甲型副伤寒杆菌ompA基因分布及重组表达产物的免疫学鉴定
发布时间：2018-08-15 11:57
【摘要】： 背景和目的近年我国南方部分地区发生了甲型副伤寒杆菌(Salmonellaparatyphi A)感染引起的甲副伤寒暴发流行。由于甲型副伤寒杆菌无荚膜多糖,故目前采用的荚膜多糖Vi疫苗无预防甲型副伤寒的效果。因此,寻找甲型副伤寒杆菌表面抗原,研制甲型副伤寒疫苗,对于预防和控制甲型副伤寒的流行具有重要意义。而外膜蛋白(outer membrane proteins,OMP)通常是沙门菌等革兰阴性菌的主要表面抗原成分。 本研究中,我们构建了甲型副伤寒杆菌ompA基因原核表达系统,鉴定了重组表达产物rOmpA的免疫原性,检测了我国甲型副伤寒杆菌临床菌株ompA基因携带率,采用小鼠感染模型初步了解了rOmpA的免疫保护作用,以期为rOmpA作为甲型副伤寒杆菌基因工程疫苗的候选抗原提供依据。 内容和方法采用PCR从甲型副伤寒杆菌临床株JH01中扩增ompA基因,T-A克隆后测序并构建ompA基因原核表达系统。采用SDS-PAGE和BioRad凝胶图象分析系统检测rOmpA表达情况及其产量,采用免疫扩散法、微量肥达试验和Western blot鉴定其抗原性和免疫反应性。采用PCR检测98株甲型副伤寒杆菌临床菌株ompA基因携带率。采用小鼠感染模型,了解rOmpA对甲型副伤寒杆菌50001株感染的免疫保护作用。 结果与报道的相关序列比较,所克隆的ompA基因核苷酸和氨基酸序列相似性均为100%。rOmpA表达量约为细菌总蛋白的65%。rOmpA免疫家兔可产生抗体,并能与其兔抗血清和甲型副伤寒杆菌全菌抗血清产生阳性Western杂交信号。94.9%(93/98)甲型副伤寒杆菌临床菌株含有ompA基因。100μg和200μgrOmpA对感染小鼠的免疫保护率分别为41.7%(5/12)和58.3%(7/12)。rOmpA免疫小鼠或保护试验存活小鼠血清对各副伤寒杆菌H抗原的凝集效价为1:5～1:40。 结论ompA基因在甲型副伤寒杆菌临床菌株中分布广泛。rOmpA有良好的免疫原性和一定的免疫保护作用,可作为甲型副伤寒杆菌基因工程疫苗候选抗原。
[Abstract]:Background and objective A paratyphoid fever outbreak caused by paratyphoid A (Salmonellaparatyphi A) infection has occurred in some parts of southern China in recent years. Because paratyphoid A has no capsule polysaccharide, the current vaccine Vi has no preventive effect on paratyphoid A. Therefore, the search for surface antigen of paratyphoid A and the development of paratyphoid A vaccine are of great significance in preventing and controlling the epidemic of paratyphoid A. Outer membrane protein (outer membrane proteinsOMP) is usually the main surface antigen of gram-negative bacteria such as Salmonella. In this study, we constructed the prokaryotic expression system of ompA gene of paratyphoid A, identified the immunogenicity of the recombinant expression product rOmpA, and detected the carrying rate of ompA gene of paratyphoid A clinical strain in China. In order to provide evidence for rOmpA as a candidate antigen for genetic engineering vaccine of paratyphoid A, the immune protective effect of rOmpA was preliminarily understood by using mouse infection model. Content and methods ompA gene was amplified from JH01 of paratyphoid A clinical strain by PCR and sequenced, and the ompA gene prokaryotic expression system was constructed. The expression of rOmpA and its yield were detected by SDS-PAGE and BioRad gel image analysis system. The antigenicity and immunoreactivity of rOmpA were identified by immunodiffusion assay, micro-Wheatty test and Western blot. The ompA gene carrying rate of 98 clinical strains of paratyphoid A was detected by PCR. To investigate the protective effect of rOmpA on 50001 strains of paratyphoid A infection in mice. Results compared with the reported sequences, the nucleotide and amino acid sequence similarity of the cloned ompA gene was that 65%.rOmpA, which expressed about the total bacterial protein, could produce antibodies in rabbits. And it can produce positive Western hybridization signal with rabbit antiserum and paratyphoid A antiserum. 94.9% (93 / 98). The protective rates of ompA gene. 100 渭 g and 200 渭 grOmpA in infected mice were 41.7% (5/ 12) and 58.3% (7 / 12), respectively. ROmpA was small immunized by clinical strains of paratyphoid A (93 / 98). The agglutination titer of serum against H antigen of paratyphoid bacillus in mice or survival mice was 1: 5: 1: 40. Conclusion ompA gene is widely distributed in paratyphoid A clinical strains. ROmpA has good immunogenicity and protective effect. It can be used as candidate antigen of paratyphoid A gene engineering vaccine.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2184129
[Abstract]:Background and objective A paratyphoid fever outbreak caused by paratyphoid A (Salmonellaparatyphi A) infection has occurred in some parts of southern China in recent years. Because paratyphoid A has no capsule polysaccharide, the current vaccine Vi has no preventive effect on paratyphoid A. Therefore, the search for surface antigen of paratyphoid A and the development of paratyphoid A vaccine are of great significance in preventing and controlling the epidemic of paratyphoid A. Outer membrane protein (outer membrane proteinsOMP) is usually the main surface antigen of gram-negative bacteria such as Salmonella. In this study, we constructed the prokaryotic expression system of ompA gene of paratyphoid A, identified the immunogenicity of the recombinant expression product rOmpA, and detected the carrying rate of ompA gene of paratyphoid A clinical strain in China. In order to provide evidence for rOmpA as a candidate antigen for genetic engineering vaccine of paratyphoid A, the immune protective effect of rOmpA was preliminarily understood by using mouse infection model. Content and methods ompA gene was amplified from JH01 of paratyphoid A clinical strain by PCR and sequenced, and the ompA gene prokaryotic expression system was constructed. The expression of rOmpA and its yield were detected by SDS-PAGE and BioRad gel image analysis system. The antigenicity and immunoreactivity of rOmpA were identified by immunodiffusion assay, micro-Wheatty test and Western blot. The ompA gene carrying rate of 98 clinical strains of paratyphoid A was detected by PCR. To investigate the protective effect of rOmpA on 50001 strains of paratyphoid A infection in mice. Results compared with the reported sequences, the nucleotide and amino acid sequence similarity of the cloned ompA gene was that 65%.rOmpA, which expressed about the total bacterial protein, could produce antibodies in rabbits. And it can produce positive Western hybridization signal with rabbit antiserum and paratyphoid A antiserum. 94.9% (93 / 98). The protective rates of ompA gene. 100 渭 g and 200 渭 grOmpA in infected mice were 41.7% (5/ 12) and 58.3% (7 / 12), respectively. ROmpA was small immunized by clinical strains of paratyphoid A (93 / 98). The agglutination titer of serum against H antigen of paratyphoid bacillus in mice or survival mice was 1: 5: 1: 40. Conclusion ompA gene is widely distributed in paratyphoid A clinical strains. ROmpA has good immunogenicity and protective effect. It can be used as candidate antigen of paratyphoid A gene engineering vaccine.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 朱水荣,张政,莫顺堂,陈秀英;浙江省伤寒、甲型副伤寒沙门氏菌耐药性监测分析[J];疾病监测;2003年04期
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