Development Of Recombinant Fowlpox Viruses Expressing Different Lengths Of Glycoprotein B From Infectious Laryngotracheitis Virus And Their Protective Efficacies

Infectious laryngotracheitis(ILT)is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus(ILTV),which has the characteristics of rapid spread,high mortality,low egg production rates in infected laying hens,and ILT is becoming prevalence in our country.Nowadays,most of vaccines to prevent ILT are modified live attenuated viruses derived from sequential passage in cell cultures or chicken embryos.However,the live attenuated vaccines are at the risk of becomingvirulent again,and some of the attenuated live ILT vaccines have shown side effects such as spreading vaccine virus to nonimmunized animals,finally leading to the outbreak and epidemic of ILT.Therefore,there is an urged need to develop safer and more efficacious ILTV vaccines.The fowlpox virus vector has unique advantages including large capacity for foreign genes expression,efficient immune respons against foreign genes,strict host range,no risk of spreading virus to the environment and an ability to distinguish infected and immunized animals.Therefore,we constructed three transfer vectors expressing full or partial of gB genes of ILTV,obtained six recombinant fowlpox viruses(rFPVs)by transfection with two fowlpox virus(wt-FPV282E4 and wt-FPVLP),and evaluated their immune response.This study laid the foundation for the development of recombinant fowlpox vaccine against ILTV1.Prokaryotic expression of gB gene of ILTV and preparation of polyclonal antibodiesAccording to the nucleotide sequence of gB gene of ILTV published by GeneBank,we used DNAstar software to analyze the hydrophilicity,antigenic index and surface probability of the amino acid sequence.The fragments of 861 bp and 972 bp were selected,which encod the main antigenic domains of gB.The amplified gB genes were cloned into expression vector pET-32a(+)to form recombinant prokaryotic plasmids pET32a-gBa and pET32a-gBb,respectively.The expressed proteins were induced by IPTG.The result of SDS-PAGE showed that gBa and gBb were expressed in form of inclusion body.The antisera against gB proteins were prepared by immunized mice with purified proteins.The results of Western-Blot showed that the antisera were reactive well with ILTV-infected LMH,indicating that the antisera can be detection reagent for gB protein.2.Construction of recombinant fowlpox viruses expressing different lengths of glycoprotein B from ILTVThe two sites of FPV transcription termination signal sequence on gB gene were mutated synonymously using the mutation kit.The full length and partial gB gene of ILTV were cloned into the insertion vector p12LS to form the transfer vectors p12LS-gB,p12LS-gB1 and p12LS-gB2,respectively.Then the vectors were used to transfect into chicken embryo fibroblast(CEF)by Liposome,which were pre-infected with flowpox virus wt-FPVLP or wt-FPV282E4,respectively.The purified rFPVLP-gB,rFPVLP-gB1,rFPVLP-gB2,rFPV282E4-gB,rFPV282E4-gB1 and rFPV282E4-gB2 were obtained by blue-white plague selection.The DNA of recombinant fowlpox viruses were extracted and subjected to PCR for confirming the insertion of gB genes in the chromosomal DNA of FPV.The results of indirect immunofluorescent assay(IFA)and Western-blot indicated that gB genes were expressed correctly in the rFPVs.3.Immunogenicity test of recombinant fowlpox virus expressing different lengths of glycoprotein B of ILTV28-day-old SPF chickens were randomly divided into 8 groups,10 in each group,and immunized with 0.2mL inoculum containing 105 PFU of rFPVLP-gB,rFPVLP-gB2,rFPV282E4-gB,rFPV282E4-gB2,wt-FPVLP,and wt-FPV282E4,respectively.The commercialized rFPV vaccine was set as positive control,and PBS as negative control.These chichens were challenged with ILTV-119 strain(105 EID50/chicken)by laryngeal inoculation on day 21 post-vaccination.The results showed that both the protection indexes induced by rFPVLP-gB and rFPV282E4-gB were 100%,which were higher than that by the commercialized rFPV vaccine(83.3%),and the protection indexes induced by rFPVLP-gB2 and rFPV282E4-gB2 were 33.3%and 16.7%,which were lower than that by the commercialized rFPV vaccine.In summary,rFPVLP-gB or rFPV282E4-gB has the best expression of gB protein and the best immune effect among the rFPVs,and can be used as vaccine candidates for ILTV.