通过差减文库筛选山羊羔肠道淋巴集结特异表达基因

通过差减文库筛选山羊羔肠道淋巴集结特异表达基因

张磊, 杨永杰, 陈小鹏, 梁奉军, 张燕君^*

山东大学生命科学学院, 山东济南250100)

收稿日期:2005-10-09 修回日期:1900-01-01 出版日期:2006-10-24 发布日期:2006-10-24
通讯作者: 张燕君

Screening of Goat Peyer's patch differentially expressed genes

ZHANG Lei,YANG Yong-jie, CHEN Xiao-peng,LIANG Feng-jun and ZHANG Yan-jun^*

School of life Sciences, Shangdong University, Jinan 250100, Shandong, China

Received:2005-10-09 Revised:1900-01-01 Online:2006-10-24 Published:2006-10-24
Contact: ZHANG Yan-jun

摘要/Abstract

摘要： 羊羔肠道淋巴集合淋巴结（Peyer's patch，以下简称PP），是B淋巴细胞的主要来源地和发育、成熟场所，兼具中枢淋巴器官和周围淋巴器官的功能.采用抑制性差减杂交（suppression subtractive hybridization，SSH）方法，构建山羊羔肠道淋巴集结与其非PP肠壁组织的差减 cDNA 文库，以期找到在山羊羔肠道淋巴集结中特异表达的与免疫相关的基因. 分别从山羊羔PP与非PP肠壁组织提取总RNA，反转录成cDNA，以PP作为待检组织(tester)，非PP肠壁作为驱动组织(driver)，经过两轮杂交和抑制性PCR扩增，产物与T载体连接，经蓝白斑筛选，提取质粒,经EcoRI酶切鉴定插入片段并测序,由此构建了两种组织间差异表达基因的差减cDNA文库. 对其中160个克隆测序，得到几个可能与免疫相关的基因，为进一步从中挑选和表达活性蛋白基因用于生产免疫增强剂奠定了基础.

关键词: 山羊, 肠道淋巴集结, 特异表达基因 , 差减cDNA文库

Abstract: Goat Peyer's patch is both central and peripheral immune organ where Blymphocytes origin, develop and maturate. In order to obtain Peyer's patch specially expressed genes, we constructed a subtractive cDNA library using suppression subtractive hybridization from Peyer's patch (PP) and nonPP tissue. Lamb PP tissue was designated as the experimental group (the tester) and the adjacent intestinal nonPP tissue as the control group (the driver). Tester and driver RNA were extracted and then transcribed to cDNA reversely. Two tester populations were created with different adaptors while driver cDNA had no adaptors. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. Templates for PCR amplification were generated from differentially expressed sequences. Using suppression PCR, only differentially expressed sequences were amplified exponentially. Background was reduced and differentially expressed sequences were further enriched. The PCR products were ligated to TVector. 160 positive clones were obtained and sequenced. Homology analysis showed that a Bcell lineage specific activator protein (BSAP) was among them. The subtractive library and the obtained ESTs will be useful for identifying immuneassociated genes and constructing active protein expression vector.

Key words: differentially expressed genes , subtractive cDNA library, Peyer's patch, goat

中图分类号:

Q781

张磊,杨永杰,陈小鹏,梁奉军,张燕君* . 通过差减文库筛选山羊羔肠道淋巴集结特异表达基因[J]. J4, 2006, 41(1): 140-144 .

ZHANG Lei,YANG Yong-jie,CHEN Xiao-peng,LIANG Feng-jun and ZHANG Yan-jun* . Screening of Goat Peyer's patch differentially expressed genes[J]. J4, 2006, 41(1): 140-144 .

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