IL-2), interleukin-4 (IL4), interferon-␥ (IFN-␥) and tumor growth factor-␤ (TGF-␤) in serum were detected by utilizing an indirect ELISA with the “chick cytokine ELISA Quantitation Kits” (catalog numbers: I04201, 1042-02, 1042-03 and 1042-04 for IL-2, IL-4, IFN-␥ and TGF-␤ respectively; GBD Laboratories, USA) in duplicate, according to manufacturer’s instructions. 2.7. Sequence analysis Positive clone, confirmed by the last screening was sequenced by Invitrogen Biotech (Shanghai, PR China). Sequence similarity was investigated using GenBank (GenBank ID: BA123456). The signal peptide, secondary structure and protein motifs were predicted using approaches accessible on the Internet: SignalP (CBS

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ID: 122.96.158.26) and Motifscan and second structure (UniProt ID: Q9H0H5) respectively. 2.8. Construction of prokaryotic expression vector of cSZ-JN1 The ORF of E. acervulina cSZ-JN1 gene was amplified utilizing the positive plasmid identified by sequence analysis as template by PCR. The forward primer and reverse primer were designed by Primer 5.0 software based on the cSZ-JN1 gene sequences. The primer set contained restriction enzyme sites EcoR I and Xho I in forward primer (5 -GGCGAATTCATGGTTACGATTACTAGC-3 ) and reverse primer (5 -AGCCTCGAGTCAAGATAGTTCAGGACTT-3 ) (sites for digestion by EcoR I and Xho I are underlined), respectively. The amplification product was purified using “AxyPrepTM DNA Gel Extraction kit” (Axygen, USA) according to manufacturer’s instructions. And then it was ligated to pMD18-T cloning vector (Takara Co., Ltd., Dalian, China) according to manufacturer’s instructions. The positive clone, confirmed by digestion with EcoR I and Xho I, was sequenced by Invitrogen Biotech (Shanghai, PR China). The correct cSZ-JN1 gene was then cloned into the EcoR I/Xho I sites of pET28a(+) vector (Novagen, USA) to generate plasmid pET28acSZ-JN1. The recombinant plasmid was sequenced to confirm that the cSZ-JN1 insert was in the proper reading frame. 2.9. Expression and purification of recombinant cSZ-JN1 The correct recombinant pET28a-cSZ-JN1 plasmid was transferred into competent E. coli BL21 (DE3) cells and the recombinant protein was induced and expressed by addition of 0.8 mM isopropyl-␤-d-thiogalactopyranoside (IPTG; Sigma–Aldrich, USA) to the cell culture after the OD600 of the culture reached 0.6 at 37 ◦ C. The cells were incubated at 37 ◦ C for 5 h after the addition of IPTG. Then cell lysates were prepared by sonication and analyzed by 12% (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified by Ni2+ -nitrilotriacetic acid (Ni-NTA) column (GE Healthcare, USA) according to the manufacturer’s instructions. Purity of the protein was detected by 12% SDS-PAGE and the concentration of purified protein was determined according to the Bradford procedure [29], using bovine serum albumin (BSA) as a standard. The protein was stored at −80 ◦ C in 50% glycerol for later use.

