双阳型梅花鹿脾脏细胞cDNA文库的构建和四个EST的发现

doi:国家自然科学基金资助课题

• 基础研究 • 下一篇

双阳型梅花鹿脾脏细胞cDNA文库的构建和四个EST的发现

陈越^1△，王莉¹，卫红飞²，杨煜¹，于永利^1*/sup>

1. 吉林大学基础医学院免疫学教研室，吉林长春130021；2. 吉林大学基础医学院分子生物学教研室，吉林长春130021

收稿日期:2003-12-02 修回日期:1900-01-01 出版日期:2004-05-28 发布日期:2004-05-28
通讯作者: 于永利

Construction of Sika deer spleen cell cDNA libraryand discovery of four expressed sequence tags

CHEN Yue^1△,WANG Li¹, WEI Hong-fei²,YANG　Yu¹, YU Yong-li^1*

1. Department of Immunology, School of Basic Medical Sciences, Jilin University,Changchun 130021,China; 2. Department of Molecular Biology, School of BasicMedical Sciences, Jilin University, Changchun 130021,China

Received:2003-12-02 Revised:1900-01-01 Online:2004-05-28 Published:2004-05-28
Contact: YU Yong-li

摘要/Abstract

摘要： 目的：开发和利用中国梅花鹿的基因资源，以期拥有一批梅花鹿基因的专利，并在专利保护下生产具有自己知识产权的基因工程药物。方法：用Trizol试剂提取梅花鹿脾脏细胞中的总RNA，分离得到mRNA后，用逆转录方法合成与mRNA互补的cDNA单链，再以此cDNA单链为模板，得到与该cDNA互补的另一条DNA链，将得到的DNA双链分子通过合适的Adapter连入pSPORT1质粒，将此重组质粒通过电转化方法导入E.coli DH5α菌，得到脾脏细胞的cDNA文库。筛选出阳性重组子，测序并分析得到的ESTS序列。结果：成功地构建了双阳型梅花鹿脾脏细胞cDNA文库；并对部分库容cDNA进行了克隆和测序，在这一过程中发现4个与已知基因同源的表达序列标签(ESTS)，其中2个EST分别是梅花鹿视网膜母细胞瘤的同源基因和梅花鹿维生素K依赖性蛋白前体cDNA的部分序列。结论：通过构建cDNA文库的方法成功地得到了一批有价值的梅花鹿的基因片段。

关键词: 表达的序列标记, 梅花鹿

Abstract: Objective To explore and protect the gene resources of Sika deer in China in oroder to develop recombinant-peptide-drugs derived from Sika deer genes. Methods The total RNA was isolated from Sika deer spleen cells by using the Trizol reagent and the total mRNA was purified, complement DNA(cDNA) and the second DNA were obtained by means of reverse transcriptase-PCR(RT-PCR) and this cDNA was used as the template respectively. The two strains of DNA was subcloned into the pSPORT1. The recombinant plasmids of pSPORT1 were transformed into the E.coli by means of electric transcription. The cDNA library was obtained. The positive recombinant-clone was selected and the sequences of ESTS were analyzed. Results Sika deer spleen cell cDNA library was established successfully; the clones in it was sequenced, during the process some expresion sequence tags were discovered that shared high homology with known cDNAs deposited in the GenBank. EST-1 was homologous to human retinoblastoma binding protein 4. The protein sequence of EST-2 was 26% homologous to human vitamin K-dependent protein precursor. Conclusion Some valuable gene fragments from Sika deer spleen cell cDNA library were obstained.

Key words: expressed sequence tags, ika deer

中图分类号:

Q785

陈越，王莉，卫红飞，杨煜，于永利. 双阳型梅花鹿脾脏细胞cDNA文库的构建和四个EST的发现[J]. J4, 2004, 30(3): 325-328.

CHEN Yue,WANG Li, WEI Hong-fei,YANG　Yu, YU Yong-li. Construction of Sika deer spleen cell cDNA libraryand discovery of four expressed sequence tags[J]. J4, 2004, 30(3): 325-328.

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双阳型梅花鹿脾脏细胞cDNA文库的构建和四个EST的发现

Construction of Sika deer spleen cell cDNA libraryand discovery of four expressed sequence tags

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