Preparation Of Duck Tembusu Virus E Protein And NS1 Protein Subunit Vaccines

Duck Tembusu virus disease is a contagious disease caused by Tembusu virus(TMUV)infection in duck populations,which is mainly characterized by ovarian bleeding and decreased egg production rate in egg-laying ducks.At present,there are whole virus inactivated vaccines and subunit vaccines in clinical application in China,and the immune effect of vaccines in different duck flocks is quite different.In order to supplement the results of the current TMUV vaccine,this study analyzed the domain I,Ⅱ and Ⅲ sequences of the main virulence gene E gene of TMUV and the sequence of the non-structural protein NS1 gene,and the prokaryotic expression of the optimized sequence of E gene and NS1 gene,and then isolated,purified and identified the expressed protein.Vaccines were prepared with the obtained protein of interest and animal immunoprotection tests were performed.The research content is mainly divided into the following three aspects:1.Construction of prokaryotic expression plasmids for duck tembusuvirus E protein and NS1 protein genes.In this study,based on the duck Tembusu virus isolated and preserved in the laboratory,the gene sequence information of duck Tembusu virus was obtained by sequencing,from which the Domain I,Ⅱ and Ⅲ sequences of the E protein gene and the NS1 protein gene sequence were screened,and the E protein and NS1 protein gene primers were designed,and the E gene fragment and NS1 gene fragment of DTMUV were successfully obtained by synthesizing the primers and amplifying the target gene fragment,and then the obtained E gene fragment was XhoI and BamH I The obtained NS1 gene fragment was digested with BamH I.and Hind Ⅲ.restriction enzymes,and then the digested gene fragment of the target gene was electrophoresis,and the agarose gel block containing the gene fragment of interest was recovered at the end of electrophoresis to obtain the gene fragment of interest.The acquired gene fragments of interest were ligated with the vector pET28-a(+)recovered by the same enzyme digestion,and then the ligation products were converted into BL21-expressing bacteria by heat shock,and the transformed bacterial solution was evenly coated on solid LB plates,and single colonies with good growth were selected for liquid expansion culture after overnight culture.The cultured bacterial solution was PCR amplified.PCR amplification-positive bacterial plasmids were extracted,enzyme digestion was used for identification,and PCR amplification products were sequenced and identified.The results showed that fragments of about 1353 bp and4700 bp,1056 bp and 4700 bp were obtained after digestion of the two plasmids,which were consistent with the expected results,and the PCR sequencing results were correct,proving that the plasmids pET-28a-DTMUV-E and pET-28a-DTMUV-NS1 had been successfully constructed.2.Prokaryotic expression and purification of duck tembusuvirus E protein and NS1 protein.Prokaryotic recombinant plasmids pET-28a-DTMUV-E and pET-28a-DTMUV-NS1 were converted to E.coli.BL21,IPTG was used to induce expression,and the optimal induction concentration and induction time of IPTG were determined.The experimental results showed that after 6 h induction with IPTG at a concentration of 1 mmol/L at 37℃,the two proteins were successfully expressed as inclusion body proteins,and protein bands of about 49 KDa and 39 KDa in size could be obtained by SDS-PAGE electrophoresis,respectively,and the band sizes were consistent with expectations.The inclusion body proteins were separated and purified by nickel column affinity chromatography,and the concentrations of E protein and NS1 protein were 1.5 mg/mL and 1.7 mg/mL,respectively,by BCA protein quantification.After the purification of the protein,the Western Blotting assay was performed,and two clear and specific bands of interest were exposed to exposure.3.Preparation and immune effect of duck tembusu virus E protein and NS1 protein subunit vaccine.The purified recombinant E protein and NS1 protein were diluted to 1 mg/mL,and then the E protein,NS1 protein,E protein and NS1 protein(1:1 mixed)were mixed with the vaccine adjuvant ISA71 VG water-in-oil(W/O)at a ratio of 3:7,and the vaccine was prepared by stirring and emulsification to test its safety and stability.The test ducklings were divided into 6 groups,namely E protein immune group(I.group),NS1 protein immune group(Ⅱ.group),E+NS1 protein immune group(1:1mixed)(Ⅲ.group),duck tembusu virus inactivated vaccine immunization group(IV.group),duck tembusu virus challenge group(V.group),normal control group(VI.group).Recombinant protein emulsified vaccine and inactivated vaccine were injected intramuscularly into four immune groups at the age of 14 days and 28 days,starting from the first immunization,blood was collected from the duck veins of each experimental group every 7 days,and the antibody titer in serum after vaccine immunization was detected by ELISA method.When the ducks were 42 days old,10 ducklings were randomly selected in each group for challenge test,the mental state of the duck group after the challenge was observed,the spleen was collected to measure the change of liver and spleen index,pathological sections were prepared,and the remaining 10 ducklings continued to collect blood to detect antibody titer.The test results showed that the E protein immune group,NS1 protein immune group,E+NS1protein immune group and inactivated vaccine immune group could induce ducklings to produce higher antibodies,among which the highest antibody level was the E+NS1protein immune group,followed by E protein immune group,NS1 protein immune group and inactivated vaccine immune group.After the DTMUV challenge test on the ducklings,the immunized ducklings survived well and had no obvious clinical symptoms,while the unvaccinated ducklings successively developed mental depression and death after the challenge.There was no obvious change in the hepatospleen index of the young ducks after immunization and the control group,and the shape of the liver and spleen was relatively normal,and there were no obvious pathological changes.However,the ducklings in the unvaccinated challenge group had different degrees of swelling and bleeding in the liver and spleen after the challenge,and the spleen sections showed obvious lesion characteristics.In summary,the E protein and NS1 protein vaccines prepared in this experiment can induce higher DTMUV antibody levels in ducklings,which has a good protective effect,and the mixed immune strategy of E protein and NS1 protein can cause higher antibody levels in ducklings,which provides more ideas and options for subsequent research on DTMUV vaccines.