| 王丽梅1 师长宏2 柏银兰1 张海2 康健1 张薇1 徐志凯1.结核杆菌Hsp65 与人IL-2 融合蛋白在耻垢杆菌表达[J].,2011,11(18):3405-3407 |
| 结核杆菌Hsp65 与人IL-2 融合蛋白在耻垢杆菌表达 |
| Expression of Heat Shock Protein 65 of Mycobacterium Tuberculosis andHuman IL-2 Fused Gene in Mycobacterium Smegmatis |
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| DOI： |
| 中文关键词: 结核分枝杆菌 HSP65 IL-2 耻垢分枝杆菌 |
| 英文关键词: Mycobacterium tuberculosis HSP65 IL-2 Mycobacterium Smegmatis |
| 基金项目:国家自然科学基金项目（No. 30801055）；" 十一五" 传染病重大专项项目（No. 2008ZX10003-013） |
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| 中文摘要: |
| 目的：构建能够分泌表达结核分枝杆菌热休克蛋白65（Hsp65）与人IL-2 融合蛋白的重组耻垢分枝杆菌（recombinant
Mycobacterium Smegmatis, rMs）。方法：用EcoRⅤ和Hind Ⅲ双酶切含Hsp65-IL-2 融合基因的pPRO-hsp65-IL-2 载体，回收目的基
因片断Hsp65-IL-2，并将其亚克隆入同样双酶切的大肠埃希菌- 分枝杆菌穿梭分泌表达载体pDE22 中。重组质粒
pDE22-hsp65-IL-2 酶切鉴定正确后，电穿孔转化MS 感受态，潮霉素抗性压力筛选阳性rMs。Western-blot 鉴定rMs 培养上清蛋白
中目的蛋白的表达。结果：重组pDE22-hsp65-IL-2 质粒酶切后可获得约2000 bp 片段，与预期大小一致。Western-blot 结果表明，
rMs 培养上清蛋白中有特异性反应条带，大小为78kD，与Hsp65-IL-2 融合蛋白大小相一致。结论：成功构建了大肠埃希菌- 分枝
杆菌穿梭分泌表达载体pDE22-hsp65-IL-2，为该rMs 的免疫学特性及抗结核分枝杆菌感染的保护效果研究奠定了基础。 |
| 英文摘要: |
| Objective: To construct the recombinant Mycobacterium Smegmatis which can express the fused protein of the heat
shock protein 65 of Mycobacterium tuberculosis with human IL-2. Methods: The EcoRⅤ/HindⅢ fragment of the fused gene Hsp65-IL-2
digested from the plasmid pPRO-hsp65-IL-2 was subcloned into the E.coli-Mycobacterium shuttle vector pDE22 predigested by the same
restrictive endoenzymes. The positive pDE22-hsp65-IL-2 recombinant plasmid was identified by restriction enzymatic digestion and
then transformed into Ms cells by electroporation. The expression of the fusion protein Hsp65-IL-2 in the culture of recombinant Ms(rMs)
was detected byWestern-blot. Results: A 2000bp fragment was obtained from the shuttle vector pDE22-hsp65-IL-2 by restriction enzyme
digestion, which was identical with the predicted. The Western-blot showed that there was a specific reactive band at the size of 78kD in
the culture filtrate proteins of rMs, which was identical with the size of Hsp65-IL-2 fusion protein. Conclusion: The shuttle expression
vector pDE22-hsp65-IL-2 was constructed successfully, which provided the foundation for the study of the immunogenicity and protective
efficacy of rMs against MTB infection. |
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