【摘要】：目的： 1研究1999-2010年浙江省麻疹流行株F基因的變異規(guī)律和進化特征。 2應用生物信息學軟件對麻疹流行株和疫苗株F基因的抗原表位進行預測和篩選，選取二者有差異的抗原表位合成多肽，研究F基因抗原表位上氨基酸的變異對抗原性的影響。 方法： 選擇浙江省疾病預防控制中心于1999-2010年麻疹疫情中分離保存的麻疹流行代表株11株，采用逆轉錄聚合酶鏈反應擴增F基因全序列，序列測定后，與我國目前使用的麻疹疫苗株滬191及其它相關毒株進行比較分析。采用生物信息學軟件對流行株和疫苗株的F基因進行抗原表位預測，合成多肽。采用間接酶聯(lián)免疫吸附試驗（ELISA）證實該對多肽的免疫原性，并用該對多肽免疫Balb/c小鼠，制備免疫血清。采用交叉ELISA法檢測二者間的抗體滴度，計算抗原比。 結果： 1浙江省麻疹流行株F基因變異分析 （1）核苷酸和氨基酸變異分析：11株浙江流行株與疫苗株滬191相比，其中核苷酸差異率最大的是2010年流行株，為5.17%；氨基酸差異率最大的分別是2005，2007，2009，2010年流行株，它們與疫苗株之間在F基因上的差異率均為3.44%。 （2）氨基酸變異位點分析：以疫苗株滬191為參考株，浙江省麻疹流行株F基因存在11個氨基酸共同變異位點。其中裂解位點上的第108位、110位氨基酸均存在變異。而F基因已知的3個重要融合功能位點，第32、64和195位氨基酸未發(fā)生改變。 （3）F基因進化分析：疫苗株滬191所屬的A基因型與浙江株所屬的H基因型之間的遺傳距離最大，達到0.046，遠高于A基因型與B、D基因型之間的遺傳距離。在F基因的系統(tǒng)進化樹上，A基因型與H基因型也分別位于不同分支，距離較遠。從F基因的熵值分析，F基因上存在3個易突變位點，分別為第1位、第152位和第174位，其中熵值最大的是第152位氨基酸，其余位點相對保守。F基因未受到明顯的正向選擇壓力，dn/ds=0.1467＜1。分析進化率得出：F基因核苷酸進化率有逐年增快的趨勢，目前已達到5.73%。 2抗原表位預測及抗原性分析 （1）抗原表位預測：疫苗株與流行株的共同抗原表位有5個，分別為第25~38，102~116，266~280，，420~449，478~485位氨基酸。選取第420~449位氨基酸進行多肽的合成。 （2）抗原性分析：兩條多肽均具有與麻疹IgG陽性血清的結合能力，具有較好的免疫原性。通過交叉ELISA試驗得出，多肽間的抗原比為2.828，二者間有明顯的抗原差異。 結論： 1、浙江省麻疹流行株與疫苗株之間在F基因上已存在一定的差異，但還未受到明顯的正向壓力，其變異屬于隨機漂移。 2、F基因的抗原表位上，流行株部分氨基酸的變異已造成了病毒抗原性的改變，提示這可能也是影響當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

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