Construction And Combined Immunization Effect Of The Recombinant Dna Vaccine Expressing Iltv Gb Gene And Chicken Il-18

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. 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 from 1990, 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-γ, IL-6, and IL-18 etc.A plasmid DNA vaccine pcDNA3.1/gB expressing gB protein of infectious laryngotracheitis virus(ILTV) was constructed, To evaluate the prophylactic efficacy of the DNA vaccine. 3-week-old chickens were immunized with pcDNA3.1/gB, pcDNA3.1/IL-18 and pcDNA3.1/gB. After immunization, peripheral blood lymphocytes were detected by flow cytometry and the sera were collected to examine antibodies of chickens each week by ELISA. The chickens were challenged with 100ELD50 ILTV on week 3 after immunization. The results indicated that chickens vaccinated with pcDNA3.1/IL-18 and pcDNA3.1/gB had an insignificancy increase in the percentage of CD3+,CD3+CD4+,CD3+CD8+ subgroups of peripheral blood. Chickens vaccinated with pcDNA3.1/IL-18 and pcDNA3.1/gB were 87% protected against challenge with ILTV. The results demonstrate that the combined immunization can elicit remarkably cell immune response and improves the protective effect against ILTV infection.The pIRES bicistronic plasmid which was constructed and identified previously, was used to construct monocistronic and bicistronic DNA vaccines. This plasmid contained two MCS which were inserted into the multiple cloning sites (MCS) located on either side of the internal ribosome entry site from the encephalomyocarditis virus (ECMV). The ILTV gB gene was subcloned from recombinant plasmid pGEM-T-gB into MCS A of pIRES between Mlu I and XhoI restriction sites, and generated the plasmid pIRES/gB. IL-18 gene was subcloned from recombinant plasmid pGEM-T- IL-18 into MCS B of pIRES between Sal I and Not I restriction sites. Then,the gB gene of avian infectious laryngotracheitis virus (ILTV) and interleukin-18 ( IL-18) gene from chicken were inserted into the bicistronic pIRES plasmid. The pIRES/gB and pIRES/gB/IL-18 plasmid encoding gB and IL-18 gene were constructed.