Research Of Bicistronic DNA Vaccine And Molecular Characteristic Of MRNA3 Of Avain Infectious Bronchits Isolated In China

1.Chicken interleukin-2 and interleukin-18 gene was amplified by polymerase chain reaction (PCR)from pDNAIL2 and pDNAIL18 plasmids,then ligated into pMD18-T vector.The pMD18-IL2,pMD18-IL18,pIBVS1,pIBVM and pIBVN recombinant plasmids which contain the IL-2,IL-18,S1,M and N gene were sequenced.The sequence analysis results showed that the open reading frame of IL-2,IL-18,S1,M and N gene was 432bp,536bp,1671bp,678bp and 1230bp,respectively.The correctness of target gene provides a reliable material for further experiments.In this study,we have constructed a bicistronic plasmid pIRES-EGFP/Red by using pDsRed-Express-N1 vector for the skeleton.This plamid contained two fluorescent protein genes which were inserted into the multiple cloning sites(MCS)located on either side of the internal ribosome entry site.Then the pIRES-EGFP/Red was transfected into Vero cells,green fluorescence and red fluorescence could be observed in transfected cells by using fluorescence microscope,indicating that the modified expression vector could correctly express two heterologous proteins at the same time.It is the first time to construct a bicistronic expression vector pIRES-EGFP/Red which introduced kanamycin resistance as a selection marker.Compared to the commercialization vector pIRES,it has a higher bio-safety.It provides a new research tool for the development of bivalent DNA vaccines of various pathogenic microorganism.2.The S1,M,N gene of avian infectious bronchitis virus and interleukin-2,interleukin-18 gene were inserted into the pIRES-EGFP/Red plasmid,respectively,and generated the bicistronic plasmids pIRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2,pIRES-S1/IL18,pIRES-M/IL18 and pIRES-N/IL18.Identification of the bicistronic plasmids was performed by restriction enzyme digestion and DNA sequencing.The plasmids were transfected into Vero cells by lipofectamine, and the expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay.The results of restriction enzyme digestion and DNA sequencing showed that the bicistronic expression plasmids were constructed successfully.The mRNA transcription of S1,M,N gene which were in the upstream of IRES and IL-2,IL-18 gene which were in the downstream of IRES,could be detected by RT-PCR in the Vero cells which were transfected with plasmids.The expression of S1,M and N protein could be detected by indirect immunofluorescent assay in the Vero cells which were transfected with plasmids.In my knowledge there was no reports about the construction of co-expression vectors of IBV S1,M,N andIL-2,IL-18.before in the world.This study might provide materials for the further development of DNA vaccine against IBV.3.In this study,the IRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2,pIRES-S1/IL18, pIRES-M/IL18,pIRES-N/IL18,pIRES-S1,pIRES-M,pIRES-N,pIRES-IL2 and pIRES-IL18 plasmids used for vaccination were extracted from JM109,and purified by a Chinese invention patent method which was established previously in our lab.The diluted plasmids(1mg/mL)were encapsulated by liposome(10mg/mL)in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After three weeks later,the chickens were boosted by DNA vaccine.Two weeks after boosting,chickens were challenged by virulent IBV strain.Fluorescence activated cell sorter and indirect ELISA methods were used to detect the number of CD3+, CD4+and CDS+ T lymphoctyes and anti-IBV antibody.The results of specific antibody show that there was a significant difference(P＜0.05)in ELISA antibody levels elicited by pIRES-S1/IL2, pIRES-M/IL2,pIRES-N/IL2 than that of pIRES-S1,pIRES-M,pIRES-N DNA vacccines since the 14th-day after first inoculation.There was a significant difference(P＜0.05)in ELISA antibody levels elicited by pIRES-M/IL18,pIRES-N/IL18 than that of pIRES-M,pIRES-N DNA vacccines since the 14th-day after first inoculation.ELISA antibody levels elicited by pIRES-S1/IL2, pIRES-S1/IL18 was higher than that of pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18,but there was no significant difference(P＞0.05).The percentage of CD3+,CD4+,CD8+ T-lymphocytes from chickens immunized with pIRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2 was significantly higher(P＜0.05)than pIRES-S1,pIRES-M,pIRES-N at the 7-28d after the first vaccination.The percentage of CD3+,CD4+,CD8+ T-lymphocytes significantly higher(P＜0.05)was observed from chickens immunized with pIRES-S1/IL18,pIRES-M/IL18,pIRES-N/IL18 than pIRES-S1, pIRES-M,pIRES-N at the 14-28d after the first vaccination.The