Screening and Cloning of the Target Genes Down-regulated by β-interferon plasmid Using Suppression Subtractive Hybridization Technique

ZHONG Yan-Wei; CHENG Jun; QU Jian-Hui; ZHANG Li-Ying; GUO Jiang; LI Xiao-Dong

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February 2006

ZHONG Yan-Wei, CHENG Jun, QU Jian-Hui, ZHANG Li-Ying, GUO Jiang and LI Xiao-Dong. Screening and Cloning of the Target Genes Down-regulated by β-interferon plasmid Using Suppression Subtractive Hybridization Technique[J]. Virologica Sinica, 2006, 21(1): 21-23.

Citation: ZHONG Yan-Wei, CHENG Jun, QU Jian-Hui, ZHANG Li-Ying, GUO Jiang, LI Xiao-Dong. Screening and Cloning of the Target Genes Down-regulated by β-interferon plasmid Using Suppression Subtractive Hybridization Technique .VIROLOGICA SINICA, 2006, 21(1) : 21-23.

SSH技术筛选β干扰素真核表达载体转染细胞的下调基因

1.
解放军第302医院传染病研究所病毒性肝炎研究室, 北京 100039

通讯作者： 钟彦伟, ;

摘要

筛选β-干扰素质粒转染下调相关基因。以β-干扰素表达质粒pcDNA3.1(-)-IFNβ转染HepG2细胞;同时以空载体pcDNA3.1(-)为对照；制备转染后的细胞裂解液;从中提取mRNA并合成cDNA;经RsaI酶切后将来自pcDNA3.1(-)转染的cDNA分成两组;分别与两种不同的接头adaptor 1和adaptor 2衔接;再与来自pcDNA3.1(-)-IFNβ转染的cDNA进行两次消减杂交及两次抑制性PCR;将产物与T/A载体连接;构建cDNA消减文库;并转染大肠杆菌进行文库扩增;随机挑选克隆PCR后进行测序及同源性分析。成功构建人β-干扰素质粒转染下调相关基因差异表达的cDNA。所获得的50个克隆中;随机挑选37个克隆均含有插入片段;将这些克隆进行序列测定;并通过生物信息学分析获得其全长基因序列;结果共获得22种编码基因;其中3种为未知功能的基因。筛选到的cDNA序列;包括与细胞生长调节、物质代谢和细胞凋亡密切相关的一些蛋白编码基因。
- β-干扰素
- , 下调
- , 抑制性消减杂交技术(SSH)

Screening and Cloning of the Target Genes Down-regulated by β-interferon plasmid Using Suppression Subtractive Hybridization Technique

1.
Center for Viral Hepatitis Research, Institute of Infectious Diseases, Chinese PLA 302 Hospital, Beijing 100039, China

Corresponding author: ZHONG Yan-Wei,

Abstract

The suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes down-regulated byβ-interferon in HepG2. The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)IFNβand pcDNA3.1(-) empty vector, respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA from pcDNA3.1(-) empty vector transfected cell was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the products were subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search. The subtractive library of genes down-regulated byβ-interferon was constructed successfully. The amplified library contained 50 clones, of which 37 clones had 200-1000 bp inserts. Sequence analysis indicated that 22 clones containing the coding sequences , of which 19 had homology in the GenBank and 3 were unknown. The obtained sequences may be target genes regulated by β-interferon, and some genes encode proteins involved in cell cycle regulation, metabolism, and cell apoptosis.
- β-interferon
- , Down-regualated
- , Suppression subtractive hybridization (SSH)

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Screening and Cloning of the Target Genes Down-regulated by β-interferon plasmid Using Suppression Subtractive Hybridization Technique

Corresponding author: ZHONG Yan-Wei,

1. Center for Viral Hepatitis Research, Institute of Infectious Diseases, Chinese PLA 302 Hospital, Beijing 100039, China

Abstract: The suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes down-regulated byβ-interferon in HepG2. The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)IFNβand pcDNA3.1(-) empty vector, respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA from pcDNA3.1(-) empty vector transfected cell was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the products were subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search. The subtractive library of genes down-regulated byβ-interferon was constructed successfully. The amplified library contained 50 clones, of which 37 clones had 200-1000 bp inserts. Sequence analysis indicated that 22 clones containing the coding sequences , of which 19 had homology in the GenBank and 3 were unknown. The obtained sequences may be target genes regulated by β-interferon, and some genes encode proteins involved in cell cycle regulation, metabolism, and cell apoptosis.