Construction And Biological Activity Study Of The Recombinant Marek's Virus Expressing Reticuloendotheliosis Virus Env Gene

Reticuloendotheliosis(RE)and Marek's disease(MD)were two important immunosuppressive diseases that threatened the development of the poultry industry in China.The previous studies that detected RE in chickens in our province and thus in the whole country showed that the RE infections has increasingly occurred among chickens.However,there were no effective vaccine for now to prevent and control RE(Su,et al).constructed the meq(Oncogene of MDV)-deleted vaccine SC9-1 that could provide complete immunoprotection for immunized chickens,with the 100% protection rate which was significantly higher than that of the MD commercial vaccine CVI988/Rispens(The protection rate was 80-90%).In this study,recombinant MDV with expressing the REV(Ret iculoendotheliosi s virus)env gene was constructed using SC9-1 while its biological activity was studied.1.The recombinant MDV SC9-1 which was capable of expressing env gene was constructed and its pathogenicity and immunoprotective effect were studied.Using the DNA of REV SNV strain as the template,the env gene was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1(+).Using the vector as the template,the entire genomes of the REV env eukaryotic expression cassette was amplified by PCR.Expression cassettes and kanamycin-resistance genes were cloned into the pUC18vector;pUC18-env-kana was used as the template to amplify the env and kanamycin resistance genes using primers containing homologous arms on both sides of the meq gene locus of the MDV SC9-1 strain.The nuclear expression cassette and the kanamycin resistance gene were inserted into the meq site of SC9-1 using homologous recombination and positively selected.Finally,arabinose was used to induce the expression of the flp recombinase and the kanamycin resistance gene was knocked out.The positive recombinant plasmid was named SC9-env BAC.Positive recombinant plasmid SC9-env BAC was selected and transfected into CEF cells to rescue the recombinant MDV.MDV monoclonal antibody H19 and REV monoclonal antibody 11B118 were detected positive by indirect immunofluorescence assay and named SC9-env.Furthermore,the level of replication on CEF cells was analyzed and the animal experiments were conducted to evaluate the pathogenicity of SC9-env against SPF chickens and the protection effect of SC9-env on SPF chickensinfected with MDV and REV.Challenging with SC9-env on SPF chickens did not cause death and tumorigenesis;SC9-env provided immune protection on 92% of the SPF chickens infected with MDV rMd5,which was not significantly different from that of SC9-1;SC9-env could reduce the weight loss and the decrease of antibody levels in inactivated vaccines induced by SNV infections in SPF chickens.2.The use of homologous recombination technology to construct different recombinant MDV expressing REV env and gp90Using the DNA of REV SNV strain as the template,the env gene and gp90 gene were amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1;then the entire eukaryotic expression cassettes of REV env and gp90 genes were amplified by PCR using the pCAGGS vector as the template.Furthermore,env and gp90 genes were cloned into the pUC18 vector with kanamycin-resistance gene respectively,and the pUC18-env-kana was used as a template to PCR-amplify the env eukaryotic expression cassette and kanamycinresistance genes using primers containing homologous arms on both sides of US10 gene locus of the MDV SC9-1 strain.PCR amplification of the REV gp90 eukaryotic expression cassette and kanamycin-resistance genes were performed with the template of pUC18-gp90-kana and the primers containing homologous arms on both sides of US10 gene locus of MDV SC9-1 strain,then the eukaryotic expression cassette and kanamycin resistance gene were inserted into the US10 locus of SC9-1 using homologous recombination and screened positively.PCR amplification of the REV gp90 eukaryotic expression cassette and kanamycin-resistance genes were performed with the template of pUC18-gp90-kana and the primers containing homologous arms on both sides of meq gene locus of MDV SC9-1 strain,then the eukaryotic expression cassette and kanamycin resistance gene were inserted into the meq locus of SC9-1 using homologous recombination and screened positively.Finally,arabinose was used to induce the expression of flp recombinase,the kanamycin resistance gene was knocked out,and the positive recombinant plasmid was named as SC9-gp90-2 BAC,SC9-env-3 BAC and SC9-gp90-4 BAC.SC9-gp90-2 BAC,SC9-env-3 BAC and SC9-gp90-4 BAC plasmids were extracted and transfected into CEF cells to rescue five MDV recombinants.The rescued recombinants were detected as positive by the indirect immunofluorescence test using MDV monoclonal antibody H19 and REV monoclonal antibody 11B118 and named as SC9-gp90-2,SC9-env-3 and SC9-gp90-4.This study laid the foundation for the further construction of recombinant MDV expressing the env gene of the reticuloendotheliovirus(REV)and evaluating its immunoprotective effect.The results showed that the recombinantMDV SC9-env and SC9-env-3 constructed by homologous recombination technology can stably express the REV env protein,and SC9-gp90-2 and SC9-gp90-4 can stably express the REV gp90 protein.The level of replication of recombinant MDV SC9-env on CEF cells was similar to that of parental virus SC9-1.3.Comparison of REV env expression on CEF cells by eukaryotic recombinant plasmids pcDNA-env,pCAGGS-env and gB-env with different promotersThe expression effects of the recombinant plasmids pcDNA-env,pCAGGS-env and gBenv with different promoters on the cells were compared and the results showed that the activity of ?-actin promoter was stronger than that of the CMV promoter,while the activity of CMV promoter was stronger than the activity of gB promoter.Chickens were immunized with recombinant gp90 protein could produce specific antibodies and no adverse reactions were found,which indicated that recombinant subunit vaccine was safe and reliable for chicks and breeders.The results showed that the gp90 protein has a good antigenicity,RE subunit vaccine could induce high-level antibody responses,and has good immune effects on chicks and breeders.The present study constructed a MDV recombinant expressing the REV env and gp90 genes and studied its biological activity.In this study,the serotype I MDV meq-deleted strain SC9-1 was constructed as a vector.The homologous recombination technology was used to insert the REV env and gp90 genes into the SC9-1 to construct a MDV recombinant and the exogenous gene can be stably expressed with the SC9-1 vector.The recombinant MDV SC9-env has shown no pathogenicity to SPF chickens while provided certain immune protective effect for SPF chickens infected with MDV and REV.This study provides ideas for the construction of other MDV recombinants as well as foundation for the prevention and control of REV infections.