percentage of CD3+,CD4+, CD8+ T-lymphocytes was higher in the pIRES-S1/IL2,pIRES-S1/IL18 than in the pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18 at the 14-28d after the first vaccination,but there was no significant difference(P＞0.05).The protection rate of bicistronic vaccines was between 65%and 85%.The protection rate of pIRES-S1/IL2,pIRES-S1/IL18 vaccine groups which was 85%,75%,was higher than pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18 vaccine groups which were 70%,60%.The studies on immunogenicity of IBV bicistronic DNA vaccines co-expression IBV S1,M,N and IL-2,IL-18 had no reports at home and abroad.It can provide a new way to enhance the IBV DNA vaccine immunogenicity and reduce costs.4.IBV E gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR). The PCR product of E gene was digested with restriction endonuclease and ligated into similarly digested pIRES-M/Red plasmid,then generated the bicistronic plasmid pIRES-M/E.Identification of the recombinant plasmid pIRES-M/E was performed by double enzymes digestion and DNA sequencing.The plasmid was transfected into Vero cells by lipofectamine,the mRNA transcripts of M and E gene were detected by RT-PCR.Restriction enzyme digestion and DNA sequencing showed that the bicistronic expression plasmid was constructed successfully,and the mRNA transcripts of M and E gene could be detected by RT-PCR in the Veto cells after transferred with pIRES-M/E plasmid,pIRES-M/E plasmid used for vaccination was extracted from JM109,and purified by a Chinese invention patent method which was established previously in our lab.The diluted plasmids(1mg/mL)were encapsulated by liposome(10mg/mL)in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After three weeks later,the chickens were boosted by DNA vaccine.Two weeks after boosting,chickens were challenged by virulent IBV strain.Fluorescence activated cell sorter and indirect ELISA methods were used to detect the number of CD3+,CD4+and CD8+ T lymphoctyes and anti-IBV antibody.The results of specific antibody and the percentage of CD3+,CD4+,CD8+ T lymphocytes in the pIRES-M/E group was higher than in the pIRES-M,but there was no significant difference(P＞0.05).The studies on immunogenicity of IBV bicistronic DNA vaccine co-expression IBV M and E gene had no reports at home and abroad.So it is the first time to explore the synergetic immune response of IBV M and E gene by co-expression.5.The 3a3b3c gene was amplified by RT-PCR from avian infectious bronchitis virus,and cloned into the pMD18-T vector.Then the molecular characters of 3a3b3c DNA fragments were analyzed after sequencing.The prediction of RNA secondary structure of 3a3b sequence was carried out,and compared with the RNA secondary structure of encephalomyocarditis virus internal ribosome entry site.Further the bicistronic p3a3b-EGFP/Red was constructed for test the function of internal ribosome entry site of 3a3b sequence.The sequence analysis results showed that the length of 3a3b3c DNA fragment was 734bp.3a3b3c fragment which include three complete ORF of 3a,3b,3c(E)gene was 170bp,190bp,429bp in length.When compared with the corresponding sequence of 58 IBV strains published on the GenBank,the homologous analysis of 3a3b3c nucleotide sequence showed that the homology rate is between 78.2%and 96.1%.SAIBk strains has the highest rates of identity with CK/CH/LGD/04II and CK/CH/LSC/99I strains(96.1%and 95.9%),and has the lowest rate of identity with AJ310640 (78.2%).Phylogenetic analysis showed that SAIBk strain has the recent relationship with CK/CH/LGD/04II and CK/CH/LSC/99I strains which were isolated in China.This study indicated that the molecular characteristics of 3a363c sequence can be used to study the genetic evolution of IBV.The results of RNA secondary structure analysis showed that the 3a3b has the same stem-loop structure as encephalomyocarditis virus IRES sequence.Free energy values of RNA secondary structure of 3a3b and encephalomyocarditis virus IRES sequences was -45.5 kkal/mol and -129.6 kkal/mol,respectively,indicate that encephalomyocarditis virus IRES has more stable secondary structure than 3a3b's.After transfer the p3a3b-EGFP/Red plasmid to the Vero cells,the green fluorescent protein could be detected but not the red fluorescent protein,it showed that the internal ribosomal functions of 3a3b is too weak to detect by fluorescent gene expression methods. This is the first time for us to evaluate the internal ribosome entry site function of 3a3b,it can be used to study the function of IBV genome and screening a new IRES sequence for the development of vaccine vector.