近十年浙江省麻疹病毒F基因氨基酸变异与抗原性的关系研究
发布时间：2019-05-15 12:47
【摘要】：目的： 1研究1999-2010年浙江省麻疹流行株F基因的变异规律和进化特征。 2应用生物信息学软件对麻疹流行株和疫苗株F基因的抗原表位进行预测和筛选，选取二者有差异的抗原表位合成多肽，研究F基因抗原表位上氨基酸的变异对抗原性的影响。 方法： 选择浙江省疾病预防控制中心于1999-2010年麻疹疫情中分离保存的麻疹流行代表株11株，采用逆转录聚合酶链反应扩增F基因全序列，序列测定后，与我国目前使用的麻疹疫苗株沪191及其它相关毒株进行比较分析。采用生物信息学软件对流行株和疫苗株的F基因进行抗原表位预测，合成多肽。采用间接酶联免疫吸附试验（ELISA）证实该对多肽的免疫原性，并用该对多肽免疫Balb/c小鼠，制备免疫血清。采用交叉ELISA法检测二者间的抗体滴度，计算抗原比。 结果： 1浙江省麻疹流行株F基因变异分析 （1）核苷酸和氨基酸变异分析：11株浙江流行株与疫苗株沪191相比，其中核苷酸差异率最大的是2010年流行株，为5.17%；氨基酸差异率最大的分别是2005，2007，2009，2010年流行株，它们与疫苗株之间在F基因上的差异率均为3.44%。 （2）氨基酸变异位点分析：以疫苗株沪191为参考株，浙江省麻疹流行株F基因存在11个氨基酸共同变异位点。其中裂解位点上的第108位、110位氨基酸均存在变异。而F基因已知的3个重要融合功能位点，第32、64和195位氨基酸未发生改变。 （3）F基因进化分析：疫苗株沪191所属的A基因型与浙江株所属的H基因型之间的遗传距离最大，达到0.046，远高于A基因型与B、D基因型之间的遗传距离。在F基因的系统进化树上，A基因型与H基因型也分别位于不同分支，距离较远。从F基因的熵值分析，F基因上存在3个易突变位点，分别为第1位、第152位和第174位，其中熵值最大的是第152位氨基酸，其余位点相对保守。F基因未受到明显的正向选择压力，dn/ds=0.1467＜1。分析进化率得出：F基因核苷酸进化率有逐年增快的趋势，目前已达到5.73%。 2抗原表位预测及抗原性分析 （1）抗原表位预测：疫苗株与流行株的共同抗原表位有5个，分别为第25~38，102~116，266~280，，420~449，478~485位氨基酸。选取第420~449位氨基酸进行多肽的合成。 （2）抗原性分析：两条多肽均具有与麻疹IgG阳性血清的结合能力，具有较好的免疫原性。通过交叉ELISA试验得出，多肽间的抗原比为2.828，二者间有明显的抗原差异。 结论： 1、浙江省麻疹流行株与疫苗株之间在F基因上已存在一定的差异，但还未受到明显的正向压力，其变异属于随机漂移。 2、F基因的抗原表位上，流行株部分氨基酸的变异已造成了病毒抗原性的改变，提示这可能也是影响当前疫苗免疫效果的原因之一。
[Abstract]:Objective: 1 to study the variation and evolution characteristics of F gene of measles epidemic strain in Zhejiang Province from 1999 to 2010. (2) the antigenic epitopes of F gene of measles epidemic strain and vaccine strain were predicted and screened by bioinformatics software. The antigenicity of amino acid variation on F gene epitope was studied by selecting the antigenic epitopes of F gene with different antigenic epitopes. Methods: eleven strains of measles epidemic representative strains isolated and preserved by Zhejiang Center for Disease Control and Prevention from 1999 to 2010 were selected. The complete sequence of F gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). The measles vaccine strain Shanghai 191 and other related strains used in China were compared and analyzed. The antigenic epitope of F gene of epidemic strain and vaccine strain was predicted by bioinformatics software, and polypeptide was synthesized. The immunogenicity of the polypeptide was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), and the immune serum was prepared by immunizing Balb/c mice with the polypeptide. The antibody titers between the two were detected by cross ELISA and the antigen ratio was calculated. Results: 1 Analysis of F gene variation of measles epidemic strain in Zhejiang Province (1) Analysis of nucleotides and amino acid variation: compared with vaccine strain Shanghai 191, 11 strains of Zhejiang epidemic strain had the highest difference rate of nucleotides in 2010. 5.17%; The difference rate of amino acids was the highest in 2005, 2007, 2009 and 2010, and the difference rate between them and vaccine strains was 3.44%. (2) Amino acid variation site analysis: taking vaccine strain Shanghai 191 as reference strain, there were 11 common amino acid variation sites in F gene of measles epidemic strain in Zhejiang Province. Among them, the 108th and 110th amino acids at the cracking site varied. However, there were no changes in amino acids at position 32, 64 and 195, which were known to be three important fusion functional loci of F gene. (3) Evolutionary analysis of F gene: the genetic distance between genotype A of vaccine strain Shanghai 191 and genotype H of Zhejiang strain was the largest, reaching 0.046, which was much higher than that between genotype A and genotype B. In the phylogenetic tree of F gene, genotype A and genotype H were also located in different branches, and the distance was far away. According to the entropy value analysis of F gene, there are three mutable sites in F gene, which are position 1, position 152 and position 174, and the highest entropy value is amino acid at position 152. The other loci were relatively conserved. F gene was not subject to significant positive selection pressure, dn/ds=0.1467 < 1. The results showed that the nucleotides evolution rate of F gene was increasing year by year, which had reached 5.73%. 