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结核分枝杆菌CFP10-ESAT-6融合基因
的表达及其多克隆抗体的制备
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唐宇龙1，2，刘湘新1 ，杨华2，丁元生2，胡忠义2
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(1.湖南农业大学 动物医学院,湖南 长沙410128; 2.上海市结核重点实验室,上海200433)
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摘要：
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以结核分枝杆菌标准株H37Rv的基因组DNA为模板，经重叠PCR扩增获得CFP10-ESAT-6融合基因片段，克隆于pET21a表达载体中，获得了重组质粒pET21a-CFP10-ESAT-6，将其转化大肠杆菌BL21细胞，经IPTG诱导表达，得到了25ku的目的蛋白。表达产物经纯化和定量分析后免疫新西兰大白兔，收集兔血清进行抗体ELISA检测。结果显示，成功构建了CFP10-ESAT-6融合基因的原核表达质粒，获得了CFP10-ESAT-6融合蛋白，以此蛋白免疫动物可产生高效价的抗体。 |
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关键词：
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结核分枝杆菌；融合蛋白CFP10-ESAT-6；表达；多克隆抗体 |
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中图分类号：S 852.618：Q 786文献标识码：
A 文章编号：1673-4696(2006)10-0811-04 |
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Expression of CFP10-ESAT-6 fusion gene of Mycobacterial tuberculosis
and preparation of polyclonal antibody against the expressed protein |
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TANG Yu-long1，2，LIU Xiang-xin1，YANG
Hua2，DING Yuan-sheng2， HU Zhong-yi2 |
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(1.College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China;
2.TB Key Laboratory of Shanghai, Shanghai 200433, China) |
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Abstract: The gene encoding protein CFP10-ESAT-6 was amplified from Mycobacterium tuberculosis H37Rv chromosomal DNA by PCR. The PCR product was cloned into pET21a and recombinant pET21a-CFP10-ESAT-6 was transformed into E.coli BL21. The target protein of 25ku was produced by induction of IPTG. The protein was injected into New Zealand rabbits after it was purified and analysed quantitatively. Then sera from the vaccinated rabbits were analysed by ELISA. ELISA result showed that high titer polyclonal antibody against CFP10-ESAT-6 fusion protein was obtained from the rabbits immunized with the protein. |
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Key words: Mycobacterium tuberculosis；fusion protein CFP10-ESAT-6；expression；polyclonal antibody |
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