IRM-2小鼠全长cDNA文库的构建及鉴定

目的 分离和鉴定IRM-2小鼠辐射抗性相关基因。方法 采用SMART技术构建IRM-2小鼠全长cDNA文库,提取雄性IRM-2小鼠脾脏总RNA,以此为模板,通过逆转录酶PowerScript反转录合成第1链cDNA,通过长距离PCR合成并扩增双链 cDNA。该扩增产物经纯化、Sfi Ⅰ酶切、去除小于500 bp片段后,将cDNA连接到Sfi Ⅰ消化过的PDNR-LIB质粒载体中,用电转化法将重组质粒转化到大肠杆菌DH5α,得到IRM-2小鼠cDNA原始文库并用PCR法对文库的质量进行鉴定。随机从cDNA文库挑选130个阳性克隆进行测序,与GenBank基因库进行同源性比较。结果 构建的cDNA文库的容量为2.25×10⁶个克隆,重组率达95%,平均插入片段长度约1.2 kb,全长基因比例为55%。从cDNA文库挑选的阳性克隆中有21条EST序列与小鼠已知基因不同源,在GenBank的EST数据库的注册号为DW474856~DW474876。结论 成功构建了IRM-2小鼠全长cDNA文库,21条EST提示IRM-2小鼠体内可能存在辐射抗性相关基因,为进一步分离和鉴定辐射抗性相关基因奠定了基础。

Construction and characterization of cDNA library for IRM-2 mice

Objective To screen and isolate the radioresistance related genes of IRM-2 mice.Methods cDNA library of IRM-2 mice was constructed by SMART technique.Total RNA was isolated from spleens of IRM-2 male mice.The first-strand cDNA was synthesized by using PowerScript reverse transeriptase,and double-strand cDNA was synthesized and amplified by long PCR.The PCR products were purified,digested with restriction enzyme Sfi I.The ds-cDNA fragment lessthan 500 bp was fractionated and ligated to the Sfi I-digested pDNR-LIB vector.The ligation mixture was transformed into E.coil DH5α by electroporution transformation to generate the unamplified cDNA library.The quality of cDNA library was identified by PCR technique.130 clones from cDNA library were sequenced and compared with GenBank database.Results The cDNA library contained 2.25 x 106 independent clones with an average insert size of 1.2 kb.The ratio of recombination and full-length was 95% and 55%,respectively.21 pieces of EST sequences from cDNA library were not the same as the known mice genes and registered into GenBank EST database,with registered number DW474856-DW474876.Conclusions cDNA library of IRM-2 mice has been constructed successfully.21 pieces of EST implies that radioresistance correlative genes may be in IRM-2 mice,which will lay a foundation for isolating and identifying radioresistance related genes in further study.