中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
2期
152-156
,共5页
蒋锦琴%孙一帆%岳文燕%严杰%阮萍
蔣錦琴%孫一帆%嶽文燕%嚴傑%阮萍
장금금%손일범%악문연%엄걸%원평
甲型副伤寒沙门菌%spaO-ompA 融合基因%重组表达%免疫原性%免疫保护性
甲型副傷寒沙門菌%spaO-ompA 融閤基因%重組錶達%免疫原性%免疫保護性
갑형부상한사문균%spaO-ompA 융합기인%중조표체%면역원성%면역보호성
Salmonella paratyphi A%spaO-ompA fusion gene%Recombinant expression%Immunogenicity%Immunoprotection
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Western blot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E.coli BL21 DE3pET42a-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 μg或200 μgrSpaO-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.
目的 構建甲型副傷寒沙門菌spaO-ompA融閤基因及其原覈錶達繫統,確定重組錶達產物rSpaO-OmpA免疫保護作用.方法 採用柔性肽序列連接spaO與ompA基因,構建spaO-ompA人工融閤基因及其原覈錶達繫統.採用SDS-PAGE和Bio-Rad凝膠圖像分析繫統檢測目的重組錶達產物rSpaO-OmpA錶達情況及其產量.採用免疫擴散法、微量肥達和Western blot法鑒定rSpaO-OmpA 抗原性和免疫反應性.採用小鼠感染模型瞭解rSpaO-OmpA對甲型副傷寒沙門菌緻死性感染的免疫保護作用,實驗中採用重組錶達的SpaO( rSpaO)及OmpA(rOmpA)作為對照.結果 所構建的spaO-ompA融閤基因與spaO或ompA單基因覈苷痠和氨基痠序列相似性均為100％.所構建的原覈錶達繫統E.coli BL21 DE3pET42a-spaO-ompA能高效錶達重組融閤蛋白rSpaO-OmpA.rSpaO-OmpA能與甲型副傷寒沙門菌全菌抗血清結閤併產生暘性雜交信號,免疫傢兔後可產生特異性抗體.100 μg或200 μgrSpaO-OmpA對甲型副傷寒沙門菌感染小鼠的免疫保護率分彆為66.7％ (8/12)和83.3％ (10/12),明顯高于等量rSpaO及rOmpA( P＜0.05).rSpaO-OmpA免疫小鼠血清與不同副傷寒沙門菌H抗原的凝集效價為1∶5 ～ 1∶40、與rSpaO和rOmpA及rSpaO-OmpA免疫雙擴散效價為1∶1 ～1∶16.結論 人工融閤重組抗原rSpaO-OmpA免疫原性和免疫保護作用較等量rSpaO或rOmpA更彊.
목적 구건갑형부상한사문균spaO-ompA융합기인급기원핵표체계통,학정중조표체산물rSpaO-OmpA면역보호작용.방법 채용유성태서렬련접spaO여ompA기인,구건spaO-ompA인공융합기인급기원핵표체계통.채용SDS-PAGE화Bio-Rad응효도상분석계통검측목적중조표체산물rSpaO-OmpA표체정황급기산량.채용면역확산법、미량비체화Western blot법감정rSpaO-OmpA 항원성화면역반응성.채용소서감염모형료해rSpaO-OmpA대갑형부상한사문균치사성감염적면역보호작용,실험중채용중조표체적SpaO( rSpaO)급OmpA(rOmpA)작위대조.결과 소구건적spaO-ompA융합기인여spaO혹ompA단기인핵감산화안기산서렬상사성균위100％.소구건적원핵표체계통E.coli BL21 DE3pET42a-spaO-ompA능고효표체중조융합단백rSpaO-OmpA.rSpaO-OmpA능여갑형부상한사문균전균항혈청결합병산생양성잡교신호,면역가토후가산생특이성항체.100 μg혹200 μgrSpaO-OmpA대갑형부상한사문균감염소서적면역보호솔분별위66.7％ (8/12)화83.3％ (10/12),명현고우등량rSpaO급rOmpA( P＜0.05).rSpaO-OmpA면역소서혈청여불동부상한사문균H항원적응집효개위1∶5 ～ 1∶40、여rSpaO화rOmpA급rSpaO-OmpA면역쌍확산효개위1∶1 ～1∶16.결론 인공융합중조항원rSpaO-OmpA면역원성화면역보호작용교등량rSpaO혹rOmpA경강.
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 prokaryotic expression system of spaO-ompA fusion gene was subsequently generated.SDS-PAGE and Bio-Rad Agarose Image Analyzer were applied to examine the expression as well as the yield of the target recombinant protein rSpaO-OmpA.The antigenicity and immunoreactivity of rSpaO-OmpA were determined using immunodiffusion 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 BL21DE3pET42a-spaO-ompA expressed the recombinant protein rSpaO-OmpA.rSpaO-OmpA combined with the antiserum against wholecell 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) immunoprotective 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 different S.paratyphi species,and 1∶1-1∶16 immunodiffusion titers to rSpaO,rOmpA and rSpaO-OmpA proteins,respectively.Conclusion The artificially fusion antigen,rSpaO-OmpA,has more powerful immunogenicity and immunoprotection that the equal rSpaO or rOmpA.
