Establishment Of A New Detection Method For PRV And Study On The Immunogenicity Of Its Subunit Vaccines

Pseudorabies(Pseudorabies,PR)caused by Pseudorabies virus(Pseudorabies virus,PRV)is an acute infectious disease characterized by invading the nervous system and reproductive system of animals.In recent years,there have been reports of human infection with PRV,so more and more researchers pay attention to PRV.In order to better prevent and control the disease,this study designed a dual nano-PCR detection method for PRV g B gene and g E gene,which increased the detection rate of the virus.At present,the methods against PRV still focus on prevention.Therefore,in this study,a subunit vaccine was prepared against PRV g D protein and its immunogenicity needed to be further studied The main research contents for these two parts include:1.Development of a dual nano PCR assay for PRV g B and g E genesAccording to the PRV strain published in Gen Bank(accession number:KX451219.1),specific detection primers for PRV were designed.By optimizing the PCR system and conditions,a dual nano PCR method was established for the detection of PRV g B and g E genes.There was no cross-reaction between PRV and common swine origin viruses,indicating that the established method had good specificity.The detectable limits of p MD18-T-PRV-g E and p MD18-T-PRV-g B positive plasmids were 1.6×10~2 copies/?L and 1.96×10~2 copies/?L,and its sensitivity was 100 times higher than that of common PCR.Positive plasmids were tested repeatedly,and the results were all positive,indicating their good reproducibility.Eighty suspected samples from different animals were tested and the concordance rate between the method established in this study and the common PCR assay was 98.8%.The results indicated that the assay established in this study providesd efficient technical support for the detection of the disease with a much smaller sample size.2.Study on immunogenicity of subunit vaccinesIn this study,the eukaryotic expression plasmid(p CAGGS-PRV-g D)was constructed by codon optimization of the extracellular domain of the PRV g D gene,and the recombinant protein was purified.BALB/c mice were immunized subcutaneously three times,and blood samples were collected at 0 d,14 d,28 d,and42 d after immunization,and serum was collected.Mice were sacrificed at 42 d,and lymph node lymphocytes were isolated.Mouse serum Ig G was measured and serum neutralization tests were performed to determine the lymphocyte proliferation rate,proportion of cell subtypes,and cytokines(IFN-?and IL-4).The results showed that the experimental group produced higher level of Ig G antibodies,the neutralizing titer of g D group was 1:132;the CD3~+CD4~+T lymphocytes in g D group increased by37.53%compared with those of PBS group;the CD3~+CD8~+T lymphocytes of g D group increased by 49.95%compared with those of PBS group;the proliferation rate of lymphocytes in g D group was increased by 184.18%compared with that of PBS group by MTT assay;the mean value of cytokine detection IFN-?level was 844.04pg/m L and the mean value of IL-4 level was 174.37 pg/m L in g D group.The values were significantly different compared with the PBS group The test groups in this trial had different degrees of immune effects,and in a comprehensive view,the constructed eukaryotic expressed proteins had better immune effects.This study may lay the foundation for later stage preparation of subunit vaccines for clinical application,but it also needs further investigation.In this study,HEK-293F cell supernatant was used for expression,and the purification was more simple and the effect was better.