Detection Method Establishment And Preliminary Construction Of Nucleic Acid Vaccine Of Porcine Astrovirus Type 4

Astrovirus is an RNA virus with many hosts.The common symptoms are apart from diarrhea and also neurological symptoms,respiratory symptoms,hepatitis,nephritis,intussusception.Multiple studies proved that astrovirus have become a potential zoonotic pathogen which is prone to cross-species transmission and genetic recombination.Porcine astrovirus(PAstV)is divided into 5 genotypes.Although the genotypes prevailing in different regions are different,they all have a high infection rate,and there are multiple genotypes co-infected.However,there are still few studies on the detection technology and prevention methods of porcine astrovirus.In this study,homology analysis and phylogenetic tree analysis were performed for a porcine astrovirus strain in Hebei,and the SYBR Green I fluorescence quantitative PCR method and indirect ELISA detection method for detecting porcine astrovirus 4 were established.A preliminary investigation was conducted on the genetic structure and prevalence in Hebei.This study preliminarily constructed a eukaryotic expression vector used as a nucleic acid vaccine and conducted immunogenicity studies,which laid the foundation for the future research work of porcine astrovirus vaccine.In this study,porcine astrovirus was detected from swine diarrhea samples in Hebei province,and named PAstV4 CH-HB-SJZ.Subsequently,primers were designed to amplify the gene sequence and conducted evolutionary tree analysis and homology analysis.In the experiment,a 6733 nt sequence was successfully amplified,including the whole viral CDSs fragments and most of the 5'UTR,3'UTR,and Poly A tails.Nucleotide homology analysis showed that the reference sequence homology between CH-HB-SJZ strain and 25 strains of porcine astrovirus was between 41.7%?80.5%,the highest homology with CH-HB-SJZ strain is porcine astrovirus 4(73.1%?80.5%).And the homology with other genotypes of porcine astrovirus is below 50%.The phylogenetic tree of 32 strains of different species shows that the CH-HB-SJZ strain is in the same branch as the other 8 porcine astrovirus 4 strains,of which the closest relationship is with the WBAstV-1 strain.The pET32a-nsp prokaryotic expression recombinant vector was constructed,and the nsplab protein with a size of about 37.3 kDa was obtained by induction and purification.An indirect ELISA method for detecting IgG antibodies was successfully established using PAstV4 nsplab protein as antigen coating.After optimizing the ELISA method,the optimal protein coating concentration is 1?ig/mL.The blocking solution is 5%skim milk.The primary and secondary antibody dilutions are 1:500 and 1:50000,respectively,and the incubation time is 1 h.There was no cross-reaction among CSFV,PRRSV,PRV,PEDV and PDCoV.The coefficient of variation of the intragroup repeatability test was<10%.The cut-off value of was 0.323 calculated from 30 PAstV4 negative sera.300 sera sample collected from Hebei province in 2018-2019 were tested and a positive diagnosis of 14%(42/300).The above results show that the indirect ELISA method established in this study can be used as a detection method for monitoring clinical antibodies of porcine astrovirus.A pair of primers was designed based on the relatively conserved ORFla gene of porcine astrovirus 4,and then the real-time quantitative PCR was established for detecting porcine astrovirus 4.The results show that the minimum detection limit of this method is 50.3 copies/?L,which has good sensitivity.No cross-reaction was detected to CSFV,PRRSV,PRV,PEDV and PDCoV,which has good specificity.The coefficient of variation between groups is less than 2%,proving that the repeatability is good.Clinical test results showed that the positive rate of porcine astrovirus 4 was 18.6%(8/43).In the study,fluorescent quantitative PCR detection method for porcine astrovirus was established,which providing a new technical means for clinical detection of porcine astrovirus.In this study,the eukaryotic expression vector pcDNA3.1-ZH was successfully constructed.The mice were immunized three times with the recombinant vector and the IgG antibodies in the serum were tested by specific ELISA.The results showed that the antibody content in the serum of mice injected with pcDNA3.1-ZH group was significantly higher than that of the control group,and the antibody level rise with the increase of the number of immunizations,which proved that the recombinant vector can stimulate mice to produce specific antibodies.