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skimmed milk powder in TBS-Tween 20 (TBST), the membranes were incubated with the primary antibodies (rat antisera and chicken antisera, respectively) for 1 h at 37 ◦ C (dilutions 1:100 to rat antisera, 1:100 to chicken antisera). Horseradish peroxidase (HRP)-conjugated goat anti-rat IgG, and HRP-conjugated donkey anti-chick IgG (Sigma, USA) were added, respectively. Finally, The bound antibody was detected using 3,3 -diaminobenzidine tetrahydrochloride (DAB) kit (Boster Bio-technology) according to manufacturer’s instructions. 2.12. Observations of the expressions of cSZ-JN1 in sporozoites and meroziotes by immunofluorescence Purified sporozoites and meroziotes were smeared and airdried on a glass slide (Beyotime) before fixation. Sporozoites and meroziotes were fixed with 2% paraformaldehyde in TBS for 10 min at RT, permeabilized with 1% Triton X-100 in TBS for 10 min, washed three times in TBS containing 0.05% Tween-20 (TBS-T), and blocked with TBST containing 5% (w/v) BSA for 2 h at 37 ◦ C. After three washes in TBST, rat antisera against cSZ-JN1 (1:50 dilution) was added and allowed to incubate for 1 h at 37 ◦ C. After three washes in TBST, goat anti-rat IgG (H + L chain specific) FITC-conjugated antibody (1:1000 dilution; Beyotime) was added to the glass slides. After incubation for 5 min and TBST wash, fluorescent mounting media (Beyotime) was added and cells were examined by fluorescent microscopy (Olympus). 2.13. Construction of cSZ-JN1 DNA vaccine In order to construct pVAX1.0-cSZ-JN1 vector, the 615 bp ORF of cSZ-JN1 was cloned into the eukaryotic expression vector to evaluate its immunogenicity. The pMD-18 T plasmid containing cSZ-JN1 ORF and the pVAX1.0 vector (Invitrogen, Life Technologies) were treated with EcoR I and Xho I enzyme and the cSZ-JN1 ORF was directionally cloned into the pVAX1.0 vector. Recombinant vector pVAX1.0-cSZ-JN1 was digested with the same restriction enzymes and sequenced by Invitrogen biotech (Shanghai, PR China) for identification. The recombinant plasmids pVAX1.0-cSZ-JN1 acting as DNA vaccines were prepared using Qiagen Plasmid DNA Mid Kit (Qiagen, USA), according to the manufacturer’s instructions. The eluted products were dissolved in TE (pH 7.4) at a concentration of 1 mg/ml, and stored at −20 ◦ C until required.

2.10. Polyclonal sera against recombinant cSZ-JN1 protein and against E. acervulina

2.14. Detection of the expressions of proteins encoded by plasmids DNA in vivo by RT-PCR assay and western blot analysis

To generate polyclonal antibodies, about 0.3 mg of the purified recombinant cSZ-JN1 protein was mixed with Freund’s complete adjuvant of a 1:1 mixture and injected into SD rats (Qualitied Certificate: SCXK 2008-0004; Experimental Animal Center of Jiangsu, PR China) subcutaneously in multiple places. Two weeks later, a booster was given in the same conditions. And then, the rats were re-boosted three times at intervals of 1 week. Finally, the serum was collected and stored until used. Sera collected before protein injection was used as negative sera [30]. The polyclonal sera against E. acervulina (chicken antisera) were collected from chickens experimentally infected with E. acervulina one week post the infection.

Chickens were injected intramuscularly (IM) in leg muscle with 100 ␮g of recombinant plasmids pVAX1.0-cSZ-JN1. One week postinoculation, injected tissues were collected and total RNA was extracted. To remove contaminating genomic DNA or plasmids injected, all RNA samples were treated with RNase-free DNase I (TaKaRa, China). RT-PCR assays were performed with cloning primer pairs of cSZ-JN1 gene. The PCR products were detected by electrophoresis on 1% agarose gel. Western blot analysis was performed as described previously [31]. Briefly, seven days after vaccination, injected muscles were grinded and treated with ice-cold RIPA solution (0.1 M phenylmethylsulfonyl fluoride, 150 mM sodium chloride, 1% Nonnidet P-40, 0.1% SDS, 50 mM Tris–HCl). Meanwhile, the same site muscles from non-injected and pVAX1.0 plasmid injected chickens were collected as controls. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Millipore, USA). The membrane was incubated with rat anti-rcSZ-JN1 polyclonal antibody as primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Sigma) as secondary antibody.

2.11. Immunoblot for the recombinant cSZ-JN1 and native protein of cSZ-JN1 Samples including crude somatic extracts of E. acervulina sporozoites and the recombinant protein were separated by SDS-PAGE. Then the protein was transferred to nitrocellulose membrane (Millipore, USA). After being blocked with 5% (w/v)

