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Objectives1Study the variation and evolutionary characteristics of measles F gene in Zhejiang provincefrom1999to2010.2Apply the bioinformatics software to do prediction and screening on measles epidemicstrains and vaccine strains of F gene epitope, and select of two different antigen epitopepolypeptide for synthesis; study the original effect of F gene epitopes on the amino acid variation.MethodsSelect measles epidemic strains representing11individual plant from Zhejiang ProvincialCenter for Disease Control and prevention that was separated and preserved in1999-2010measlesoutbreaks, use the reverse transcriptase polymerase chain reaction amplification of F gene, conductsequencing, compare and analyze our current use of measles vaccine strain Shanghai191and otherrelated strains. Use the bioinformatics software for the epidemic strains and vaccine strains of Fgene epitope prediction, synthetic polypeptide. Use indirect ELISA method confirmed theimmunogenic polypeptides, and use the Balb/c mice of immune polypeptides, for preparation ofimmune serum. Use a cross-ELISA assay for detection of antibody titers, then for the calculationof antigens.Results1Variation analysis of measles epidemic strain on F gene in Zhejiang province(1) Analysis of variation on the nucleotide and amino acid: by comparing11strains ofZhejiang epidemic and vaccine strains to191nucleotide, the largest difference of the epidemicstrain is strains of2010, of which the difference rate is5.17%; amino acid difference rate wereepidemic strains of years2005,2007,2009,2010, with the variation rate of3.44%between vaccinestrains in the F gene.(2) Amino acid mutation analysis: take vaccine strains of Shanghai191for reference strains,there are11amino acids common variants about strain F gene of measles prevalence in Zhejiangprovince. The cleavage site on108th,110th amino acids are variant. While three known importantintegration of functional sites about the F gene,32th,64th and195th amino acids have not changed. (3) Analysis of F gene evolution: The largest genetic distance lies in vaccine strains ofShanghai191belonging to the A with the Zhejiang genotype strains belonging to the H genotype,reaching0.046, much higher than the genetic distance between A genotype and B, D genotype. Inthe F gene phylogenetic tree, A genotype and H genotype were also located in different branches,existing the distance. From the entropy analysis of F gene, there are3easy mutation in F gene,respectively1st,152and174, in which the maximum Entropy is152amino acids, the remainingsites are relatively conservative. The F gene was not under obvious positive selection pressure, dn/ds=0.1467<1. Analysis of evolutionary rates obtained: the trend is that the F gene nucleotideevolutionary rate is increasing year by year, which has now reached5.73%.2Epitope prediction and analysis of its antigenicity(1) Epitope prediction: there are five common epitope of vaccine and the epidemic strains,respectively25~38,102~116,266~280,420~449,478~485amino acids. Conduct polypeptidesynthesis from420th to449th amino acid.(2) Antigenic analysis: two peptides with IgG positive serum binding capacity can be of goodimmunogenicity. By crossing the ELISA tests, the antigen peptide ratio is2.828, with distinctantigenic differences.Conclusions1There exist some differences about F gene between measles epidemic of Zhejiang provinceand vaccine strains, but has not been under obvious positive pressure, and its variation belongs torandom drift.2On F gene epitopes, epidemic strains of amino acid variability has resulted in virusantigenic change, suggesting that this may be one of the reasons that is affecting the immune effectof vaccine.
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