| Infectious laryngotracheitis(ILT) is an acute respiratory infection of chickens that can result insevere production losses characterized by signs of respiratory depression, gasping, expectoration ofbloody mucus, and high mortality and decressed egg production. The causative agent of the disease isan alphaherpesvirus, infectious laryngotracheitis virus (ILTV), classified as gallid herpesvirus I.Thedisease occurs worldwide and is one of the major health and economic problems in the poultryindustry. To date gB is known to be a main immunogen of ILTV and gC, gD may also play a importantrole in inducing protective immunity. Present vaccines for ILT are all modified live viruses. Asimmunogenicity of ILTV is usually correlated with its virulence, almost all modified live ILT vaccinesremained insufficient attenuation and have shown a variety of side effects including spread of vaccinevirus to nonvaccinates. Therefore, there is an urgent demand for safer vaccines which could be used tocontrol and possibly eradicate ILTV. Gene vaccination against ILTV was carried out in this researchwith recombinant eukaryotic expression plasmids containing the immunogen genes of ILTV strainWanggang.vaccination has generally been used only in areas where the disease is endemic.Since gene immunization was found from 1990, different gene of different virus has beendelivered into different animal and good results has been obtained. DNA vaccine now become thefocus of the vaccine field. The gene immunization is usually that the gene encoding theimmunogenic proteins is inserted into an appropriate eukaryotic expression plasmid that can bereplicated in bacteria, purified and then directly inoculated by various methods into the animal to bevaccinated, the plasmid insert is then expressed by the host cells and the protein produced initiatesan immune response.In the study, the genes gB and gD cloned from Infectious Laryngotracheitis virus (ILTV) WangGang (WG) strain were cloned into the EcoRI site of the eukaryotic expression vector pCAGGS,respectively. The positive recombinant plasmids were screened by transformed into E. coli DH5αcells, which were named pCAGG-gB and pCAGG-gD respectively after identification by restrictionendonuclease analysis and sequencing analysis. Then the recombinant plasmids were amplified andpurified and transfected into 293T cells in vitr. The indirect immunofluorescence Analysis was usedto detect the expression of target proteins.To test immune efficiency of pCAGG-gB and pCAGG-gD, SPF chickens were randomlydivided into seven groups:(A) Control, (B) pCAGGS, (C) rFPV-ILTVgB, (D) pCAGG-gB,(E)pCAGG-gB+pCAGG-gD, (F) pCAGG-gB+rFPV-ILTVgB, (G) pCAGG-gD.30-day-old chickens ingroups were immunized by four sites intramuscular injection, the dose of each injected plasmid for achicken is 50μg. Plasmids with the same dose (50μg) for each in group E were mixed beforeinjection. Chickens of B,D,E and G groups were intramuscally immunized two times with twoweek-interval with 50μg of respective plasmid DNA each time. Chickens of C and F groups wereimmunized with 0.1 ml rFPV-ILTVgB at the second time. Two weeks after the second immunization,all chickens were challenged by nasal drop with 0.4 ml ILTV WG strain virus. Antibody responseswere assayed using indirect ELISA, the percentages of CD4+T,CD8+ T and TCR+ T lymphocytes inperipheral blood of immunized chickens were assayed by flow cytometer and cytokines IFN-γandIL-18 were assayed by real-time PCR after vaccination and challenge. Antibody responsesimmunized chickens were increased constantly, the percentages of CD4+,CD8+ and TCR+ Tlymphocytes were higher than control groups and expression level of cytokine IFN-γimmunizedwith gD gene (E and G) and IL-18 immunized with gD gene (D and E) were all higher than otherimmunized groups. while the control groups showed no changes after vaccination. After challenge,the protective immunity was observed, The results showed that all chickens in vector and controlgroup dieased and began to die after the second day, chickens immunized with rFPV-ILT...
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