| 用于时间分辨荧光免疫分析的结核分枝杆菌培养滤液蛋白10参照品的制备 |
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| 作者姓名： | Guo FF Zou LL Wu YS Hu ZM Li JL Lv JX Gao JM |
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| 作者单位： | 1. 南方医科大学生物技术学院生物治疗研究所,广东,广州,510515
2. 温州医学院浙江省医学遗传学重点实验室/浙江省模式生物技术与应用重点实验室,浙江,温州,325035 |
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| 基金项目： | 国家科技重大专项(2008ZX10301)~~ |
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| 摘 要： | 目的通过基因工程技术制备结核分枝杆菌培养滤液蛋白10(CFP10)、链亲和素(SA)/CFP10融合蛋白(包括SA-CFP10、CFP10-SA),筛选灵敏度最高、稳定性最好者作为时间分辨荧光免疫分析法检测(TRFIA)的参考标准品。方法以结核分枝杆菌H37Rv标准株基因组为模板,扩增CFP10基因,将其克隆到pET24b、pET24b-SA、pET21a-SA三种载体中,转化到大肠杆菌Rosetta中进行表达,表达产物经镍离子亲和层析(Ni-NTA)柱纯化,透析法复性。三种重组蛋白用双抗体夹心法TRFIA从灵敏度、稳定性两方面进行评价。结果成功构建了pET24b-CFP10、pET24b-SA-CFP10和pET21a-CFP10-SA三种表达载体,重组蛋白CFP10主要为可溶形式表达,融合蛋白CFP10-SA、SA-CFP10均以包涵体形式存在,Ni-NTA柱纯化后透析复性,纯度均可达90%以上。三种重组蛋白经双抗体夹心TRFIA,CFP10-SA灵敏度最高(0.02μg/L),稳定性最好。结论得到了灵敏度高、稳定性好的TRFIA检测CFP10的参照品CFP10-SA融合蛋白,为研制该结核分支杆菌时间分辨荧光诊断试剂盒,并应用于临床打下了基础。
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| 关 键 词： | 结核分枝杆菌 培养滤液蛋白10 时间分辨荧光分析法 |
Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay |
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| Guo FF,Zou LL,Wu YS,Hu ZM,Li JL,Lv JX,Gao JM.Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay[J].Journal of Southern Medical University,2011,31(6):955-959. |
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| Authors: | Guo Fang-fang Zou Li-lin Wu Ying-song Hu Zhi-ming Li Jin-long Lv Jian-xin Gao Ji-min |
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| Institution: | GUO Fang-fang1,ZOU Li-lin2,WU Ying-song1,HU Zhi-ming1,LI Jin-long1,Lü Jian-xin2,GAO Ji-min2 1School of Biotechnology,Southern Medical University,Guangzhou 510515,China,2Zhejiang Provincial Key Lab of Medical Genetics,Wenzhou Medical College,Wenzhou 325035 |
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| Abstract: | Objective To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA). Methods The CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refol... |
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| Keywords: | mycobacterium tuberculosis culture filtrate protein 10 time-resolved fluoroimmunoassay |
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