甲型副伤寒沙门菌spaO—ompA融合基因构建及其表达产物免疫保护作用

Generation of spaO-ompA fusion gene of Salmonella paratyphi A and the immunoprotection of expres- sion product of the fusion gene

目的构建甲型副伤寒沙门菌spaO—ompA融合基因及其原核表达系统，确定重组表达产物rSpaO—OmpA免疫保护作用。方法采用柔性肽序列连接spaO与ompA基因，构建spaO-ompA人工融合基因及其原核表达系统。采用SDS-PAGE和Bio—Rad凝胶图像分析系统检测目的重组表达产物rSpaO—OmpA表达情况及其产量。采用免疫扩散法、微量肥达和Westernblot法鉴定rSpaO—OmpA抗原性和免疫反应性。采用小鼠感染模型了解rSpaO—OmpA对甲型副伤寒沙门菌致死性感染的免疫保护作用，实验中采用重组表达的SpaO（rSpaO）及OmpA（rOmpA）作为对照。结果所构建的spaO-ompA融合基因与spaO或ompA单基因核苷酸和氨基酸序列相似性均为100％。所构建的原核表达系统EcoliBk21DE3pET42a-spaO-ompA能高效表达重组融合蛋白rSpaO-OmpA。rSpaO-OmpA能与甲型副伤寒沙门菌全菌抗血清结合并产生阳性杂交信号，免疫家兔后可产生特异性抗体。100汕g或2006xgrSpaO—OmpA对甲型副伤寒沙门菌感染小鼠的免疫保护率分别为66．7％（8／12）和83．3％（10／12），明显高于等量rSpaO及rOmpA（P〈0．05）。rSpaO—OmpA免疫小鼠血清与不同副伤寒沙门菌H抗原的凝集效价为1：5～1：40、与rSpaO和rOmpA及rSpaO—OmpA免疫双扩散效价为1：1～1：16。结论人工融合重组抗原rSpaO—OmpA免疫原性和免疫保护作用较等量rSpaO或rOmpA更强。

Objective To generate the spaO-ompA fusion gene of Salmonella paratyphi A and its prokaryotic expression system, and to determine the immunoprotection of the recombinant expression product rSpaO-OmpA. Methods A flexible peptide sequence was used to link spaO and ompA genes and a prokary- otic expression system of spaO-ompA fusion gene was subsequently generated. SDS-PAGE and Bio-Rad Agar- ose Image Analyzer were applied to examine the expression as well as the yield of the target recombinant pro- tein rSpaO-OmpA. The antigenicity and immunoreactivity of rSpaO-OmpA were determined using immunodif- fusion test, Western Blot assay and micro-Widal＇s test. By a mouse infection model, the immunoprotection of rSpaO-OmpA against the lethal challenge of S. paratyphi A was determined. In the animal protective test, the recombinant expressed SpaO （rSpaO） and OmpA （rOmpA） were used as the controls. Results The generated spaO-ompA fusion gene had 100% nucleotide and amino acid sequence identities compared to the single spaO or ompA gene. The constructed prokaryotic expression system IPTG E. coli EcoliBk21DE3pET42a-spaO-ompAexpressed the recombinant protein rSpaO-OmpA, rSpaO-OmpA combined with the antiserum against whole- cell of S. paratyphi A to present positive hybridization signal and induced specific antibody in the immunized rabbits. Immunization with 100 or 200 μg rSpaO-OmpA contributed 66.7% （8/12） or 83.3% （10/12） im- munoprotective rates in mice when the animals were attacked with S. paratyphi A. The immunoprotective rates produced by rSpaO-OmpA were significantly higher than that of equal rSpaO or rOmpA（P〈0.05 ）. The sera from rSpaO-OmpA immunized mice presented 1：5-1：40 agglutination titers to the H antigens of differentS. paratyphi species, and 1：1-1：16 immunodiffusion titers to rSpaO, rOmpA and rSpaO-OmpA proteins, re- spectively. Conclusion The artificially fusion antigen, rSpaO-OmpA, has more powerful immunogenicity and immunoprotection that the equal rSpaO or rOmpA.