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 library and discovery of four expressed sequence tags

作　　者：陈越[1] 王莉[1] 卫红飞[2] 杨煜[1] 于永利[1] 

机构地区：[1]吉林大学基础医学院免疫学教研室 [2]吉林大学基础医学院分子生物学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2004年第3期325-328,共4页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题 (30 0 70 70 5 )

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 m RNA后 ,用逆转录方法合成与 m RNA互补的 c DNA单链 ,再以此 c DNA单链为模板 ,得到与该 c DNA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 p SPORT1质粒 ,将此重组质粒通过电转化方法导入 E.coliDH5 α菌 ,得到脾脏细胞的 c DNA文库。筛选出阳性重组子 ,测序并分析得到的 ESTS序列。结果 :成功地构建了双阳型梅花鹿脾脏细胞 c DNA文库 ;并对部分库容 c DNA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 (ESTS) ,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 c DNA的部分序列。结论 :通过构建 cObjective 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.

关 键 词：基因文库 表达的序列标记 梅花鹿 

分 类 号：Q785[生物学—分子生物学]