中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (2): 289-293.doi: 10.3969/j.issn.1673-8225.2010.02.023

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

Bcl-x微基因及其突变微基因的构建与鉴定

黄  波1，王小中1，王彩纹1，李  静2，章海斌1，熊火梅1，肖  芸1　　   

  1. 1南昌大学第二附属医院检验科，江西省南昌市  330006；2南昌大学第一附属医院检验科，江西省南昌市  330006
  • 出版日期:2010-01-08 发布日期:2010-01-08
  • 通讯作者: 王小中，副教授，南昌大学第二附属医院检验科，江西省南昌市 330006 wangxzlj@126.com
  • 作者简介:黄 波☆，男，1983年生，江西省鄱阳县人，汉族，南昌大学在读硕士，主要从事疾病分子机制及其诊断研究。 jxmuhb666@sohu.com
  • 基金资助:

    国家自然科学基金项目(3070033 8)*

Construction and identification of Bcl-x minigene and its mutated minigene

Huang Bo1, Wang Xiao-zhong1, Wang Cai-wen1, Li Jing2, Zhang Hai-bin1, Xiong Huo-mei1, Xiao Yun1   

  1. 1 Department of Laboratory Medicine, Second Affiliated Hospital of Nanchang University, Nanchang   330006, Jiangxi Province, China; 2 Department of Laboratory Medicine, First Affiliated Hospital of Nanchang University, Nanchang   330006, Jiangxi Province, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Wang Xiao-zhong, Associate professor, Department of Laboratory Medicine, Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China wangxzlj@126.com
  • About author:Huang Bo★, Studying for master’s degree, Department of Laboratory Medicine, Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China jxmuhb666@sohu.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30700338*

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被引次数

1

摘要：

背景：Bcl-xL/Bcl-xS比值的增高与肿瘤的发生密切相关，其机制尚未阐明。Bcl-x微基因是研究其选择性剪接机制的重要工具。
目的：构建人类凋亡相关基因Bcl-x的微基因及其突变微基因，为进行Bcl-x基因选择性剪接方面的研究打下基础。
方法：首先采用PCR法从人白血病细胞K562基因组DNA中扩增出Bcl-x基因外显子2及内含子2的5’部分序列片段，定向插入真核表达载体pcDNA3.1(-)中，然后扩增出Bcl-x基因内含子2的3’端及外显子3部分序列，定向插入上一片段下游，构建成 pcDNA3.1(-)-bcl-x微基因，测序鉴定无误后,用pcDNA3.1(-)-bcl-x微基因瞬时转染HL-60，通过RT-PCR方法对其在细胞内表达作进一步鉴定。另外，以pcDNA3.1(-)-bcl-x微基因为模板，利用反向PCR法构建突变微基因pcDNA3.1(-)-bcl-x-CRCE1(M)。
结果与结论：以人白血病细胞K562基因组DNA为模板，P1和P2为引物，经PCR扩增得约686 bp的目的片段；以P3和P4为引物扩增出约178 bp的目的片段。微基因pcDNA3.1(-)-bcl-x 分别经Xba Ⅰ和Eco RⅠ双切、Xba Ⅰ和Xho Ⅰ双切鉴定均得到预期的片段，突变微基因 pcDNA3.1(-)-bcl-x-CRCE1(M)经EcoR Ⅰ单酶切后，均得到预期片段，测序结果正确，表明成功构建人类凋亡相关基因Bcl-x的微基因及其突变微基因。

关键词: 微基因, 突变微基因, 选择性剪接, Bcl-x, 顺式作用原件

Abstract:

BACKGROUND: It is not elucidated about its mechanism of the rising ratio of Bcl-xL/Bcl-xS linked closely with the development of tumors. The Bcl-x minigene is an important tool to study its mechanism of alternative splicing.
OBJECTIVE: To construct the human apoptosis-related Bcl-x minigene and its mutated minigene, and to provide basis for studying the alternative splicing of Bcl-x gene.
METHODS: At First, the fragment (P1P2) including exon 2 and part of 5’ sequence of intron 2 in bcl-x gene were amplified by PCR from human leukemia cell K562 genomic DNA, then directionally inserted into the eukaryotic expression vector pcDNA3.1(-), called pcDNA3.1(-)-p1p2; secondly, the fragment (P3P4) including 3’ sequence of intron 2 and part of exon 3 in bcl-x gene were amplified, then directionally inserted into the downstream of fragment p1p2, called pcDNA3.1(-)-p1p2-p3p4, it is pcDNA3.1(-)-bcl-x minigene. By sequencing and identifying, pcDNA3.1(-)-bcl-x minigene was introduced into HL-60 by transient transfection, so as to identify its expression by RT-PCR. In addition, pcDNA3.1(-)-bcl-x minigene was utilized as a template to construct its mutated minigene called pcDNA3.1(-)-bcl-x-CRCE1 (M) by inverse PCR.
RESULTS AND CONCLUSION: Taking human leukemia cell K562 genome DNA as the template, P1 and P2 as primers, approximately 686-bp target fragment were obtained by PCR amplification; taking P3 and P4 as primers, approximately 178-bp target fragment could be obtained. Through XbaⅠ and Eco RⅠdouble enzyme digestion, Xba Ⅰ and Xho Ⅰ double enzyme digestion, the anticipated fragment could be obtained from minigene pcDNA3.1(-)-bcl-x, also from mutated minigene pcDNA3.1(-)-bcl-x-CRCE1(M) through Eco RI single enzyme digestion, the sequence result was correct. Results showed that we successfully construct the minigene and mutated minigene of human apoptotic-related gene bcl-x. 

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