| Infectious laryngotracheitis (ILT) is an acute respiratory infection of chickens that can result in severe production losses characterized by signs of respiratory depression, gasping, expectoration of bloody mucus, and high mortality and decressed egg production. The disease occurs worldwide and is one of the major health and economic problems in the poultry industry. The causative agent of ILT is infectious laryngotracheitis virus (ILTV), a member of the alphaherpesvirinae subfamily of the herpesviridae. To date gB is known to be a main immunogen of ILTV. At present ILT is controlled by the use of attenuated live viruses. As immunogenicity of ILTV is usually correlated with its virulence, almost all modified live ILT vaccines remained insufficient attenuation and have shown a variety of side effects including spread of vaccine virus to nonvaccinates. Therefore, there is an urgent demand for safer vaccines which could be used to control and possibly eradicate ILTV.Since gene immunization was found from1990, different genes of different viruses have been delivered into different animal and good results have been obtained. DNA vaccines now become the focus of the vaccine field. However, DNA vaccines induce animals to produce low antibody levels which partially protect animals from a lethal dose pathogen challenge. Therefore, there is an urgent demand how enhances immunization protection. Many studies showed immunogenicity of an antigen could be enhanced by cytokines, including IL-2, IFN-y, IL-6, and IL-18etc. Co-administration of a cytokine enhances antigen-specific immune responses following vaccination with DNA encoding a pathogen. Therefore, gB gene of ILTV HN1strain isolated from Henan province was amplified in this study and cloned into eukaryotic express vector pcDNA3.1(+) to generate a recombinant expression plasmid pcDNA3.1/gB(pgB). Gene vaccination against ILTV was carried out with recombinant eukaryotic expression plasmids containing the immunogen genes of ILTV HN1strain and chicken IL-18. The research contents are as follows.1. One pair of primers was designed and synthesized according to ILTV gB gene nucleotide sequence (M64927) repulished in the GenBank. gB gene of ILTV HN1strain isolated from Henan province was amplified by PCR, then was cloned and sequenced. The results indicated gB gene nucleotide sequence of ILTV HN1strain is2629bp in length, which includes one open-reading frame (2622bp), encoding873amino acid residues, there are eight potential N-glycosalation sites and twelve cysteines in deduced amino acid sequence. When compared with ILTV632strain, SA2strain, Throne strain, yantai strain, and liaoning strain in GenBank, the similarity of gB gene nucleotide sequence of these strains shared99.0%above. When compared with ILTV SA2strain, ILTV HN1strain had one base deletion of G at89nt and one base insertion of A at102nt within gB gene resulting five amino acid frame shift mutation.2. Another pair of primers was designed according to the nucleotide sequence of gB gene of ILTV HN1strain. gB gene was amplified by PCR. The PCR product was digested with EcoR I and Xho I, then inserted into eukaryotic express vector pcDNA3.1(+) between to generate a recombinant expression plasmid pcDNA3.1/gB(pgB). Then the recombinant plasmids were amplified and purified and transfected into chicken embryo fibroblasts (CEF) cells in vitro. gB glycoprotein mRNA expression was found in CEF cells. Glycoprotein encoded by gB gene was detected by indirect immunofluoresent test. The result showed that the protein had successfully been expressed in CEF cells.3. One pair of primers was designed and synthesized according to chicken IL-18gene sequences published in GenBank. Luoman chicken IL-18gene was amplified by RT-PCR from chicken splenocyte total RNA extract without induction of PHA, then was cloned into pGEM-T Easy vector to generate a recombiant plasmid pGEM-IL-18and sequenced. The result showed that the full length sequence of luoman chicken IL-18gene was consisted of597bp. When the nucleotide sequence of luoman chicken IL-18gene was compared with that of chicken IL-18genes published in GenBank, the nucleotide sequence was just the same as published by Schneider. Recombinant plasmid pGEM-IL-18was digested with EcoR. I and Xho I, then Chicken IL-18gene was inserted into pcDNA3.1(+) between EcoR I and Xho I sites to generate an expression plasmid pcDNA3.1/IL-18(pIL-18), and transformed into Escherichia coli JM109. The recombinant colonies were identified by the methods of restriction enzyme digestion, PCR and sequencing. After the transfection of pIL-18into Cos7cells, chicken IL-18mRNA expression was found in Cos7cells. The results of MTT assay showed that the expression of chicken IL-18protein in Cos7cells could induce significantly transformation of chicken T lymphocytes. The result showed that chicken IL-18had successfully been expressed in Cos7cells.4. To test immune efficiency of pgB and pIL-18, SPF chickens were randomly divided into seven groups:(A) pgB,(B) pIL-18,(C) pgB and pIL-18,(D) ILTV live vaccines and pIL-18,(E) ILTV live vaccines,(F) pcDNA3.1(+),(G) Control.21-day-old chickens in groups were immunized by four sites intramuscular injection, the dose of each injected plasmid for a chicken is100μg. Plasmids with the same dose100μg for each in group C were mixed before injection. Chickens of A, B, C, D and F groups were intramuscally immunized two times with two week-interval with100μg of respective plasmid DNA each time. Chickens of D and E groups were immunized with ILTV live vaccines after two weeks. Antibody responses were assayed using indirect ELISA, the percentages of CD4+T, CD8+T and TCR+T lymphocytes in peripheral blood of immunized chickens were assayed by flow cytometer and cytokines IFN-y and IL-2were assayed by real-time PCR after vaccination. Antibody responses immunized chickens were increased constantly, the percentages of CD4+,CD8+and TCR+T lymphocytes were higher than control groups and expression level of cytokine IFN-y and IL-2immunized with gB and chicken IL-18gene were all higher than the group immunized with gB gene; while the control groups showed no changes after vaccination. Challenge protection test after co-immunization of eukaryotic expression vector of immunity infectious laryngotracheitis virus UN1strain gB and chicken IL-18was evaluated. After challenge, the protective immunity was observed. The results showed that all chickens in vector and control group dieased and began to die after the second day, chickens immunized with plasmids have been partially protected, i.e. pIL-18(28), pgB(79%), pgB and pIL-18(86%). These results indicated that DNA immunization can induce immune response and partially protect chickens from homologous ILT virus challenge and chickn IL-18enhanced immunization of DNA vaccine. |
PDF Full Text Request