Position: Home > Articles > Prokaryotic Expression and Purification of Mature Pulmonary Surfactant Protein B of Rats
Progress in Veterinary Medicine
2016,37
(4)
18-23
大鼠成熟的肺表面活性蛋白B的原核表达及纯化
作 者：
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
单 位：
重庆理工大学药学与生物工程学院;第三军医大学大坪医院野战外科研究所第四研究室
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
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。
译 名：
Prokaryotic Expression and Purification of Mature Pulmonary Surfactant Protein B of Rats
作 者：
ZHANG Xiao-jun;WANG Wan-neng;ZHOU Ji-hong;LIU Qin;GU Shi-ling;ZHOU Dai-jun;QIU Jun;DAI Wei;YUAN Dan-feng;LIU Da-wei;School of Pharmacy and Bioengineering,Chongqing University of Technology;Department 4,Institute of Surgery Research,Daping Hospital,Third Military Medical University;
关键词：
surfactant protein B;;prokaryotic expression;;Western blot;;protein purification;;rat
摘 要：
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 optimized,and cloned into linear pGEX4T-1after PCR.Secondly,the recombinant plasmid was transformed into 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 expression and purification of SP-B could be the foundation of pulmonary surfactant alternative drugs.
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