CAJ | 학술논문

目的将A族链球菌表面蛋白Fba分段克隆、表达,免疫动物并攻毒,以鉴定各区段诱导的保护性免疫并确定保护性最强的区段.方法根据Fba蛋白的结构特点将其划分为4个重叠区域,以A族链球菌(GAS)的SSI-9为模板,PCR扩增4段蛋白的基因,克隆、表达后,Western印迹鉴定蛋白表达.4段蛋白纯化后分别免疫小鼠,同时设PBS对照组.ELISA检测各免疫组血清IgG水平,并用致死量的GAS(M+Fba+)攻击免疫3次后的小鼠,计算各组保护率.数据行方差检验和Fisher精确概率法.结果成功构建原核表达质粒pGEX-2T/FbaAl、pGEX-2T/FbaA2、pGEX-2T/FbaA3和pGEX-2T/FbaA4,成功表达了4段蛋白.Fba蛋白免疫小鼠后,实验组血清IgG随免疫次数的增加呈逐渐增高趋势.第3次免疫后,与PBS对照组比较,FbaA2蛋白组效价升高最为明显,达1∶102 400(F=1290.2,P<0.01).攻毒后,FbaA2蛋白组8只小鼠中有4只得到保护,FbaAl、FbaA3及FbaA4蛋白组各8只小鼠中,仅2只得到保护(P<0.05).结论成功构建、表达了Fba分段蛋白;初步确定FbaA2段可诱导较强的保护性免疫应答.
목적 장A족련구균표면단백Fba분단극륭、표체,면역동물병공독,이감정각구단유도적보호성면역병학정보호성최강적구단.방법 근거Fba단백적결구특점장기화분위4개중첩구역,이A족련구균(GAS)적SSI-9위모판,PCR확증4단단백적기인,극륭、표체후,Western인적감정단백표체.4단단백순화후분별면역소서,동시설PBS대조조.ELISA검측각면역조혈청IgG수평,병용치사량적GAS(M+Fba+)공격면역3차후적소서,계산각조보호솔.수거행방차검험화Fisher정학개솔법.결과 성공구건원핵표체질립pGEX-2T/FbaAl、pGEX-2T/FbaA2、pGEX-2T/FbaA3화pGEX-2T/FbaA4,성공표체료4단단백.Fba단백면역소서후,실험조혈청IgG수면역차수적증가정축점증고추세.제3차면역후,여PBS대조조비교,FbaA2단백조효개승고최위명현,체1∶102 400(F=1290.2,P<0.01).공독후,FbaA2단백조8지소서중유4지득도보호,FbaAl、FbaA3급FbaA4단백조각8지소서중,부2지득도보호(P<0.05).결론 성공구건、표체료Fba분단단백;초보학정FbaA2단가유도교강적보호성면역응답.
Objective The truncated fibronectin-binding proteins A (Fba protein) were cloned and expressed, then animals were immunized with Fba protein and subsequently challenged with group A streptococcus (GAS) to further investigate protective antibodies induced by each domain and determine the most immunogenic domain. Methods Fba proteins, which were divided into four overlaps based on the structural domains, were truncated and expressed. The fba truncated genes were amplified by polymerase chain reaction (PCR) with SSI-9 of GAS as template, and cloned into prokaryotic expression plasmid pGEX-2T and expressed in E. coli BL-21. The products were confirmed by Western blot and purified by affinity chromatography column. Female BALB/c mice were immunized with the four truncated proteins respectively, with phosphate buffered solution (PBS) as control. The serum IgG of mice was detected by enzyme-linked immunosorbent assay (ELISA). After the third immunization, mice were challenged with fatal dose of GAS (M+ Fba+ ) to evaluate the protective rates in each group. The data were compared by analysis of variance and Fisher's exact test.Results Prokaryotic expression plasmids of pGEX-2T/FbaAl, pGEX-2T/FbaA2, pGEX-2T/FbaA3 and pGEX-2T/FbaA4 were successfully constructed and the four truncated proteins were expressed and purified successfully. Serum levels of IgG in each experimental group gradually increased with immunization with Fba protein more times. After the third immunization, the IgG titer against FbaA2 1290.2, P<0. 01). After GAS challenge, four out of eight mice were protected in FbaA2 protein group, while two out of eight mice in FbaAl protein, FbaA3 protein and FbaA4 protein groups,respectively (P<0. 05). Conclusions Four truncated Fba proteins are constructed and expressed successfully. Truncated FbaA2 protein could be able to induce strongest protective immune response.