Genome Sequencing And Prokaryotic Expression Of Major Immunogen Genes Of Atypical Porcine Pestivirus

Congenital Tremors(CT),also known as contagious congenital tremor,"piglet shivering disease" is a contagious disease characterized by the occurrence of systemic or local muscle paroxysmal contracture in newborn piglets.In the clinical reports,some of the piglets are infected by whole group,some of them are infected by part of group,and even the symptoms can be extended to the nursery pigs and the pre-fertilizer pigs,which seriously affect the growth and survival rate of the pigs,causing greater economic losses to the pig industry.Atypical Porcine Pestivirus(APPV)is a newly discovered prion member that causes CT.But up to now,no reports of successful separation by cells of APPV have been reported,and its biological characteristics,rapid detection methods and prevention and control are still the focus of this research.In this study,the rapid detection method of APPV,genome sequence,virus isolation and protein expression of major immunogenic genes were studied to lay the foundation for further research and prevention of APPV and CT.1.Establishment and application of APPV RT-PCR detection methodA pair of primers were designed according to the APPV genome sequence in GenBank as a reference sequence.RNA was used as a template extracted from clinically suspected CT pigs.The RT-PCR method for detecting APPV was established by optimizing conditions and sequence determination.The results showed that the fragments identical to the expected fragment size were amplified from the lymph nodes of CT dead pigs,and the nucleotide sequence similarity between the nucleotide sequence of the amplified fragment and the corresponding fragment of APPV in GenBank was above 89%,determined by sequence analysis and alignment analysis.The established RT-PCR method could only amplify the APPV fragment,while the other several common viruses were negative,the results of the three repeated tests were basically the same,and the lowest detectable cDNA concentration is 184.11 ng/?L.The detection of the clinically sent samples,the detection rate of suspected CT cases reached 100%.The results showed that the established RT-PCR method has good specificity,reproducibility and sensitivity,and can be used for rapid detection of CT caused by clinical suspected APPV,as well as the virus identification and molecular epidemiological investigation.2.Sequencing and analysis of the whole genome of one strain of APPV and sequence analysis of part of the second strain of APPVUsing APPV positive disease material as the sample to be tested,according to the APPV genome sequence in GenBank,7 pairs of primers were designed,RT-PCR,sequencing and splicing were conducted,and the phylogenetic tree was drawn and the similarity analysis was performed by MEGA7.0 software and DNA Star software.Theresults showed that 1 of these samples were amplified into 7 gene fragments,and 1 APPV genome sequence was obtained by splicing,named JX-JM01-2018A01(GenBank accession number: MG792803.1),and only A fragment of approximately 8000 nt was amplified in the other one case,and designated as JX-02,containing the complete major immunogen E0 and E2 genes,and the prion virus non-conserved NS2-3 gene sequence.The whole genome of JX-JM01-2018A01 is 11.5 kb in length,which is in the same evolutionary branch as APPV in GenBank,while other animal prions such as BVDV and BDV are in another evolutionary branch;the sequence homology of APPV reference genome in GenBank is 82.5%.~93.2%;the nucleotide sequence of E0,E2 and NS2-3 of JX-JM01-2018A01 and JX-02 and the corresponding sequence of APPV of GenBank are:82.5%~93.2%,82.3%~93.1 %,80.3%~92.9%;the amino acid sequence homology is estimated to be: 38.4%~51.6%,33.0%~37.2%,36.0%~39.0%.The phylogenetic tree analysis of E0,E2 and NS2-3 showed that JX-JM01-2018A01 and JX-02 are located in the same evolutionary branch with close evolutionary distance and located in different evolutionary branches from other animal prions such as BVDV and BDV.3.Preliminary separation and identification of APPVThe samples tested as strong positive were inoculated into PK-15 cells and IBRS-2cells,and the primary cells of APPV-positive newborn piglets were prepared and inoculated with the treated material suspension.The results showed that the toxic PK-15 cells were blindly transmitted for 20 generations,and only the F1 and F6 cell fluids had detected the nucleotide fragments with the same size as the target fragments.Sequence sequencing results also showed that the fragment was APPV,and other cell generations were negative;IBRS-2 cells were blindly transmitted for 10 generations and were tested as negative.The primary cells of APPV-positive newborn piglets were cultured for 7 days in vitro,and APPV test was conducted as negative.After APPV cultured,the cells were cultured and passaged for 3 times,and APPV is negative.4.Prokaryotic expression of E0 and E2 proteins of APPV immunoprotective geneAccording to the complete genome sequence of JX-JM01-2018A01,primers were designed to amplify the complete major protective antigen genes E0 and E2,and the recombinant prokaryotic expression vectors pET-E0,pET-E2,pGEX-E0 and pGEX-E2 were constructed and optimized.The expression results showed that it could not express the target protein product.The E0 and E2 gene sequences were codon-optimized,and the recombinant expression vectors pET-E0 y,pET-E2 y,and pGEX-E0 y were constructed.The induction conditions was optimized and SDS-PAGE assays was conduct,which found that it could express proteins with the same size as the target;The mouse was immunized with recombinant protein GST-E0 y,and the recombinant protein HIS-E0 y was used as the detection antigen.After the detection,the immunized mice could produce high levels ofantibodies.