B型流感嗜血杆菌的荚膜多糖抗体水平及基因分型研究

发布时间：2018-05-26 12:48

本文选题：B型流感嗜血杆菌 + 荚膜多糖　；参考：《重庆医科大学》2011年硕士论文

【摘要】：目的: 本研究旨在关注引发小儿严重侵袭性疾病病原体B型流感嗜血杆菌(Haemophilus influenzae type b,Hib),以动物为模型,评价Hib特异性荚膜多糖的免疫原性及其与抗体水平之间的剂量关系,评估PCR技术在检测小儿呼吸道疾病中Hib的应用价值,探讨与荚膜剂量对应的基因分型在反映菌株侵袭力方面的应用潜力及可行性,指导儿童临床用药及接种疫苗,为研发新疫苗提供一定的实验基础。方法: 1.以SD大鼠为模型,分别给予不同剂量荚膜多糖的B型流感嗜血杆菌PRP-T结合疫苗免疫大鼠,设生理盐水为对照,ELISA法测定血清中Hib多糖抗体浓度,用SAS 8.1统计软件处理数据,分析其Hib荚膜多糖的免疫原性及剂量效应。 2.搜集2009-2010年重庆儿童医院呼吸道感染患儿深部痰标本分离的流感嗜血杆菌菌株159份。针对Hib荚膜基因合成型特异性引物及编码转运荚膜多糖基因的引物,应用PCR技术对临床分离菌株进行Hib检测及亚型鉴定,对阳性标本进行PCR产物序列测定分析。结果: 1.末次免疫后给予不同剂量荚膜多糖Hib结合疫苗的各实验组诱导产生PRP IgG抗体平均浓度均高于0.15μg/ml。免疫后1、2、4周各实验组内PRP IgG抗体水平逐渐升高,其差异有显著性统计学意义(P0.01)。 2.给予不同剂量荚膜多糖的Hib结合疫苗各实验组诱导大鼠产生抗PRP IgG抗体水平随着荚膜多糖剂量的升高而升高。将免疫前及免疫后1、2、4周同一时间点的各实验组平均抗体水平进行组间比较,免疫前各实验组组间无显著性差异(p0.01)。免疫后1周0.8μg多糖组与2.5μg多糖组差异无统计学意义(P0.01),其余各实验组组间抗体水平差异有统计学意义(p0.01)。 3.159株流感嗜血杆菌中,肺炎患儿中分离Hi 90株,占56.6%;支气管炎患儿39株,占24.5%。 4.应用bex A基因特异性扩增159株流感嗜血杆菌,4株阳性。其中69号临床分离菌株经Hi-b、HiHcsA-I特异性编码基因扩增阳性,PCR产物序列测定结果与GenBank数据库内基因序列进行比对分析,一致性分别为98.5%、99%。 5.159株流感嗜血杆菌中有4株bex A基因扩增阳性,为荚膜型流感嗜血杆菌,占流感嗜血杆菌2.52%,不可分型流感嗜血杆菌占97.48%;69号临床菌株Hi-b及HiHcsA-I基因扩增条带阳性,经基因序列比对分析鉴定为B型流感嗜血杆菌I型。结论: 1.Hib结合疫苗中荚膜多糖能诱导大鼠产生较好的免疫应答,其抗体水平随时间及荚膜多糖含量的升高而升高,有良好的剂量效应。 2. Hi是引起小儿呼吸道疾病的主要病原体之一,应用PCR技术对临床菌株进行Hib检测及分型具有较高灵敏度及特异性。针对HiHcsA基因的Hib分型可能反映临床菌株的侵袭力。
[Abstract]:Objective: The aim of this study was to evaluate the immunogenicity of Hib specific capsule polysaccharides and their dose-dependent relationship with antibody levels in animals. To evaluate the value of PCR in the detection of Hib in children with respiratory diseases, to explore the potential and feasibility of genotyping corresponding to capsule dose in reflecting the invasiveness of strains, and to instruct children to use drugs and vaccinations in clinic. To provide a certain experimental basis for the development of new vaccines. Methods: 1. SD rats were immunized with different doses of Haemophilus influenzae type B (Haemophilus influenzae B) combined with PRP-T vaccine. The concentration of Hib polysaccharides antibody in serum was determined by using normal saline as control Elisa, and the data were processed by SAS 8.1 software. The immunogenicity and dose effect of Hib capsule polysaccharides were analyzed. 2. A total of 159 Haemophilus influenzae strains isolated from deep sputum samples of children with respiratory tract infection in children's hospitals in Chongqing were collected from 2009-2010. According to the specific primers of Hib capsule gene synthesis and the primers encoding the translocation of capsule polysaccharide gene, the Hib and subtype identification of clinical isolates were carried out by PCR technique. The positive samples were sequenced by PCR products. Results: 1. After the last immunization, the average concentration of PRP IgG antibody was higher than 0.15 渭 g / ml in each experimental group treated with different doses of capsule polysaccharide Hib conjugated vaccine. The level of PRP IgG antibody increased gradually in each experimental group at 4 weeks after immunization, and the difference was statistically significant (P 0.01). 2. The level of anti PRP IgG antibody induced by Hib conjugated vaccine with different doses of capsule polysaccharide increased with the increase of the dose of capsule polysaccharide. The average antibody levels of each experimental group at the same time point of 4 weeks before and after immunization were compared. There was no significant difference between the experimental groups before immunization and at the same time point (p0.01). At 1 week after immunization, there was no significant difference between 0.8 渭 g polysaccharide group and 2.5 渭 g polysaccharide group, but there was significant difference in antibody level among other experimental groups. Among the 3.159 Haemophilus influenzae strains, 90 strains (56.6%) were isolated from pneumonia and 39 strains (24.555%) from bronchitis. 4. Nine strains of Haemophilus influenzae were specifically amplified by bex A gene and 4 strains of Haemophilus influenzae were positive. The results of PCR products sequencing of 69 clinical isolates were compared with those in GenBank database by Hi-bSA-I specific coding gene amplification. The consistency was 98.5% and 99g, respectively. Of the 5.159 Haemophilus influenzae strains, 4 were positive for bex A gene amplification, accounting for Haemophilus influenzae, accounting for 2.52% of Haemophilus influenzae, 97.48% for Haemophilus influenzae, and 97.48% for Haemophilus influenzae. Haemophilus influenzae type B was identified by gene sequence alignment analysis. Conclusion: Capsule polysaccharides in 1.Hib conjugated vaccine could induce a good immune response in rats, and the antibody level increased with the increase of time and the content of capsule polysaccharides, which had a good dose effect. 2. Hi is one of the main pathogens causing respiratory tract diseases in children. PCR technique has high sensitivity and specificity in the detection and typing of clinical strains by Hib. The Hib typing of HiHcsA gene may reflect the invasiveness of clinical strains.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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