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The bound antibody was detected using 3,3 -diaminobenzidine (DAB). 2.15. Immunization and challenge infection Two-week-old chickens were randomly divided into five groups of 30 each as shown in Table 1. According to the permission for inoculating the chickens with the DNA vaccine issued by the Animal Care and Ethics Committee of Nanjing Agricultural University (Approval No. 201009022). Experimental group chickens were respectively inoculated with 100 ␮g plasmids pVAX1.0-cSZJN1 and recombinant cSZ-JN1 protein. Plasmid control group was given 100 ␮g of pVAX1.0 plasmid alone each chicken. Challenged control group and unchallenged control chickens were injected with sterile PBS at the same injection site. A booster immunization was given 1 week later with the same amount of components as the first immunization. Seven days post second injection, 20 chickens in each group except the unchallenged control group were challenged orally with 1.2 × 105 sporulated oocysts of E. acervulina. Unchallenged control chickens were given PBS orally. All of the chickens were euthanized following protocols approved by the Animal Care and Ethics Committee of Nanjing Agricultural University (Approval No. 200709005) to determine the effects of immunization on the 6th day post-challenge. The other 10 chickens in each group were killed by cardiac puncture to collect blood serum for determination of cytokine and antibody levels 10 days after the second immunization. 2.16. Evaluation of immune protection The efficacy of immunization was evaluated on the basis of lesion score, body weight gain, oocyst output, oocyst decrease ratio and anti-coccidial index (ACI). Body weight gain of chickens in each group was determined by the body weight of the chickens at the end of the experiments subtracting the body weight at the time of challenge. Lesion scores were evaluated as described previously [32]. Additionally, the duodenal content for each group was collected separately and oocysts per gram of content (OPG) were determined using McMaster’s counting technique. Oocyst decrease ratio was calculated as follows: the number of oocysts from the challenged control chickens − vaccinated chickens/the challenged control chickens × 100%. ACI was a synthetic criterion for assessing the protective effect of a medicine or vaccine and calculated as follows: (relative rate of weight gain + survival rate) − (lesion value + oocyst value). The IgG antibody levels were tested by the methods described as 2.6 except that the antigen used to coat 96-well microtitre plates was purified recombinant cSZ-JN1 protein in sodium carbonate buffer. The methods to test the concentration of IL-2, IL-4, IFN-␥ and TGF-␤ in serum were the same to 2.6. 2.17. Statistical analysis Statistical analysis was performed using the SPSS statistical package (SPSS for Windows 16, SPSS Inc., Chicago, IL, USA). Differences among groups were tested with the one-way ANOVA Duncan test, and p < 0.05 was considered to indicate a significant difference. 3. Results 3.1. Construction of E. acervalina cDNA library A cDNA expression library of sporozoites of E. acervalina JS strain was constructed in a eukaryotic expression vector, pVAX1.0. The results of restriction analysis of the library showed that the library

Fig. 1. Screening sublibraries M-7 of cDNA library. Each subpool contains a single clone. The concentrations of IL-4 and TGF-␤ (mean ± SD) in the serum are expressed in pg/ml. The IgG titers are expressed as mean ± SD with respect to absorbance at 450 nm. Bars with different lower-case letters are significantly different (p < 0.05). (a) IL-4 concentration; (b) TGF-␤ concentration and (c) the IgG titers.

was successfully constructed with a size of 3 × 103 clones and all of the clones analyzed by enzyme digestion comprised cDNA inserts of 0.5–2 kb in size. 3.2. Library screen and DNA sequence analysis A total of 15 pools (termed pools 1–15), each comprising approximately 200 individual clones, were originally screened. The results showed that pool 1 and pool 2 induced highly significant level of IgG antibody and cytokines in chickens compared with that of the control groups. Pool 1 and pool 2 were fractionated into 12 subpools, each containing 12 clones, for the second-level screening. The screening results indicated that only pool M-7 induced a significant level of IgG antibody and high concentration of IL-2, IFN-␥ and TGF-␤ in chickens compared with that of the vector control chickens (p < 0.05). 12 individual clones of pool M-7 were used for the last screening. The results showed that the clone of M-7-11 resulted in significant high levels of IL-4 and TGF-␤ of the chickens (p < 0.05) (Fig. 1a and b). However, M7-7 significantly enhanced the levels of TGF-␤ and IgG (IgY) of the chickens compared to that of the control chickens (p < 0.05) (Fig. 1b and c). In the present work, M-7-11 was selected for further research.

