Expression and purification of turkey coronavirus nucleocapsid protein in Escherichia coli.
Expression and purification of turkey coronavirus nucleocapsid protein in Escherichia coli.
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DOI：
10.1016/j.jviromet.2003.11.006
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发表时间：
2004-03-15
影响因子：
3.1
通讯作者：
Schrader D
中科院分区：
文献类型：
--
作者：
Loa CC;Lin TL;Wu CC;Bryan TA;Hooper T;Schrader D
纯化火鸡冠状病毒（TCoV）核衣壳(N)蛋白，该蛋白在原核表达系统中以组氨酸标记的融合蛋白表达。采用蔗糖梯度超离心法从感染的火鸡胚肠中分离得到部分火鸡冠状病毒，并提取RNA。通过逆转录-聚合酶链反应从提取的RNA中扩增出N蛋白基因并进行克隆。通过聚合酶链反应和序列分析鉴定重组表达构建体（pTri-N）。Western blotting检测组氨酸标记融合N蛋白的表达，其分子量为57 kd。经镍-琼脂糖柱层析纯化，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定，表达的N蛋白接近均匀性。从100 ml细菌培养物中，蛋白质回收率可达2.5 mg。经Western blotting检测，纯化的N蛋白可被TCoV抗体识别。在酶联免疫吸附试验（ELISA）中，重组N蛋白能够区分感染TCoV的火鸡阳性血清和正常火鸡血清。这些结果表明，表达的N蛋白是开发检测TCoV抗体的抗体捕获ELISA的优越抗原来源。
Purification of turkey coronavirus (TCoV) nucleocapsid (N) protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. Turkey coronavirus was partially purified from infected intestine of turkey embryo by sucrose gradient ultracentrifugation and RNA was extracted. The N protein gene was amplified from the extracted RNA by reverse transcription-polymerase chain reaction and cloned. The recombinant expression construct (pTri-N) was identified by polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion N protein with a molecular mass of 57 kd was determined by Western blotting analysis. By chromatography on nickel-agarose column, the expressed N protein was purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The protein recovery could be 2.5 mg from 100 ml of bacterial culture. The purified N protein was recognized by antibody to TCoV in Western blotting assay. The capability of the recombinant N protein to differentiate positive serum of turkey infected with TCoV from normal turkey serum was evident in enzyme-linked immunosorbent assays (ELISA). These results indicated that the expressed N protein is a superior source of TCoV antigen for development of antibody-capture ELISA for detection of antibodies to TCoV.
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影响因子：
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