一种亨廷顿舞蹈症体外药物筛选细胞模型的建立

中国生物工程杂志

技术与方法

一种亨廷顿舞蹈症体外药物筛选细胞模型的建立

王爱娥张睢扬于新荣王东霞王英马建新梅开城闫春莲

中国人民解放军第二炮兵总医院呼吸内科信阳师范学院生命科学学院信阳师范学院生命科学学院信阳师范学院生命科学学院信阳师范学院生命科学学院信阳师范学院生命科学学院信阳师范学院生命科学学院

Establishment of the Huntington's Disease in vitro Drug Screening Cell Model

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摘要： 为建立以多聚谷氨酰胺（polyQ）为靶点的亨廷顿舞蹈症体外药物筛选细胞模型，以随机引物PCR法克隆了不同长度的 CAG片段，经序列测定正确后，分别融合到已经建立的氯霉素抗性融合蛋白表达系统pCAR中CAT的N端，重组质粒转化大肠杆菌，并在其中诱导表达，SDS-PAGE 和氯霉素抗性平板试验对目的蛋白可溶性与氯霉素活性进行测定。结果显示长度在40以上的polyQ为不溶性包涵体表达，表现为低水平的氯霉素抗性，40以下的polyQ则以可溶形式表达，表现为高水平氯霉素抗性，从而建立起能够模拟亨廷顿舞蹈症病理过程的体外细胞模型。通过检测模型细胞的氯霉素抗性，可以定性、定量地反映polyQ在体内的折叠状态和可溶性，故可以借助该模型对促溶药物或生物活性物质进行高通量筛选，为亨廷顿舞蹈症预防、诊断、治疗提供了新思路。

关键词： 蛋白溶解性; 氯霉素乙酰基转移酶; 细胞模型; 多聚谷氨酰胺（polyQ）; 亨廷顿舞蹈症（HD）

Abstract: To develop a Huntington's disease(HD) cell model in vitro to screen drugs targeting the aggregation of polyQ，different length of CAG repeat fragments were amplified by random primer PCR, identified by DNA sequencing and were fused to the N-terminus of CAT in the pCAR system respectively which had been constructed and identified before. Recombinant plasmids were transformed into and induced to express in the host E.coli. SDS-PAGE and chloramphenicol resistance test were done to determine the solubility of the polyQ and chloramphenicol resistance levels of the fusions. With different length of CAG repeat fragments cloned and expressed in the CAT-fusion protein reporting system, we found that when the length of the fragments increased over 40, their encoding polyQ expressed as insoluble protein and chloramphenicol resistance levels are lower, while under 40, the polyQ expressed as soluble ones and chloramphenicol resistance levels are higher. A in vitro HD model that could minimize the pathological process of the HD thus has been developed. With which by measure the recombinant bacteria's resistance to chloramphenicol we can determine the polyQ' solubility and folding state in vitro by quality and quantity. Thus this model can be used to screen drugs or bioactivity materials that can inhibit aggregation of the polyQ, which thereby shedding new light on the prevent, diagnosis and therapy of HD.

Key words: Huntington's disease (HD) protein solubility chloramphenicol acetyltransferase (CAT) cell model polyglutamine (polyQ)

收稿日期: 2006-06-02 出版日期: 2006-10-25

通讯作者: 王爱娥

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	张睢扬
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	王英
	于新荣
	王爱娥
	梅开城
	闫春莲

引用本文:

王爱娥,张睢扬,于新荣,王东霞,王英,马建新,梅开城,闫春莲. 一种亨廷顿舞蹈症体外药物筛选细胞模型的建立[J]. 中国生物工程杂志, .

. Establishment of the Huntington's Disease in vitro Drug Screening Cell Model. China Biotechnology, .

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2006/V26/I10/18

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