CAJ | 학술논문

目的分析重组沙门菌表达的结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分泌性蛋白ESAT-6诱导的特异性免疫应答.方法将ESAT-6蛋白编码基因导入原核表达载体pYA3333中,构建重组质粒pYA33-esat.通过电穿孔法转化减毒鼠伤寒沙门菌X4550,获得重组菌X4550(33-esat).以每只105CFU剂量的重组菌滴鼻免疫C57BL/6小鼠,间隔18 d,在第2次免疫后10 d取免疫小鼠脾脏、肺脏、肠系膜淋巴结(mesenteric lymph node,MLN)及派伊尔淋巴集结(Peyer's patch,PP)细胞,以ESAT-6多肽作为刺激原,检测特异性的IFN-γ分泌细胞和IL-4分泌细胞.同时,运用CFSE方法榆测了体内抗原特异性CTL效应.结果经沙门菌表达并运送的Mtb抗原ESAT-6能诱导特异性的免疫应答.在肺脏及PP细胞巾,检测到较高水平的IFN-γ和IL-4分泌细胞,免疫应答以Th1型为主.而在脾脏和MLN中,免疫应答呈现Th1/Th2混合应答.此外,体内CTL试验表明,重组菌能够诱导抗原特异的CTL效应,且特异性杀伤率为69.9%.结论以滴鼻方式接种重组沙门菌,不仅能够诱导ESAT-6蛋白特异性的细胞免疫应答,还能激发特异的CTL效应,为结核病的防控提供了新的认识.
목적 분석중조사문균표체적결핵분지간균(Mycobacterium tuberculosis,Mtb)분비성단백ESAT-6유도적특이성면역응답.방법 장ESAT-6단백편마기인도입원핵표체재체pYA3333중,구건중조질립pYA33-esat.통과전천공법전화감독서상한사문균X4550,획득중조균X4550(33-esat).이매지105CFU제량적중조균적비면역C57BL/6소서,간격18 d,재제2차면역후10 d취면역소서비장、폐장、장계막림파결(mesenteric lymph node,MLN)급파이이림파집결(Peyer's patch,PP)세포,이ESAT-6다태작위자격원,검측특이성적IFN-γ분비세포화IL-4분비세포.동시,운용CFSE방법유측료체내항원특이성CTL효응.결과 경사문균표체병운송적Mtb항원ESAT-6능유도특이성적면역응답.재폐장급PP세포건,검측도교고수평적IFN-γ화IL-4분비세포,면역응답이Th1형위주.이재비장화MLN중,면역응답정현Th1/Th2혼합응답.차외,체내CTL시험표명,중조균능구유도항원특이적CTL효응,차특이성살상솔위69.9%.결론 이적비방식접충중조사문균,불부능구유도ESAT-6단백특이성적세포면역응답,환능격발특이적CTL효응,위결핵병적방공제공료신적인식.
Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.