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Table 1 Effects of cSZ-JN1 against E. acervulina challenge. Groups

Average body weight gains (g)

Unchallenged control Challenged control pVAX1 control pVAX1-cSZ-JN1 Recombinant cSZ-JN1 protein

144.32 72.5 70.5 128.78 123.35

± ± ± ± ±

1.52a 5.35d 4.63d 2.98b 0.56c

Mean lesion scores (mean ± SD)

Oocyst output (×105 ) (mean ± SD)

0a 2.85 ± 0.46c 2.78 ± 0.38c 1.4 ± 0.40b 1.58 ± 0.18b

0.00 2.73 2.68 0.61 0.70

± ± ± ± ±

0.00a 0.42b 0.23b 0.03c 0.01c

Oocyst decrease ratio (%)

Anti-coccidial index

100a 0.00b 2.15b 77.41c 74.34c

200 102.79 107.28 169.28 161.25

Note: in each column, significant difference (p < 0.05) between numbers with different letters and no significant difference (p > 0.05) between numbers with the same letter.

The positive clone, M-7-11, was named as cSZ-JN1 and the insert was sequenced and analyzed. Nucleic acid sequencing of cSZ-JN1 clone identified an insert of 870 bp, which contained a 5 untranslated region composed of 196 bp, a 615 bp open reading frame (ORF), and a 3 untranslated region containing 59 bp. The deduced primary translation product of the 615 bp ORF consisted of 204 amino acids and had a predicted molecular mass of 21.8 kDa with isoelectric point of 5.405. One N-glycosylation site presented in the protein. The cDNA sequence and inferred amino acids were submitted to GenBank, accession no. JN857359. When compared with the sequences on the GenBank database and other sequence databases, the results showed that the identity of the amino acid sequence of cSZ-JN1 to the complete sequence of Eimeria tenella annotated protein (ETH 00022005.1.pep) was 31.37% and to Toxoplasma gondii ME49 hypothetical protein (gb|EEB00972.1|) 24%, and there was no significant homology with the known genes of E. acervulina and other parasites deposited in the GenBank and other database.

3.3. Expression of recombinant cSZ-JN1 protein The ORF of E. acervulina cSZ-JN1 gene was successfully amplified by PCR described already. The recovered PCR product was purified and successfully cloned into pMD18-T cloning vector which was confirmed by sequencing. Target fragment was observed by enzyme digestion and the sequence analysis showed that the insert in the pET28a(+) vector was the ORF of cSZ-JN1. This indicated that the expression vector pET28a-cSZ-JN1 had been exactly constructed. Recombinant plasmid conceiving cDNA fragment of cSZ-JN1 was expressed in vitro in E. coli BL21 (DE3). High amount of recombinant protein cSZ-JN1 was obtained 5 h after the beginning of IPTG induction, a molecular weight of the cSZ-JN1 protein band was approximately 26 kDa on the SDS-PAGE gel. Because of the 3.8 kDa fused protein in the vector, the recombinant protein’s molecular weight was almost the same to the value (21.8 kDa) of rcSZ-JN1 calculated based on the deduced amino acid sequence.

Fig. 2. Immunoblot for the recombinant cSZ-JN1and native protein of cSZ-JN1. (Lane 1) Somatic extract of E. acervulina sporozoites probed by rat anti-rcSZ-JN1 antisera as primary antibody; (Lane 2) Recombinant protein cSZ-JN1 probed by serum from chickens experimentally infected with E. acervulina as primary antibody and (Lane 3) Recombinant protein cSZ-JN1 probed by serum of normal chickens as primary antibody.

that greater staining intensity was seen in sporozoites and merozoites. No staining was seen in the negative control sections. 3.6. Identifications of DNA vaccines and the expression of cSZ-JN1 in vivo Recombinant plasmid pVAX1.0-cSZ-JN1 produced a fragment of approximately 615 bp after digestion with EcoR I and Xho I. This indicated that DNA vaccine pVAX1.0-cSZ-JN1 was correctly constructed. The results of RT-PCR indicated that the target fragment of cSZJN1 (615 bp) was detected from muscle RNA samples of chickens injected with pVAX1.0-cSZ-JN1 (Fig. 3). No specific bands were detected in non-injected control and pVAX1.0 plasmid control samples. Western blotting of the muscle of chickens injected with pVAX1.0-cSZ-JN1 revealed a prominent band of 26 kDa, which indicated the expression of cSZ-JN1 gene (Fig. 4). In contrast, no corresponding band was detected in the muscle of chickens injected with pVAX1.0.

