The Bioinformatic Analysis Of NS1, VP2Genes And Prokaryotic Expression Of VP2Gene Of Porcine Parvovirous HB Strain

Porcine parvovirus (Porcine parvovirus, PPV) is one of the mainpathogens causing reproductive disorders, which includes stillbirths,mummifiedfetuses, early embryonic death, and infertility. Gastrointestinal, respiratory andplacenta is the main mode of transmission of porcine parvovirus. The main source ofinfection carriers of porcine parvovirus is sows and boars. Porcine parvovirus andporcine pseudorabies virus (Pseudorabies Virus, PRV), Japanese encephalitis virus(Japanese encephalitisvirus, JEV), porcine circovirus (Porcine circovirus, PCV) andother mixed infections increased its hazards. States herd serological surveys havehigher positive rate, PPV strains also had been isolated in Beijing, Shanghai, Jilin,Heilongjiang, Sichuan, Zhejiang and other places. Current research on porcineparvovirus is focused on the detection methods and the study of their VP1and NS1genes, which is less studied and predicted on their structural and non-structuralprotein. The experimental strain derived from the clinical sample is Carried out thefollowing research:According to NS1PPV gene sequences form NCBI database published, designeda pair of primers using PCR technology to detect the PPV derived from sample. Afterdouble with anti-(penicillin-streptomycin), chloroform, and other handling diseasedmembrane, PPV was incubated in ST cells and blinded passage to appear cytopathicgenerations. Nucleic acid extraction cytotoxic was connected to pMD18-T vector.after amplification sequencing. The Sequence analysis results showed that: PCRamplification of the target band around a360bp, after virus was blinded passage byST cells seven generations, cells appear slight widening net, oval and other cytopathiceffect; sequencing results showed that the nucleic acid of the virus PPV and othernucleic acid sequence homology is between98%-100%.Acquired PPV NS1gene sequence, analysised its nucleic acid composition,restriction sites, amino acid composition, physical and chemical properties,hydrophobicity, transmembrane proteins, signal peptide, secondary and tertiarystructure of the predicted amino acid. The results show, PPV NS1gene consists of1977bp, which encode658amino acids, GC base content37.28%, AT base content 62.72%. Restriction sites contained HpaⅡ andMsp,which are vulnerable to theeffects of CpG methylation.Dpn Ⅱ, Mbo Ⅰ, Alw Ⅰ, Eco0109Ⅰ, PpuMⅠaresusceptible to the influence of other methylation.In20amino acids threonine inside isthe highest proportion of10.5%, the lowest proportion of cysteine share is2.6%,compared with the hydrophilic region hydrophobic regions and more capacity thanthe hydrophilic region of strong hydrophobic regional capacity. The absence of thesignal peptide and the transmembrane protein region in protein coding region, thesecondary structure of α-helical structure representing28.88%, β-sheet structureaccounted for23.25%, representing47.87%random coil, the tertiary structure fromthe three-dimensional structure, the forecast shows that the protein is more stablemodel.The use of the same method obtained PPV VP2gene sequence and analyzed itsnucleic acids, restriction sites, amino acid composition, physical and chemicalproperties of amino acids, hydrophobic transmembrane proteins, signal peptide,secondary and tertiary structures. The results show, PPV VP2gene consists of1740bp,which encode579amino acids, GC base content38.28%, AT base content61.72%.Restriction sites Hpa Ⅱ, Mwo Ⅰ, TspM Ⅰ, AvaⅠ, MspⅠ, BsmF Ⅰ, SmaⅠ,MspA1Ⅰ, vulnerable to theeffects of CpG methylation.BaB Ⅰ, susceptible to theinfluence of other methylation. In20amino acids threonine accounted for the highestproportion of11.1%, the lowest proportion of cysteine share of0.7%, the hydrophilicregions and hydrophobic regions are not distinct. The absence of the signal peptideand the transmembrane protein region in protein coding region, the secondarystructure of α-helical structure representing11.74%, β-sheet structure accounted for22.63%, representing65.63%random coil, the tertiary structure of the protein showedthe lack of stability structural framework.In recent years, porcine parvovirus infection rate is rising.Especially the swinevaccined vaccination still occurred the disease, whether occurrence of thisphenomenon is because of the instability caused VP2protein immunogenicity andantigenicity change. The reason for the preliminary inquiry, according to porcineparvovirus VP2gene sequence, which designed a pair of specific primers to amplifyporcine parvovirus VP2gene major epitope fragment and cloned into the expressionvector pET-28a, transformed into BL21bacterial expression, the expression of theprotein was induced by IPTG, SDS-PAGE electrophoresis experiments confirmed thatlay the foundation for the next step to detect the current prevalence of porcine parvovirus disease.