婴幼儿人巨细胞病毒临床分离株糖蛋白H基因多态性研究

发布时间：2018-03-23 11:10

本文选题：人巨细胞病毒　切入点：糖蛋白H　出处：《华中科技大学》2012年硕士论文

【摘要】：目的本文主要研究婴幼儿人巨细胞病毒（HCMV）临床分离株包膜糖蛋白H（gH）的基因多态性，分析该基因不同型别在HCMV感染引起的不同疾病中的分布情况及其与不同疾病之间的关系，为进一步了解HCMV致病机理及亚单位疫苗研究提供参考。方法 1、HCMV临床分离株的分离培养：取患儿尿液标本接种于人胚肺细胞孵育8-12小时后换液，连续培养至细胞发生病变（CPE），再取上清液接种于新鲜人胚肺成纤维细胞培养至CPE达80%-90%病变时收集和保存。 2、gH基因分型：应用巢式PCR扩增HCMV临床分离株和AD169株gH基因，核苷酸纯化试剂盒纯化PCR产物，用限制性内切酶HhaI酶切，酶切片段经12%聚丙烯酰胺凝胶电泳，银染显色，比对分型。 3、gH基因序列多态性分析：将部分PCR扩增阳性的产物纯化后测序，与AD169株、Towne株序列进行比对，用DNAsis软件分析。结果 1、HCMV临床分离株的分离培养：共选择105例样本进行分离，最终CPE阳性病毒株50株，分离成功率为47.6%。 2、gH基因分型：经巢式PCR扩增，50例临床分离株中有34例（14例为婴儿肝炎综合征，12例为无黄疸型肝炎，5例为肺炎，1例为先天性CMV感染）gH基因阳性，阳性率为68%；分型显示，gH1型21株，占61.8%；gH2型13株，占38.2%；未见混合型和发现新的型别，gH基因型别在不同疾病间呈散在分布(X2=0.336,P0.05)。 3、gH基因序列多态性分析：随机挑选15株gH基因阳性标本测序分型并与AD169和Towne株序列比对，gH1型有10株，gH2型有5株，其结果与酶切分型结果完全符合。其中所选的第2、4、5、6、7、9、10、11临床株碱基序列相同，编码的氨基酸也相同，同源性为100%，与AD169株核苷酸相似性、氨基酸一致性分别为97.2%、95.52%，同源性较高；第8、12、14临床株碱基序列、氨基酸相同，相互间同源性也高达100%，与TOWNE株核苷酸相似性为99.53%，氨基酸一致性为95.52%。gH序列变异以核苷酸替换为主，也可见插入和缺失突变；与AD169株相比，核苷酸相似性为89.1%～97.2%，氨基酸一致性为74.24%～95.52%；与TOWNE株相比核苷酸相似性为91.12%～99.53%，氨基酸一致性为80.06%～98.5%。DNA变异位点分布比较散在；氨基酸变异相对集中，，与AD169相比，氨基酸变异集中在41～87位点，与TOWNE株比对则集中于33～74位点。结论 1、婴幼儿HCMV临床分离株gH基因型别以gH1为主，gH基因型别在婴儿肝炎综合征、无黄疸型肝炎、肺炎疾病中的分布未见明显差异。 2、不同HCMV临床分离株gH基因相对保守但仍存在一定的多态性。 3、未发现HCMV gH基因多态性与不同临床疾病的关联性。
[Abstract]:Purpose. In this paper, we studied the gene polymorphism of human cytomegalovirus (HCMV) in infants and young children, and analyzed the distribution of different types of the gene in different diseases caused by HCMV infection and its relationship with different diseases. It provides a reference for further understanding the pathogenesis of HCMV and the study of subunit vaccine. Method. 1 isolation and culture of HCMV clinical isolates: urine samples of children were inoculated with human embryonic lung cells for 8 to 12 hours after incubation, The supernatant was collected and stored in fresh human embryonic lung fibroblasts cultured to 80% to 90% CPE. 2G H genotyping: HCMV clinical isolates and AD169 strains were amplified by nested PCR. The PCR products were purified by nucleotide purification kit. The PCR products were digested with restriction endonuclease HhaI, and the fragments were digested by 12% polyacrylamide gel electrophoresis, silver staining and typing. Sequence Polymorphism Analysis of 3G H Gene: partial PCR amplified positive products were sequenced and compared with AD169 strain Towne strain. The results were analyzed by DNAsis software. Results. 1. Isolation and culture of clinical isolates of HCMV: a total of 105 samples were selected for isolation, 50 strains of CPE positive virus strains were isolated, the success rate of isolation was 47.6%. 2G H genotyping: among 50 clinical isolates amplified by nested PCR, 34 cases were infantile hepatitis syndrome, 12 cases were non-jaundice type hepatitis, 5 cases were pneumonia, 1 case was congenital CMV infection, the positive rate was 68%. There were 13 strains of GH2 type, 38.2%, and no mixed type and new type G H genotype were scattered among different diseases. (3) Polymorphism analysis of GH gene: 15 samples were randomly selected for sequencing and typing, and compared with AD169 and Towne strains, there were 10 strains of GH1 type and 5 strains of GH 2 type. The results were in good agreement with the results of enzyme digestion typing, in which the clinical strains of the second strain were the same in base sequence, encoding the same amino acids, the homology was 100, the nucleotide similarity with the AD169 strain and the amino acid consistency were 97.2and 95.52, respectively, with high homology, and the results showed that the nucleotide sequence of the clinical strain was the same as that of the AD169 strain, and the amino acid sequence of the clinical strain was the same as that of the other two strains. The nucleotide similarity with TOWNE strain was 99.53, the amino acid identity was mainly nucleotide substitution, insertion and deletion mutation was also seen, and compared with AD169 strain, the nucleotide similarity was 99.53. The nucleotide similarity was 89. 1 and 97. 2 and the amino acid consistency was 74. 24 and 95.52. Compared with the TOWNE strain, the nucleotide similarity was 91. 12 and 99. 53, and the amino acid identity was the distribution of the 80.06%~98.5%.DNA mutation site. The amino acid variation was relatively concentrated, and compared with the AD169, the amino acid variation was concentrated at 41. 87 sites. Compared with TOWNE strain, it was located at 3374 locus. Conclusion. 1. There was no significant difference in the distribution of G H genotype in infantile hepatitis syndrome, no jaundice hepatitis and pneumonia. 2, the gh gene of different HCMV clinical isolates is relatively conserved but still has certain polymorphism. 3. No association between HCMV gh gene polymorphism and different clinical diseases was found.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.1

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