大鼠肝再生相关基因LRRP1的克隆化  

Cloning of rat liver regeneration related gene LRRP1

作　　者：王刚[1] 刘妍[1] 牟劲松[1] 洪源[1] 邵得志[1] 张耀新[1] 李莉[1] 成军[1] 

机构地区：[1]中国人民解放军第302医院传染病研究所基因治疗研究中心,北京市100039

出　　处：《世界华人消化杂志》2002年第2期165-168,共4页World Chinese Journal of Digestology

摘　　要：目的:利用抑制性消减杂交技术构建大鼠肝脏部分切除术后差异表达基因的cDNA文库,并通过同源引物反转录PCR的方法克隆大鼠肝再生相关基因LRRP1的全长序列.方法:分别提取肝脏部分切除术后24h以及对照大鼠肝脏mRNA,以之为模板合成cDNA,称为测试和驱动.酶切消化后将测试分成两组,分别与不同的接头连接,分别与驱动进行杂交,然后混合两份杂交液,加入过量的驱动不经变性进行第二次杂交即消减杂交.之后进行两次PCR扩增,将产物克隆入质粒载体,构建差显文库,测序后以生物信息学技术进行序列同源性比较.通过同源引物反转录PCR的方法克隆基因全序列,并用软件分析其蛋白结构.结果:成功构建大鼠肝脏部分切除术后的cDNA差显文库,消减效果良好.将所得的部分基因进行测序分析,选择一个未知序列,通过同源引物反转录PCR的方法克隆到大鼠肝再生相关基因LRRP1的全长序列,并初步分析了其蛋白结构.结论:确定了大鼠肝再生相关蛋白LRRP1的基因序列,并证明其在大鼠肝再生过程中高度表达.AIM:To construct the differentially expressed cDNA library af ter liver partial hepatectomy of rats by suppression subtractive hybridization m ethod and clone genes related to liver regeneration. METHODS:The mRNAs of partial hepatectomy and control rats were e xtracted and reverse transcribed into cDNA which were named tester and driver, r espectively. After restrictive digestion, the tester was divided into two parts, linked with different adapters and hybridized with driver, respectively. The tw o parts was combined and added with overdosed driver to process the second hybri dization. The products were amplified by PCR twice and cloned into plasmids to c onstruct the library. Selected clones were sequenced and the sequences were anal yzed for homology. One unknown sequence was selected and was subjected RT-PCR a mplification by homologous primers to acquire full length cDNA. RESULTS:The cDNA library of differentially expressed genes relat ed to liver regeneration was constructed successfully. 150 clones were selected to be sequenced. A full length cDNA was acquired and named LRRP1. The 2nd structure of the protein was predicted by software. CONCLUSION:A novel protein LRRP1 was acquired and be proved over expression during rat liver regeneration.

关 键 词：肝再生 肝切除术 PCR技术 LRRP1基因 

分 类 号：R346[医药卫生—基础医学]