CAJ | 학술논문

目的：获得结核分支杆菌重组MPT63蛋白，研究其免疫学特性，评价其在结核痛血清学诊断中的价值，寻找更有效的结核病诊断试剂。方法：应用基因工程技术表达、纯化MPT63蛋白，通过Western blotting和免疫双扩散试验分析其抗原性，通过斑点免疫试验、结明试验和结核病一步法检测血清中抗结核抗体。结果：重组质粒pET24b—MPT63测序表明具有正确的编码序列，MPT63蛋白在大肠杆菌细胞内以可溶性蛋白的形式高效表达，表达量占菌体总蛋白的40％左右，分子量约16kDa，Western blotting和免疫双扩散试验检测抗结核抗体的阳性率分别为46.7％和50％，结核病一步法检测的阳性率为70％，结明试验的阳性率为80％。在34例正常血清中，MPT63和PPD斑点免疫试验检测抗结核抗体的阳性率分别为38.2％和35.3％，结核病一步法检测的阳性率为23.5％。结论：pET24b—MPT63大肠杆菌工程菌能以可溶性蛋白的形式高效表达，重组的MPT63也许可作为结核病血清学诊断的一组抗原之一。
목적：획득결핵분지간균중조MPT63단백，연구기면역학특성，평개기재결핵통혈청학진단중적개치，심조경유효적결핵병진단시제。방법：응용기인공정기술표체、순화MPT63단백，통과Western blotting화면역쌍확산시험분석기항원성，통과반점면역시험、결명시험화결핵병일보법검측혈청중항결핵항체。결과：중조질립pET24b—MPT63측서표명구유정학적편마서렬，MPT63단백재대장간균세포내이가용성단백적형식고효표체，표체량점균체총단백적40％좌우，분자량약16kDa，Western blotting화면역쌍확산시험검측항결핵항체적양성솔분별위46.7％화50％，결핵병일보법검측적양성솔위70％，결명시험적양성솔위80％。재34례정상혈청중，MPT63화PPD반점면역시험검측항결핵항체적양성솔분별위38.2％화35.3％，결핵병일보법검측적양성솔위23.5％。결론：pET24b—MPT63대장간균공정균능이가용성단백적형식고효표체，중조적MPT63야허가작위결핵병혈청학진단적일조항원지일。
Objective:To obtain the recombinant MPT63 protein,to study its immunological characteristics,to evaluate its value in the serodiagnosis of tuberculosis, and to develop more effective diagnostic reagents for tuberculosis. Methods: MPT63 protein was expressed and purified with the genetic engineering techniques. The immunogenicity of MPT63 was analyzed by Western blotting and Double immunodiffusion. 64 human sera were detected the antibodies against M. tuberculosis antigens by Dot - immunoblot assay POC - TB assay and MycoDotTM assay. Results: The recombinant plamoid pET24b- MPT63 has a correct gene sequence. The recombinant MPT63 expressed in E. coli exists in the form of soluble protein. SDS- PAGE proved that 40% or so of somatic solubel protein of MPT63 was expressed. Molecular weight was estimated about 16kDa. Western blotting and Double immunodiffusion revealed good antigenicity of MPT63. Of 30 sera from tuberculosis patients,46.7% and 50% were detected antibodies against MPT63 and PPD by DOt-immunoblot assay,respectively. 70% were deteced antibodies against M. tuberculosis by POC- TB assay. 80% were detected antibodies against LAM by MycoDotTM assay. Conclusions:The recombinant MPT63 protein could be highly expressed in the form of soluble protein in E. coli. It is likely that the reoombinant MPT63 can be used as one of multiple antigens in the serodiagnosis of tuberculosis.