Construction Of Eukaryotic Expression Vector VR1020 And PVAX1 Carrying S1, M, And N Gene Of Avian Infectious Bronchitis Virus And Study On Their Immunogenicity

Three pair primers were designed according to the sequence of avian infectious bronchitis virus strains SAIB_K which was published on GenBank(GenBank Entry Number: DQ288927).The S1,M and N gene of IBV strains SAIB_K which was isolated from chicken in Sichuan Province,P.R.C.were amplified from the SAIBk by reverse transcription polymerase chain reaction(RT-PCR),and the site-directed mutagenesis of N gene was carried out by gene splicing by overlap extension PCR(SOE-PCR).Then these genes were inserted respectively into the multiple cloning sites(MCS) of the eukaryotic expression vector VR1020.Restriction enzyme analysis and identification of PCR showed that these eukaryatic recombinant expression plasmids(VR1020-S1,VR1020-M and VR1020-N) were constructed successfully.These eukaryotic expression plasmids were ctransfected into mammalian cell COS7 and their transcriptions(mRNA) were assayed by RT-PCR. The expression products of them were analyzed by fluorescence microscope using indirect immunofluorescent assay(IFA).The S1,M and N gene were amplified from the mRNA of recombinant plasmids in COS7 cells by RT-PCR and green fluorescence in COS7 cells was observed in the group which was ctransfected respectively by VR1020-S1,VR1020-M and VR1020-N.The results confirm that these recombinant plasmids can be transcribed and translated correttly in COS7 cells.Primers were designed and the S1,M and N gene were amplified by RT-PCR from SAIBk,and these genes were inserted into the eukaryotic expression vector pVAX1,then three recombinant plasmids(pVAX1-S1,pVAX1-M and pVAX1-N) were constructed successfully.At the time,the genes encoding S1,M and N were transcribed and translated correctly as shown by RT-PCR and IFA. The eukaryotic expression recombinant plasmids(VR1020-S1,VR1020-M, VR1020-N) and(pVAX1-S1,pVAX1-M and pVAX1-N) were mixed respectively at the ratio of(1:1:1).The two ternary DNA mixture vaccines combining with liposome at the ratio of(1:10) as adjuvant were injected into health chickens.The results indicated that the titers of peculiarity antibody of the two ternary DNA mixture vaccines combining with liposome were higher than other control experiment groups and the former was significant higher than the latter(P＜0.05).However,the percentage of CO₄+ and CD₈+ T lymphocyte of the latter was higher than the former(P＜0.05).The protection rate of (VR1020-S1+VR1020-M+VR1020-N+liposome) group was 72%(13/18) and the protection rate of(pVAX1-S1+pVAX1-M+pVAX1-N+liposome) group was 78% (14/18).At present,there is no report about that DNA vaccine which carried S1,M,and N gene of SAIB_K strains was constructed by eukaryotic expression vectorVR1020 or pVAX1. The feasibility which we use VR1020 and pVAX1 as the vectors of avian DNA vaccine was substantiated,however the former was rather apt to stimulate humeral immune response,but on the other hand the latter was rather apt to stimulate cellular immune responses.This study provides some basic material and scientific gist for the further development of DNA vaccine against IBV.