| 大鼠成熟的肺表面活性蛋白B的原核表达及纯化 |
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| 引用本文： | 张潇骏,王万能,周继红,刘钦,顾仕苓,周代君,邱俊,代维,袁丹凤,刘大维.大鼠成熟的肺表面活性蛋白B的原核表达及纯化[J].动物医学进展,2016(4):18-23. |
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| 作者姓名： | 张潇骏 王万能 周继红 刘钦 顾仕苓 周代君 邱俊 代维 袁丹凤 刘大维 |
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| 作者单位： | 1. 重庆理工大学药学与生物工程学院,重庆,400054;2. 重庆理工大学药学与生物工程学院，重庆 400054; 第三军医大学大坪医院野战外科研究所第四研究室，重庆 400042;3. 第三军医大学大坪医院野战外科研究所第四研究室,重庆,400042 |
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| 基金项目： | 国家自然科学基金项目(81471865) |
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| 摘 要： | 为表达和纯化SP-B蛋白,先对SP-B基因进行稀有密码子优化,PCR反应获得SP-B片段,构建重组质粒pGEX4T-1/SP-B,转化到E.coli BL21(DE3)中诱导表达;用SDS-PAGE与Western blot进行检测;用GSTPrep FF 16/10对融合蛋白进行纯化。经双酶切鉴定证实质粒中插入基因长250bp,测序结果与大鼠SP-B cDNA序列相符;质粒转化后用IPTG进行诱导,在分子质量约34ku处出现1个新条带,与pGEX4T-1/SP-B蛋白预期大小一致,且该融合蛋白能可溶性表达并被纯化。结果表明成功构建了pGEX4T-1/SP-B重组质粒,表达、纯化得到GST/SP-B蛋白,为研究肺表面活性物质替代药物奠定了基础。
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| 关 键 词： | 肺表面活性蛋白 B 原核表达 免疫印迹 蛋白纯化 大鼠 |
Prokaryotic Expression and Purification of Mature Pulmonary Surfactant Protein B of Rats |
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| Abstract: | In order to express and purify surfactant protein B(SP-B),and the gene of SP-B from rat was cloned,constructed into the prokaryotic vector pGEX4T-1.Firstly,rare codons of SP-B gene were opti-mized,and cloned into linear pGEX4T-1 after PCR.Secondly,the recombinant plasmid was transformed in-to E.coli BL21(DE3),and the expression of SP-B was detected by SDS-PAGE and Western blot.Then,the recombinant protein was purified by GSTPrep FF 16/10.The results showed that the SP-B gene was 250 bp and the result of sequencing pGEX4T-1/SP-B was consistent with the SP-B cDNA sequence.At the same time,the E.coli BL21(DE3)was induced by IPTG,and one new band about 34 ku appeared.Those results indicated that the pGEX4T-1/SP-B recombinant plasmid was constructed successfully,and the ex-pression and purification of SP-B could be the foundation of pulmonary surfactant alternative drugs. |
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| Keywords: | surfactant protein B prokaryotic expression Western blot protein purification rat |
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