摘要
目的构建甲型副伤寒杆菌(Salmonella paratyphi A)H1a基因原核表达系统,确定表达产物rH1a免疫原性和保护作用,检测甲型副伤寒杆菌临床菌株H1a基因携带和表达情况。方法采用高保真PCR从甲型副伤寒杆菌临床株JH01中扩增H1a基因,T-A克隆后测序,构建H1a基因原核表达系统pET32a-H1a-E．coli BL21DE3。采用SDS-PAGE和BioRad凝胶图象分析系统检查rH1a表达情况,Ni-NTA亲和层析法收集rH1a。采用Western blot鉴定其免疫反应性和免疫原性。建立PCR和ELISA检测98株甲型副伤寒杆菌临床菌株H1a基因携带和表达频率。观察rH1a对甲型副伤寒杆菌50001株感染小鼠的免疫保护作用。结果与报道的相关序列比较,所克隆的H1a基因核苷酸和氨基酸序列相似性均为99．59％。rH1a表达量为细菌总蛋白的60％左右。甲型副伤寒杆菌全菌抗血清能识别rH1a并与之结合。rH1a免疫家兔可产生抗体。100％(98／98)甲型副伤寒杆菌临床菌株均含有H1a基因并表达H1a。500μgrH1a灌喂或皮下注射免疫小鼠受甲型副伤寒杆菌50001株攻击后的存活率均为50．0％,若加入5μg rLTB,存活率分别上升至75．0％和66．7％。结论本研究成功地从甲型副伤寒杆菌临床菌株中构建了H1a基因高效原核表达系统。rH1a有良好的免疫原性和一定的免疫保护作用。甲型副伤寒杆菌临床菌株广泛存在H1a基因并高频率表达。
Objective To construct a pmkaryotic expression system of Salmonella paratyph/ A H1a gene, and to determine the immunogenicity and immunoprotection of the expressed product rH1a and the frequencies of H1a gene carrying and expressing in S. paratyphi A isolates. Methods The H1a gene of a clinical S. paratyphi A strain JH01 was amplified using high fidelity PCR. The cloned H1a gene was sequenced after T-A cloning. A prokaryotic expression system of the H1a gene, pET32a-H1a-E, coli BK21DE3, was then constructed. SDS-PAGE and BioRad Agamse Image Pattern Analysis System were applied to examine the rH1a expression. Ni-NTA affinity chromatography was performed to collect rH1a. The immunogenicity of rH1a was determined by Western blot. PCR and ELISA were established to detect the carrying and expressing frequencies of the H1a genes in 98 S.paratyphi A isolates. The immunoprotective effects of rH1a in S. paratyphi A strain 50001 infected mice were observed. Results In comparison with the reported corresponding sequences, both the nucleotide and putative amino acid sequence homologies of the cloned H1a gene were 99.59%. The expression output of rH1a was approximately 60% of the total bacterial proteins. S. paratyphi A antiserum could recognize as well as combine with rH1a. rH1a could efficiently induce rabbits to produce specific antibody. 100%（98/98） of the S.paratyphi A isolates had H1a gene and expressed H1a. Half of the mice that oral-taken or subcutaneous injected 500 μg with rH1a for immunization were survival after challenged S. paratyphi A strain 50001. And when co-immunized with 5 μg rLTB, the survival rates of the mice increased to 75.0% and 66.7%, respectively. Conclusion A prokaryotic expression system with high efficiency of H1a gene from a clinical S. paratyphi strain was successfully constructed in this study, rH1a showed a fine immunogenicity and a certain immunoprotective effect. H1a is an extensively distributed and frequently expressed gene in different chnical S. paratyphi strains.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第7期614-618,共5页
Chinese Journal of Microbiology and Immunology
基金
浙江省医药卫生科学研究基金项目(编号:2006A116)
