The Construction And Immunoreactivity Of A Chimeric Multi-epitope DNA Vaccine Against IBV In Chickens

Infectious bronchitis virus(IBV) is one of the primary causes of respiratory disease in domestic fowl.Multitude serological types of IBV are occurred due to variation,and there is no cross-protection between different serological types.In addition,conventional vaccines against infectious bronchitis applied in the field have respective weakness. Therefore,it is of significance to develop a safety and effective genetically engineering vaccine such as DNA vaccine to control the disease.Based on former achievement of our research group,we analyzed and predicted the structural protein of nephritic IBV SAIBk strain,screened several epitopes from S1,S2 and N protein of IBV,and constructed a chimeric multi-epitope gene by using of the screened epitopes genes.The constructed chimeric gene was used to perform prokaryotic expression and a corresponding ELISA assay was established by using the expressing recombination protein.Moreover,the constructed chimeric multi-epitope gene was subcloned into eukaryotic expression vectors and then prepared DNA vaccines,and the immunoreactivity of the chimeric multi-epitope DNA vaccines were evaluated,as well as coadministration with avian interleukins.The aim of the study was to provide a new candidate target for IBV vaccine development.1.The dominant epitopes of S1,S2,and N protein of IBV SAIBk strain were analyzed on base of computer bioinformatics softwares and reported references,and then seven T-and B-cell epitopes from S1,S2 and N protein(S1:24-150,240-255,290-400,532-537;S2: 1-65;N:1-120,290-410) were selected.The seven multi-epitope minigenes were paiallelled as a single chimeric gene separated from each other with the GA/GP spacers with an unique open reading frame by using Splicing by Overlap Extension(SOEing) and polymerase chain reaction(PCR),and the constructs included a Kozak sequence at the N-terminus and avian specific CpG motif in the end of the chimeric gene.The analysis for the chimeric gene indicated that the coded chimeric protein showed favourable hydrophilicity,flexibility and antigenicity.The study provided foundations for the prokaryotic expression,ELISA method,eukaryotic expression,and research for DNA vaccine of the multi-epitope chimeric gene.2.The constructed chimeric multi-epitope gene of IBV was digested by BamHI and XhoI to generate chimeric gene F,which was subcloned into the prokaryotic expression vector pET-32a(+).The recombinant plasmid was transformed into E.coli Rosetta.The recombinant bacterium was induced by IPTG.The chimeric F protein was expressed fusedly in the form of cytorrhyctes.Western-bloting results showed the expressed recombinant protein occurred specific reaction with positive serum of IBV.The expression conditions of chimeric F protein were optimized with proper inducing conditions of 1 mmol/L IPTG for 3 hours at 30℃temperature.Using the puridied chimeric recombinant protein as coating antigen,the optimal concentration of F protein for coating of plate was 20μg/mL;the dilution of antibody was 1:40;the working concentration of HRP rabbit-anti-chicken IgG was 1:3000;the threshold value of ELISA assay was 0.133. According to the determination of condition of enzyme-linked immunosorbent assay (ELISA),an indirect ELISA based on the chimeric F protein was developed.The study provided new approachs for the development of subunit vaccine and detection of ELISA antibodies for IBV.3.The constructed chimeric multi-epitope gene F of IBV was subcloned into eukaryotic expression vectors pcDNA3.1,pVAX1 and VR1020,respectively.In addition,the avian interleukin-1β,interleukin-2 and interleukin-18 gene were inserted into the plasmid pcDNA3.1,respectively.Identification of the constructed eukaryotic expression plasmids were performed by restriction enzyme digestion and DNA sequencing.The positive recombinant plasmids were transfected into COS-7 cells by lipofectamine,and the expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay(IFA).The results of restriction enzyme digestion and DNA sequencing showed that the eukaryotic expression plasmids were constructed successfully, The corresponding mRNA transcripts were detected by RT-PCR in the COS-7 ceils which were transfected with plasmids pcDNA-F,pVAX1-F,VR1020-F,pcDNA-IL-1β, pcDNA-IL-2 and pcDNA-IL-18,respectively.The expression of chimeric multi-epitope recombinant protein were detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with plasmids pcDNA-F,pVAX1-F and VR1020-F,respectively. The study provided bases for the further development of chimeric multi-epitope DNA vaccine against IBV.4.The eukaryotic recombinant plasmids pcDNA-F,pVAX1-F,VR1020-F,pcDNA-IL-1β, pcDNA-IL-2 and pcDNA-IL-18 used for vaccination were extracted from DH5a,and purified by a Chinese invention patent method which was established previously in our laboratory.The diluted recombinant plasmids(1mg/mL) were encapsulated by liposome(10mg/mL) in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After two weeks,the chickens were boosted by equivalent dosage of DNA vaccine.The percentage of CD4+ and CD8+ T-lymphocytes from peripheral blood and anti-IBV specific antibodies were measured after the booster, respectively.Five weeks after booster,chickens were challenged by virulent IBV strain.In addition,the distribution in different times and possibility of integration into host genomic DNA of plasmid pVAX1-F were detected.The results of percentage of CD4+ and CD8+ T-lymphocytes showed that,the number of CD4+ and CD8+ T-lymphocytes of chickens immunized with pVAX1-F,pcDNA-F and VR1020-F were extremely significantly higher (P＜0.01) than that of PBS and control empty plasmids groups 7-21d post the booster, respectively,but showed no distinguished differences between interclass of themselves, and the number of CD4+ and CD8+ T-lymphocytes of chickens immunized with pcDNA-F+pcDNA-IL-1β,pcDNA-F+pcDNA-IL-2,pcDNA-F+pcDNA-IL-18 were significantly higher(P＜0.05) than that of the single plasmid pcDNA-F inoculated group 7-28d post the booster,respevtively.The results of specific antibodies detection showed that,the antibody titers of chickens immunized with pVAX1-F,pcDNA-F and VR1020-F were extremely significantly higher(P＜0.01) than that of PBS and control empty plasmids groups 7-28d post the booster,respevtively,but showed no noticeable difference between interclass of themselves,and the antibody titers of chickens co-administrated with pcDNA-F and pcDNA-IL-1β,pcDNA-IL-2,pcDNA-IL-18 were significantly higher (P＜0.05) than that of the pcDNA-F alone group 14-28d post the booster,respevtively.The protection rate of pVAX1-F,pcDNA-F and VR1020-F group were 80%,80%and 75%. The protection rates of groups inoculated with pcDNA-F+pcDNA-IL-1β, pcDNA-F+pcDNA-IL-2 and pcDNA-F+pcDNA-IL-18 were 85%,90%and 85%, respectively,which indicated that IL-2 showed better immune enhancement contribution than IL-1βand IL-18.The tissue section indicated that the kidneys of chickens protected by DNA vaccines were normal and physical fitness,without pathological changes.The distribution and integration detection of plasmid pVAX1-F showed that the plasmid was detected in blood and all the other checked tissues 24h post inoculation and last 90d in some tissues at least,and there was no detective phenomenon of integration during the period of experiment.The study provided a new way to enhance the immunogenicity of IBV DNA vaccine and reduce costs.