Bloom 综合症(BS) 和Rothmund-Tho-Bloom 解旋酶突变体的克隆与表达

生物技术通报 ›› 2013, Vol. 0 ›› Issue (1): 123-128.

Bloom 综合症(BS) 和Rothmund-Tho-Bloom 解旋酶突变体的克隆与表达

张金彪^1,2,3,许厚强^2,3，骆衡^2,3，许庆贺^1,2,3，陈祥^2,3，李坤^1,2,3

1. 贵州大学生命科学学院，贵阳 550025；2. 贵州大学高原山地动物遗传育种与繁殖省部共建教育部重点实验室动物科学学院，贵阳 550025；3. 贵州大学贵州省动物遗传育种与繁殖重点实验室动物科学学院，贵阳 550025

收稿日期:2012-07-06 修回日期:2013-01-30 出版日期:2013-01-30 发布日期:2013-01-30
作者简介:张金彪，男，硕士研究生，研究方向：分子细胞生物学；E-mail ：550183934@qq.com.cn
基金资助:
国家重点基础研究发展计划（973 计划）资助项目（2010CB534912），教育部博士点基金资助项目（200806570003），贵州省国际科技合作计划项目［黔科合外G 字（2011）7008 号］，贵州省优秀人才省长资金资助项目（200822）

Cloning and Expression of Bloom Helicase Mutant

Zhang Jinbiao^1,2,3, Xu Houqiang^2,3 ,Luo Heng^2,3 ,Xu Qinghe^1,2,3 ,Chen Xiang^2,3 ,Li Kun^1,2,3

1. College of Life Science，Guizhou University，Guiyang 550025;
2. Key Laboratory of Animal Genetics，Breeding and Reproduction in the Plateau Mountainous Region，Ministry of Education，College of Animal Science，Guizhou University，Guiyang 550025;
3. Guizhou Key Laboratory of Animal Genetics，Breeding Reproduction，College of Animal Science，Guizhou University，Guiyang 550025

Received:2012-07-06 Revised:2013-01-30 Published:2013-01-30 Online:2013-01-30

摘要/Abstract

摘要：

blm 基因的突变可导致Bloom 综合症（BS），Bloom 综合症是一种罕见隐性常染色体遗传疾病，患者遗传不稳定并易患多种类型癌症。临床发现BS 患者细胞中的BLM 的RecQ-Ct 区域的1 036 位半胱氨酸残基发生突变。运用生物信息学及分子生物学技术，通过两轮重组PCR，构建985 位和1 036 位氨基酸突变的BLM 解旋酶多点突变基因，克隆到载体pET-28a 中，并转化至大肠杆菌BL21（DE3）中进行表达。分离纯化获得了高纯度（>95%）的双突变型BLM 解旋酶，并对其进行了Western blotting鉴定。这些结果可为进一步研究BS 的致病机制奠定基础。

关键词: BLM , 解旋酶 , 重组PCR , 多位点突变 , 基因克隆表达

Abstract:

Bloom syndrome(BS) which is caused by mutation of BLM is an autosomal recessive disorder characterized by genomicinstability and the high rate of many types of cancer. Disease-causing missense mutations have been identified at 1 036 cysteines localized in theRecQ-Ct domain. In this work, with bioinformatics and molecular biology technology, blm gene which 985 and 1 036 amino acids were mutated was obtained by two cycles of recombinant PCR, and cloned into expression vector pET-28a, and transformed into the strain E. coli BL21(DE3).It was purified that double mutation BLM helicase is the high purity(>95%), and identified by Western blotting. They could be pivotal forunderstanding the pathogenic mechanisms of BS.

Key words: BLM helicase , Recombinant PCR , Multisite mutation , Gene cloning and expressing

张金彪, 许厚强, 骆衡, 许庆贺, 陈祥, 李坤. Bloom 综合症(BS) 和Rothmund-Tho-Bloom 解旋酶突变体的克隆与表达[J]. 生物技术通报, 2013, 0(1): 123-128.

Zhang Jinbiao, Xu Houqiang, Luo Heng, Xu Qinghe, Chen Xiang, Li Kun. Cloning and Expression of Bloom Helicase Mutant[J]. Biotechnology Bulletin, 2013, 0(1): 123-128.

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