3.4. Immunoblot for the recombinant cSZ-JN1 and native protein of cSZ-JN1 The results of the immunoblot assay (Fig. 2) indicated that the recombinant cSZ-JN1 protein was recognized by immune sera of chickens infected with E. acervulina, but no protein of anti-rcSZJN1 in negative control was identified by the serum of normal chickens. Western blot analysis also showed that rat anti-rcSZ-JN1 antiserum bound to a band of about 26 kDa in the somatic extract of E. acervulina sporozoites.

3.5. Expressions of cSZ-JN1 in sporozoites and meroziotes Using anti-rcSZ-JN1 serum, cSZ-JN1 protein expression was investigated in sporozoites and merozoites. The results showed

Fig. 3. Detection of expression of pVAX1-cSZ-JN1 in chicken muscle by RT-PCR. Key: (Lane 1) non-injected muscle; (Lane 2) pVAX1.0 plasmid injected muscle; (Lane 3) pVAX1.0-cSZ-JN1 injected muscle and (Lane M) DNA Marker.

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Fig. 4. Detection of expression of pVAX1.0-cSZ-JN1 in chicken muscle by western blotting analysis. The samples were electro-transferred to nitrocellulose membranes and probed with rat anti-rcSZ-JN1 serum as first antibody. Key: (Lane M) standard protein molecular weight marker; (Lane 1) the muscle sample from chicken injected with pVAX1.0-cSZ-JN1 and (Lane 2) the muscle sample from chicken injected with pVAX1.0 plasmid.

3.7. Protective effects of vaccination against E. acervulina challenge The immunization efficacies of the vaccines are described in Table 1. No chicken died from coccidial challenge in any group in this study. Body weight gains were significantly reduced in challenged control and pVAX1.0 control group compared with unchallenged control group (p < 0.05). Chickens immunized with recombinant cSZ-JN1 protein or DNA vaccine displayed significantly enhanced weight gains relative to chickens in challenged control group and pVAX1.0 control group (p < 0.05). The oocyst counts of immunized chickens were significantly lower than that of challenged control group and pVAX1.0 group (p < 0.05). Significant alleviations in duodenal lesions were observed in immunized chickens compared to that of challenged control group and pVAX1.0 control group (p < 0.05). Group of chickens immunized with pVAX1.0-cSZ-JN1 resulted in ACI more than 165, higher than that of recombinant cSZ-JN1 protein vaccinated groups. 3.8. IgG titers and concentration of cytokines in sera of immunized chickens As depicted in Fig. 5a, serum from chickens immunized with recombinant cSZ-JN1 protein showed significantly high level of IgG antibody (p < 0.05) compared to that of controls, whereas IgG antibody of chicken immunized with plasmid pVAX1.0-cSZ-JN1 was not significantly induced (p > 0.05). However, significantly higher levels of IL-4 and TGF-␤ were observed in chickens immunized with recombinant cSZ-JN1 protein and recombinant plasmid pVAX1.0cSZ-JN1 compared to the control groups (p < 0.05) (Fig. 5b and c). No IL-2 and IFN-␥ were detected. 4. Discussion ELI is one of the effective methods to identify novel antigens. Many protective antigens against bacteria, fungi and parasites were obtained by this method [17–23]. In this research, we used it to screen the new antigen of E. acervulina sporozoites and successfully obtained one new gene. In the ELI, screening parameters are very important. Different parameters will result in antigens with different functions. IFN-␥

Fig. 5. Serum cSZ-JN1-specific IgG and cytokine levels in chickens. Chickens were immunized intramuscularly with PBS (negative control), pVAX1.0 plasmid (pVAX1.0 control), pVAX1.0-cSZ-JN1 or recombinant cSZ-JN1 protein. The IgG titers are expressed as mean ± SD with respect to absorbance at 450 nm. The concentrations of IL-4 and TGF-␤ (mean ± SD) in pg/ml. Bars with different lower-case letters are significantly different (p < 0.05). (a) IgG titers; (b) IL-4 concentration and (c) TGF-␤ concentration.

