Construction Of Recombinant Flow Pox Virus With ChIL-2 And Glycoprotein D Of Infectious Laryngotracheitis Virus And Its Immune Efficacy

Infectious laryngotracheitis(ILT) is a highly contagious ILTV virus-induced an acute respiratory infection of chicken.Character of this disease is sign of respiratory depression, gasping,weight losses due to mortality and decreased egg production.The disease occurs worldwide and is one of the major health and economic problems in the poultry industry. ILT has been controlled efficiently by vaccination with modified live strains of ILTV.But all ILTV live vaccines remain certain virulence,and are able to establish latent infection for life time;the vaccine viruses may increase virulence during in vivo passage.Therefore, there is an urgent demand for safer vaccines which could be used to control and possibly eradicate ILTV.With development of the molecular biology,many researchers have made major commitment to rDNA-based vaccination.The fowl virus vaccine viruses are considered one of the most potent vector live vaccines expressing foreign antigens related to vaccine-induced immunity against poultry diseases.In this study,we constructed recombinant fowl virus with ChIL-2 and glycoprotein D of infectious laryngotracheitis virus of Hnchangge strain and evaluated the abilities of this recombinants to protect against mortality in SPF chickens.This study includes:1.Isolation and Identification the Infectious Laryngotracheitis Virus HNchangge strain and Sequence Analysis of gB,gC and gD GeneA virulent strain ILTV was isolated from the larynx and trachea tissue of infected chickens which was identification by double agar precipitation test.Full-length ILTV gB, gC and gD gene were amplified by PCR and then cloned into the pGEM-T vector.ILTV genes of gB,gC and gD were sequenced and the sequences were compared with the gB,gC and gD gene sequences from others strains of ILTV in GenBank.The nucleotide homology of ILTV gB,gC and gD gene were 99.7%～99.9%.It indicated that ILTV gB, gC and gD gene were conservative.2.Construction of DNA Vaccines for ILTV HNchangge strain and their immune efficiencyThree genes encoding gB,gC and gD glycoprotein of ILTV HNchangge strain were inserted into pcDNA3.1 vector and named pcDNA-gB,pcDNA-gC and pcDNA-gD.The recombinant DNA vaccines were transcribed and expressed in eukaryotic cells effectively by IFA identification.Thirty30-day-old chickens were immunized by intramuscular injection with the DNA vaccines and blank vector pcDNA3.1,the dose of each injected plasmid for a chicken is 100μg.Another group chickens were vaccinated with the commercial ILT vaccine(D-805) and the blank control group chickens were injected with normal saline.The results of MTT assay showed that recombinant plasmid DNA induced strong proliferative response of Lymphocytes compared with the control groups.The serum-neutralization antibodies induced by DNA vaccine pcDNA-gB,pcDNA-gC and pcDNA-gD were higher than those induced by pcDNA3.1 vector.The results indicated that the recombinant plasmids pcDNA-gB,pcDNA-gC and pcDNA-gD had immune efficiency.3.Expression of gD Gene of ILTV and Primary Development of Indirect ELISA Assay Based on the Expressed ProteinThe gD gene of ILTV HNchangge strain was inserted into pET-28a(+) vector and named pET28-gD.The pET28-gD was further transformed into E.coli BL21 cells and a 55ku protein was high expressed after induced with IPTG.Western-blotting analysis proved the recombinant protein has a good reactive ability against ILT positive serum.The indirect ELISA for detecting ILTV antibodies was established by using purified recombinant gD protein.The optional working circumstances for the ELISA(antigen concentration of 24.5μg·mL,serum dilution of 1:320) were tried out with chess titration. The tested serum of positive criterion of this ELISA is OD₄₅₀≧0.25,the negative serum OD₄₅₀＜0.25.4.Expression of glycoprotein D(gD) of ILTV in recombinant fowl pox virus and its immune efficacyA recombinant fowl pox expressing glycoprotein gD named as rFPV-ILTV-gD was constructed by introducing gD gene of infectious laryngotracheitis virus isolated from Henan Changge recombined into the genome of fowl pox virus.Thirty-day-old non-immunized chickens were vaccinated with the rFPV-ILTV-gD and the commercial ILT vaccine(D-805).All groups were challenged with a lethal dose of virulent ILTV 42 days later.Results showed that protective efficacy could be significantly enhanced after immunizations and the anti-ILT neutralization antibody level were significantly increased compared with the unvaccinated chickens.There is no significant difference between the commercial vaccine and the rFPV-ILTV-gD considering the immune efficacy.Additionally, the recombined virus has the similar immune effects with the commercial ILT vaccine.5.Construction of recombinant fowl pox virus with ChlL-2 and glyeoprotein D of ILTV and its immune efficacyA recombinant fowl pox named as rFPV-IL2-ILTV-gD was constructed.Four groups of 20-day SPF chickens were immunized with sterilized water,commercial ILT vaccine (D-805),recombinant virus rFPV-ILTV-gD and recombinant virus rFPV-IL2-ILTV-gD respectively.Serum of chickens in each group was collected at 7,14,21,28,35,42,49,and 56 day post inoculation,and the antibody to ILT was measured by ELISA.Parts of chickens were challenged with a lethal dose of virulent ILTV at 28 day post inoculation.Results of immune protection test showed that there was no significant difference among the recombinant virus rFPV-ILTV-gD,the recombinant virus rFPV-IL2-ILTV-gD and the commercial vaccine considering the immune efficacy(P＞0.05),and the protection rates of the chickens after the virus challenge were 92%,96%and 96%,whereas that of the control group was 8%.It indicates,therefore,the recombined virus has similar immune potential to that of the commercial ILT vaccine,and can effectively protect chicken from ILTV challenge.