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| 颗粒溶素的表达纯化及生物学活性分析 |
| 钱琤,陈孙孝,周晔,王燕,谷明莉,陈波,陈燕,邓安梅,仲人前 |
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| (第二军医大学长征医院实验诊断科,全军临床免疫中心,上海,200003) |
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| 摘要: |
| 目的:采用原核表达系统对颗粒溶素(granulysin,GNLY)进行体外表达,纯化及活性鉴定.方法:以体外培养的人外周血单个核细胞(PBMC)为模板,RT-PCR扩增编码GNLY的cDNA片段,插入pMD-18-T 载体,测序正确后再亚克隆到质粒pET28a(+) 中,构建重组表达质粒pET28a(+)-GNLY,转化大肠杆菌BL21(DE3) plysS,IPTG诱导表达融合蛋白,包涵体经变复性后用Ni亲和层析纯化,CFU法测定蛋白活性.结果:成功地将GNLY cDNA片段插入载体pET28a(+)中,构建了表达质粒pET28a(+)-GNLY.经诱导在原核表达系统中以包涵体形式高效表达了相对分子质量为9 000的融合蛋白,变复性纯化后的GNLY蛋白经CFU法检测,发现其具有细胞毒活性且细胞毒作用具有剂量依赖性.结论:本实验利用原核表达系统成功地表达了具有生物学活性的GNLY,为研究其功能、作用机制及临床应用奠定了基础. |
| 关键词: 颗粒溶素、基因表达、生物学活性 |
| DOI：10.3724/SP.J.1008.2006.00829 |
| 投稿时间：2005-12-12修订日期：2006-06-28 |
| 基金项目:国家自然科学基金(30471616)、上海市重大科技攻关基金(04DZ19116)、上海市重点科研支撑条件项目(051409012)和上海市青年科技启明星计划(QMX01423). |
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| Expression, purification and bioactivity analysis of granulysin |
| QIAN Cheng,CHEN Sun-xiao,ZHOU Ye,WANG Yan,GU Ming-li,CHEN Bo,CHEN Yan,DENG An-mei,ZHONG Ren-qian |
| (第二军医大学长征医院实验诊断科,全军临床免疫中心,上海,200003) |
| Abstract: |
| Objective：To express the granulysin protein in E. coli and to detect its bioactivity after purification. Methods： Using cultured human peripheral blood mononuclear cells as the template, we selectively amplified the fragment coding granulysin by RT-PCR. The fragment was then inserted into the prokaryotic expression vector pET28a （＋） for transforming E. coli BL21（DE3） plysS. Fusion protein expression was induced by isopropyl-Beta-D-thiogalactopyranoside （IPTG）. After renaturation, the protein was purified by affinity chromatography and its bioactivity was examined by colony-forming unit. Results： We successfully inserted granulysin cDNA fragment into vector pET28a （＋） and constructed the expression plasmid pET28a （＋）GNLY. The fusion protein, with a molecular weight of 9 000, was obtained through IPTG induction. After renaturation and purification, the recombinant protein was proven to be bioactive by CFU and its cytotoxicity was in a dose-dependent manner. Conclusion： The fusion protein obtained in the present study is bioactive and can be used for further study on its function,mechanism of action and clinical application. |
| Key words: granulysin gene expression bioactivity |
