新霉素磷酸转移酶基因nptⅡ的原核表达、蛋白纯化及其活性鉴定

doi:10.3969/j.issn.1001-7216.2011.03.015

摘要/Abstract

摘要： 通过PCR方法从pCAMBIA1305载体上克隆了新霉素磷酸转移酶基因nptⅡ的全编码序列，并插入到原核表达载体pET30a（+）中，构建了重组质粒pET30anptⅡ。将重组质粒转化大肠杆菌BL21（DE3）宿主菌，经IPTG诱导，获得了相对分子量为35 kD的表达蛋白，约占全菌总蛋白的45％。表达蛋白以可溶性和包涵体两种形式存在。用5 mmol/L 二硫苏糖醇和1％十二烷基肌氨酸钠变性溶解包涵体，经透析后复性获得了可溶性重组蛋白。将可溶的表达蛋白用Ni2+NTA亲和层析纯化，获得纯化的NPTⅡ蛋白，电泳谱带扫描分析表明蛋白纯度达95％以上。体外活性检测显示，NPTⅡ蛋白具有良好的生物活性，在浓度30 μg/mL以上时可使卡拉霉素失去抑菌活性。

关键词: 新霉素磷酸转移酶基因, 原核表达, 包涵体, 可溶性蛋白, 纯化

Abstract: Neomycin phosphotransferaseⅡ (nptⅡ) gene, cloned from a pCAMBIA1305 vector by PCR method, was inserted into a pET30a (+) vector to construct the recombinant vector pET30anptⅡ. Then the recombinant vector was transformed into an Escherichia coli strain BL21 (DE3) and the expression of the NPTⅡ protein was successfully induced by adding isopropylβDthiogalactopyranoside (IPTG). The NPTⅡ fusion protein was produced in the form of the solubility or inclusion body with molecular weight around 35 kD, and a yield of approximately 45%. The inclusion bodies were solubilized and denatured by addition of 5 mmol/L dithiothreitol (DDT) and 1% sodium lauroyl sarcosine (SKL), then renatured by dialysis. The solubilized proteins were purified by Ni2+NTA affinity chromatography at approximately 95% purity. An in vitro assay revealed that, the NPTⅡ protein can inactivate the kanamycin at concentrations up to 30 μg/mL.

Key words: neomycin phosphotransferase gene, prokaryotic expression, inclusion body, soluble protein, purification

王玲,刘连盟,傅强,黄世文. 新霉素磷酸转移酶基因nptⅡ的原核表达、蛋白纯化及其活性鉴定[J]. 中国水稻科学 2011,25(3): 326-330 . , DOI: 10.3969/j.issn.1001-7216.2011.03.015 .

WANG Ling,LIU Lian-meng,FU Qiang,HUANG Shi-wen*. Prokaryotic Expression, Purification and Activity Assay of Neomycin Phosphotransferase Ⅱ (nptⅡ) Gene[J]. Chinese Journal of Rice Science 2011,25(3): 326-330 ., DOI: 10.3969/j.issn.1001-7216.2011.03.015 .

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