| Eimeria acervulina (E. acervulina) is an important intestinal protozoon, which causes chicken coccidiosis and results in intense damage to duodenum accompanied by severe weight loss and poor feed conversion. Until now, few genes of this coccidian have been reported and tested for their immunogenicity. 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, especially DNA vaccines.In the present study, we report the results of screening an expression library of cDNA of E. acervulina sporozoites, the cDNA clones encoding for E. acervulina antigen gene were identified and the protective efficacies of the recombinant proteins and eukaryotic expression plasmid encoding this antigen were evaluated using chicken challenge experiments.1 Construction of expression library of cDNA of E. acervulina sporozoitesTotall RNA and purified mRNA was isolated from the E. acervulina JS sporozoites. A library of oligo (dT)-Xho I primed cDNA with added directional EcoR I Adaptor was constructed and ligated to the EcoR I/Xho I arms of eukaryotic expression vector, pVAX1.0. A total of 15 pools (termed pool 1 to pool 15), each comprising approximately 200 individual clones, were constructed and restriction analysis with EcoR I and Xho I showed that all eight colonies picked randomly from the cDNA library had inserts from 500 to 2000 bp.2 Screening of cDNA expression library15 primary pools (termed pool 1 to pool 15), each comprising approximately 200 individual clones, were originally tested. The plasmids in each library pool were used to inoculate chickens by intramuscular injection in the leg each animal, and 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. The results of serum from two sublibraries (pool 1 and pool 2) showed a highly significant level of IgG antibody response compared with that of the control groups (p< 0.05). Similarly, significantly higher levels of IFN-y, IL-4 and TGF-β were observed in chickens immunized with pool 1 and pool 2 compared with the control group (p< 0.05). A combination of pool 1 and pool 2 was fractionated into 12 subpools. The results showed that only Pool M-7 induced a significant level of IgG antibody compared with that of the vector control chickens (p< 0.05). High concentration of IL-2, IFN-y and TGF-β was also obtained in the animals immunized with the M-7 subpool compared with the control groups (p< 0.05). Plasmid DNA from individual clone of Pool M-7 was prepared and was used to immunise chickens of 10 each as described previously. After three rounds of screening, the three positive clones, M-7-11, M-7-12 and M-7-7, were named as cSZ-JN1, cSZ-JN2 and cSZ-JN3 respectively, and the inserts were sequenced and analyzed. The results of sequence analysis from BLASTN search revealed that cSZ-JN1 had no significant homology with the known genes of E. acervulina, but had 31.37% identity with Eimeria tenella annotated protein and 24% with Toxoplasma gondii ME49 hypothetical protein. cSZ-JN2 and cSZ-JN3 had no significant homology with any of the genes deposited in the GenBank database. Nucleotide sequences and the predicted amino acid sequences have been deposited in the GenBank/NCBI database, the accession numbers was JN857359-JN857361 respectively.3 Cloning, expression and immunoprotection of cSZ-JN1cSZ-JN1 gene which were from E. acervulina cDNA expression library was clone by PCR and characterized. The results showed that the open reading frame (ORF) of cSZ-JN1 gene was 615 bp and encoded a predicted protein of 204 amino acids with 21.8 kDa as well as N-glycosylation sites was identified in amino acids sequence. The cSZ-JN1 gene was subcloned into prokaryotic expression vector pET28a (+) and transformed into BL21 (DE3). Western blotting showed that the recombinant cSZ-JN1 was recognized strongly by serum from naturally infected chick with E. acervulina and rat anti-rcSZ-JN1 antiserum bound to a band of about 26 kDa in the somatic extract of E. acervulina sporozoites. Immunofluorescence analysis using antibody against recombinant cSZ-JN1 indicated that this protein was expressed in sporozoite and merozoite development stages of E. acervulina.Chickens were injected intramuscularly (IM) in leg muscle with 100μg of correctly constructed recombinant plasmids pVAX1.0-cSZ-JN1. Two-week-old chickens were injected intramuscularly (IM) in leg muscle with recombinant plasmids after they were identified. One week post-inoculation, vaccine injected tissues, non-injected tissues and pVAX1.0 plasmid injected tissues were collected. Expressions of proteins encoded by plasmids DNA in vivo were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. Results indicated that all DNA vaccines could be well transcripted and expressed in injected tissues.Two-week-old chickens were inoculated with DNA vaccines pVAX1.0-cSZ-JN1 and recombinant cSZ-JN1 protein by leg intramuscular injection, respectively. A booster immunization was given 1 week later. Challenged control group and unchallenged control chickens were injected with sterile TE buffer at the same injection site. pVAX1.0 plasmid alone was given to chickens as plasmid control. 