and IL-2 are marks of Th1 immune response and seem to be dominant in coccidiosis [33,34]. IL-4, a Th2-type cytokine, is known to regulate humoral immunity and to function more effectively as helper for B-cell activation [35,36]. TGF-␤, as a regulatory (iTreg) cytokine, plays important role in the immune response against Eimeria antigens [16]. So, in this research, we used these cytokines as marks to select antigens with different functions. In the screening of the expression library, the chickens immunized with M-7-11 (cSZ-JN1) showed marked rises of concentrations of IL-4 and TGF-␤. However, the antibody level of animals immunized with this antigen was not increased. In the protective experiment, the chickens immunized with DNA vaccine carrying cSZ-JN1 gene also demonstrated no significant difference of IgG antibody level compared to control groups. On the contrary, in the protective experiment, the animals immunized with the recombinant protein of cSZ-JN1 produced high antibody and IL-4 and TGF-␤ as high as that of chickens immunized with DNA vaccine. Western blotting assay showed that recombinant cSZ-JN1 could be detected by the sera of the chicks experimentally infected with E. acervulina indicating that cSZ-JN1 could stimulate antibody response in the natural infection. One possible explanation for the contradiction was that, in the screening and the DNA vaccination, the antigen was expressed and presented to the target immune cells by the

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different pathway and resulted in the different immune response. However, the exact causes of the contradiction need to be further probed. In the current work, the nucleic acid sequencing of cSZ-JN1 clone identified an insert of 870 bp, which contained a 615 bp open reading frame (ORF) with one N-glycosylation site presented in the ORF. The deduced primary translation product of the ORF consisted of 204 amino acids and had a predicted molecular mass of 21.8 kDa. The ORF contained the start cordon at the site 197–199 and the stop cordon at the site 809–811. The sera against recombinant cSZ-JN1 recognized a band of about 26 kDa in the somatic extract of E. acervulina sporozoites. These results suggested that the native cSZ-JN1 was about 26 kDa, slightly larger than the deduced size which might result from the small post-translational modification. These sequence and western blot analysis results indicated that the native cSZ-JN1 was about 26 kDa in size and underwent small post-translational modification in the nature infection and the gene we obtained was the full length of it. However, the compare of the sequences of cSZ-JN1 with proteins of other protozoa also suggested that cSZ-JN1 had 31.37% identity to E. tenella annotated protein (ETH 00022005.1.pep), especially the N-terminus of CSZ-JN1 had good homology to the Cterminus of ETH 00022005.1.pep. Which relationship is presented between CSZ-JN1 and ETH 00022005.1.pep and whether the CSZJN1 was truncated from the same gene to ETH 00022005.1.pep in E. acervulina need to be further researched. In the current study, western blotting assay showed that recombinant cSZ-JN1 could be detected by the sera of the chicks experimentally infected with E. acervulina. It indicated that cSZ-JN1 could enter into host tissues and be recognized by host immune system and induce immune response. Identification of genes expressed in the life cycle of coccidian is critical to understand the developmental biology of these parasites. In this research, we demonstrated that cSZ-JN1 could be expressed in sporozoite and merozoite stages of the life cycle. This result suggested that the protein was conserved in the life cycle of this parasite and might play some roles in the processes of host-parasite interactions in the infection. But the expressions of this antigen in other stages of E. acervulina and its localization as well as detailed function were worthy of further researches. IFN-␥ and IL-2, especially IFN-␥ is usually to be considered as marks of Th1 immune response. In this research, however, we could not detect the expressions of IFN-␥ and IL-2 induced by cSZ-JN1 in the screening and in the protective experiment. This indicated that cSZ-JN1 might be incapable to stimulate Th1 immune response. However, cSZ-JN1 induced high level of TGF-␤. That indicated this antigen might play some immune regulation functions in the infection of E. acervulina. Recently, many publications demonstrated that co-delivery of cytokines as adjuvants could enhance the potential for DNA vaccines to induce strong humoral and cellular immunity [13,15,16]. In this study, the cSZ-JN1 could stimulate high levels of IL-4 and TGF-␤ and resulted in an ACI more than 160 in the protective experiment. These results suggested that it might be an effective candidate for DNA vaccines against E. acervulina. In conclusion, cSZ-JN1 was successfully identified from the cDNA expression library of E. acervulina sporozoites by the method of cDNA expression library immunization in this study. Sequence analysis and BLASTN search revealed that the clone had no significant homology with the known genes of E. acervulina deposited in the GenBank database. Animal challenge experiment showed that recombinant cSZ-JN1 protein and the DNA vaccine carrying cSZ-JN1 gene were able to induce partial protection against homologous challenge in chickens. This suggested that cSZ-JN1 was a novel antigen of E. acervulina.

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