2 Antigenic epitope prediction and antigenic analysis (1) Antigenic epitope prediction: there were 5 common antigenic epitopes between vaccine strain and epidemic strain, which were amino acids at position 25 ~ 38102 鈮
本文编号：2477518
[Abstract]:Objective: 1 to study the variation and evolution characteristics of F gene of measles epidemic strain in Zhejiang Province from 1999 to 2010. (2) the antigenic epitopes of F gene of measles epidemic strain and vaccine strain were predicted and screened by bioinformatics software. The antigenicity of amino acid variation on F gene epitope was studied by selecting the antigenic epitopes of F gene with different antigenic epitopes. Methods: eleven strains of measles epidemic representative strains isolated and preserved by Zhejiang Center for Disease Control and Prevention from 1999 to 2010 were selected. The complete sequence of F gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). The measles vaccine strain Shanghai 191 and other related strains used in China were compared and analyzed. The antigenic epitope of F gene of epidemic strain and vaccine strain was predicted by bioinformatics software, and polypeptide was synthesized. The immunogenicity of the polypeptide was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), and the immune serum was prepared by immunizing Balb/c mice with the polypeptide. The antibody titers between the two were detected by cross ELISA and the antigen ratio was calculated. Results: 1 Analysis of F gene variation of measles epidemic strain in Zhejiang Province (1) Analysis of nucleotides and amino acid variation: compared with vaccine strain Shanghai 191, 11 strains of Zhejiang epidemic strain had the highest difference rate of nucleotides in 2010. 5.17%; The difference rate of amino acids was the highest in 2005, 2007, 2009 and 2010, and the difference rate between them and vaccine strains was 3.44%. (2) Amino acid variation site analysis: taking vaccine strain Shanghai 191 as reference strain, there were 11 common amino acid variation sites in F gene of measles epidemic strain in Zhejiang Province. Among them, the 108th and 110th amino acids at the cracking site varied. However, there were no changes in amino acids at position 32, 64 and 195, which were known to be three important fusion functional loci of F gene. (3) Evolutionary analysis of F gene: the genetic distance between genotype A of vaccine strain Shanghai 191 and genotype H of Zhejiang strain was the largest, reaching 0.046, which was much higher than that between genotype A and genotype B. In the phylogenetic tree of F gene, genotype A and genotype H were also located in different branches, and the distance was far away. According to the entropy value analysis of F gene, there are three mutable sites in F gene, which are position 1, position 152 and position 174, and the highest entropy value is amino acid at position 152. The other loci were relatively conserved. F gene was not subject to significant positive selection pressure, dn/ds=0.1467 < 1. The results showed that the nucleotides evolution rate of F gene was increasing year by year, which had reached 5.73%. 2 Antigenic epitope prediction and antigenic analysis (1) Antigenic epitope prediction: there were 5 common antigenic epitopes between vaccine strain and epidemic strain, which were amino acids at position 25 ~ 38102 鈮
本文编号：2477518
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