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 to determine the effects of immunization on the 6th day post-challenge. The efficacy of immunization was evaluated on the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and anti-coccidial index (ACI). 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. The result showed that the recombitant cSZ-JN1 protein and recombitant cSZ-JN1 plasmid could protect chickens from E. acervulina infection, presenting anti-coccidial indexs more than 160, and pVAX1.0-cSZ-JN1 could confer better protection. Serum from chickens immunized with recombinant cSZ-JN1 protein showed significantly high level of IgG antibody whereas IgG antibody of chicken immunized with plasmid pVAX1.0-cSZ-JNl was not significantly induced. However, significantly higher levels of IL-4 and TGF-(3were observed in chickens immunized with recombinant cSZ-JN1 protein and recombinant plasmid pVAX1.0-cSZ-JN1 compared to the control groups. Taken together, these results suggest that cSZ-JN1 possess better immunogenicity and may be a potential candidate E. acervalina antigen for use in the development of vaccines.4 Cloning, expression and immunoprotection of cSZ-JN2cSZ-JN2 gene which were from E. acervulina cDNA expression library was clone by PCR and characterized. The results showed that the open reading frame (ORF) of cSZ-JN2 gene was 153 bp and a putative casein kinase Ⅱ phosphorylation site located at position (3SLND6) and a WH2 domain profile (8GSSTQFAAIRQCVVLRKA25) were present in this putative protein. The result of sequence search was that cSZ-JN2 had no significant homology with any of the genes deposited in the GenBank database The cSZ-JN2 gene was subcloned into prokaryotic expression vector pET28a (+) and transformed into BL21 (DE3). Western blotting showed that the recombinant cSZ-JN2 was recognized strongly by serum from naturally infected chick and whole parasite protein and rat anti-rcSZ-JN2 antiserum bound to a band of about 10 kDa in the somatic extract of E. acervulina sporozoites. Immunofluorescence analysis using antibody against recombinant cSZ-JN2 indicated that this protein was expressed in sporozoite and merozoite development stages of E. acervulina.By the means of DNA recombination technique, the eukaryotic expression plasmid pVAX1.0-cSZ-JN2 was constructed. Two-week-old chickens were injected intramuscularly (IM) in leg muscle with recombinant plasmids after they were identified. One week post-inoculation, vaccine injected tissues, non-injected tissues and pVAX1.0 plasmid injected tissues were collected. Expressions of proteins encoded by plasmids DNA in vivo were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. Results indicated that all DNA vaccines could be well transcripted and expressed in injected tissues.Two-week-old chickens were inoculated with DNA vaccines pVAX1.0-cSZ-JN2 and recombinant cSZ-JN2 protein by leg intramuscular injection, respectively. A booster immunization was given 1 week later. Challenged control group and unchallenged control chickens were injected with sterile TE buffer at the same injection site. pVAX1.0 plasmid alone was given to chickens as plasmid control. Seven days post second injection,20 chickens in each group except the unchallenged control group were challenged orally with 1.2×10 sporulated oocysts of E. acervulina. Unchallenged control chickens were given PBS orally. All of the chickens were euthanized to determine the effects of immunization on the 6th day post-challenge. The efficacy of immunization was evaluated on the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and anti-coccidial index (ACI). 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. Animal challenge experiments demonstrated that the recombitant cSZ-JN2 protein and recombitant cSZ-JN2 plasmid could protect chickens from E. acervulina infection, presenting anti-coccidial indexs more than 165, and pVAX1.0-cSZ-JN2 could confer better protection. Serum from chickens immunized with recombinant cSZ-JN2 protein showed significantly high level of IgG antibody whereas IgG antibody of chicken immunized with plasmid pVAX1.0-cSZ-JN2 was not significantly induced. However, significantly higher levels of IL-2 was observed in chickens immunized with recombinant cSZ-JN2 protein and recombinant plasmid pVAX1.0-cSZ-JN2 compared to the control groups. Taken together, these results suggest that cSZ-JN2 may be a potential candidate E. acervalina antigen for use in the development of vaccines. |
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