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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 the prokaryotic expression system of H1a gene of Salmonella paratyphi A, determine the immunogenicity and protective effect of the expressed product rH1a, and to detect the carrying and expression of the H1a gene of Paragonimiaphi paratyphi A strain. Methods High-fidelity PCR was used to amplify H1a gene from clinical strain JH01 of paratyphoid A strain. The T-A clone was cloned and sequenced to construct the prokaryotic expression system pET32a-H1a-E of H1a gene. coli BL21DE3. The expression of rH1a was examined by SDS-PAGE and BioRad gel image analysis system, and rH1a was collected by Ni-NTA affinity chromatography. Western blot was used to identify the immunoreactivity and immunogenicity. PCR and ELISA were used to detect the frequency of H1a gene in 98 clinical isolates of Parag. To observe the immunoprotective effect of rH1a on Paramyxoviral Type 50001 infected mice. Results Compared with the reported sequences, the similarity of nucleotide and amino acid sequence of the cloned H1a gene was 99.59%. The expression level of rH1a is about 60% of the total bacterial protein. Paratyphoid A paratyphoid antisera can recognize rH1a and combined with it. Rabbits immunized with rH1a produce antibodies. 100% (98/98) Clostridium paratyphi A clinical isolates harbor H1a gene and express H1a. Survival rates of mice immunized with 500μg rH1a or subcutaneously injected with S. paratyphi A strain 50001 were 50.0%, respectively. Survival rates increased to 75.0% and 66.7%, respectively, with 5μg rLTB. Conclusions This study successfully constructed the highly efficient prokaryotic expression system of H1a gene from clinical strains of Paragonimia parathyroidus. rH1a has good immunogenicity and a certain degree of immune protection. The clinical isolates of Paratyphoid A (Salmonella Paratyphi A) are widely present in H1a gene and expressed at high frequency.