| | | | | 亲和标记DAB_(389)(Gly_4Ser)_2-αMSH重组蛋白的构建、表达及纯化 | | | 李泽鸿;张国利;岳玉环;朱平 | | | 目的:构建含有亲和标记His-tag的DAB389(Gly4Ser)2-αMSH重组蛋白,便于融合蛋白质的纯化。方法:利用含有His-tag序列、Factor Xa酶切识别序列、白喉毒素N端序列的基因作上游引物,含有αMSH全序列和(Gly4Ser)2互补序列基因作下游引物,以pET28a/DAB389(Gly4Ser)2EGF为模板,PCR扩增目的基因片段,并插入到原核表达载体pET28a中,构建了重组表达载体pET28a/亲和标记DAB389(Gly4Ser)2-αMSH,融合蛋白在BL21(λDE3)中以可溶形式表达,通过硫酸铵盐析、亲和层析纯化、脱盐、Factor Xa酶切得到重组蛋白纯品。结果:扩增的片段与理论值一致,序列分析正确;目的蛋白表达量约占菌体总蛋白量的30.6%;纯化得到纯度为94.36%的重组蛋白。结论:成功构建了含有His-tag的DAB389(Gly4Ser)2-αMSH重组蛋白,减少了纯化步骤,为进一步研究重组毒素、简便纯化途径奠定基础。
【作者单位】：吉林农业大学生命科学学院;军事医学科学院军事兽医研究所;军事医学科学院军事兽医研究所;军事医学科学院军事兽医研究所 长春130118;军事医学科学院军事兽医研究所;长春130062;长春130062;长春130062;长春130062
【关键词】：亲和标记His-tag;DAB389(Gly4Ser)2-αMSH;重组蛋白;基因表达;免疫毒素类;白喉毒素
【基金】：吉林省教育厅资助课题(吉教科合字2007第067号);吉林农业大学科学研究启动基金资助项目(2007008)
【分类号】：Q78
【DOI】：CNKI:SUN:JSYX.0.2008-03-014
【正文快照】：
免疫毒素所用的导向部分主要有单抗、单链抗体、激素分子或细胞因子等。在原核生物中表达免疫毒素时,为确保其生物活性,表达产物力求以可溶形式存在。我们以前的实验表明[1],DAB389(Gly4Ser)2-αMSH免疫毒素能在大肠杆菌中进行可溶性表达,克服了传统免疫毒素以包涵体形式表达、 | | |
| | |  推荐 CAJ下载  PDF下载 | | | CAJViewer7.0阅读器支持所有CNKI文件格式，AdobeReader仅支持PDF格式 | | | | Construction,expression and purification of recombinant protein DAB_(389)(Gly_4Ser)_2-αMSH with His-tag | | | LI Ze-Hong1;2;ZHANG Guo-Li 2;YUE Yu-Huan2;ZHU Ping2(1.College of Life Science;Jilin Agriculture University;Changchun 130118;China; 2.Institute of Military Veterinary Medicine;Academy of Military Medicine Sciences;Changchun 130062;China) | | | Objective:To construct the recombinant protein DAB389 (Gly4Ser)2-αMSH containing His-tag in order to simplify purification. Methods: Gene fragment was amplified from plasmid pET28a/DAB389(Gly4Ser)2-EGF by PCR with the upper primer containing His-tag, factor Xa distinguish sequence and diphtheria toxin complementary sequences, and the down primer containing αMSH and (Gly4Ser)2 complementary sequences. The DAB389 (Gly4Ser)2-αMSH gene fragment was amplified from plasmid pET28a/DAB389(Gly4Ser)2-EGF by PCR, cloned into the expression vector pET28a (+), resulting in plasmid pET28a/ DAB389(Gly4Ser)2-αMSH with His-tag. Recombinant plasmid was transformed into E.coli BL21(λDE3)and the recombinant toxin was expressed in a soluble protein. The protein was precipitated by ammonium sulfate, purified by Cu Chelating Sepharose Fast Flow, Superdex G-25 chromatography, then factor Xa was used for cleavage, the pure and DAB389 (Gly4Ser)2-αMSH was obtained. Results: The gene fragment was amplified right, the recombinant protein DAB389 (Gly4Ser)2-αMSH was constructed,expressed and accounted for 30.6% of the total protein.The purity of DAB389 (Gly4Ser)2-αMSH was about 94.36%. Conclusion: The recombinant protein DAB389(Gly4Ser)2-αMSH containing His-tag was successfully constructed,and the steps of purification were reduced which provides the basis for further research on the recombinant toxin and the predigest approaches.
【Keyword】：His-tag;DAB389(Gly4Ser)2-αMSH;recombinant proteins;gene expression;immunotoxins;diphtheria toxin |
| | | | | | 1 | 李泽鸿,张国利,岳玉环,邱业峰,吴广谋,朱平; 白喉毒素-(Gly_4 Ser)_2-α促黑素重组蛋白的研究 [J]; 中国肿瘤生物治疗杂志; 2005年01期 | | 2 | 李泽鸿,张国利,岳玉环,朱平; DAB_(389)(Gly_4Ser)_2αMSH重组融合蛋白的构建及表达 [J]; 中国兽医学报; 2007年04期 | | 3 | 张国利,吴广谋,李俊植,岳玉环,李树民,朱平; 白喉毒素(Gly_4Ser)_2-人表皮生长因子融合蛋白的纯化及其特异性细胞毒性研究 [J]; 中国免疫学杂志; 2004年03期 |
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| | | | | | 1 | 李泽鸿,岳玉环,吴广谋,张国利; 白喉毒素DAB_(389)-(Gly_4Ser)_2-α-促黑激素工程菌发酵条件的初步研究 [J]; 生物技术通讯; 2008年01期 | | 2 | 李泽鸿,张国利,岳玉环,朱平; DAB_(389)(Gly_4Ser)_2αMSH重组融合蛋白的构建及表达 [J]; 中国兽医学报; 2007年04期 | | 3 | 李泽鸿,岳玉环,张国利,朱平; DAB_(389)(Gly_4Ser)_2-αMSH重组蛋白的构建表达及特异性细胞毒性研究 [J]; 中国新药杂志; 2007年07期 | | 4 | 李泽鸿,李树民; 两种重组融合蛋白的分子生物学特性比较 [J]; 北华大学学报(自然科学版); 2007年06期 | | 5 | 李泽鸿,张国利,岳玉环,吴广谋; DAB_(389)(Gly_4Ser)_2-αMSH的纯化及细胞特异性毒性 [J]; 中国兽医学报; 2007年06期 |
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