中华放射医学与防护杂志
中華放射醫學與防護雜誌
중화방사의학여방호잡지
Chinese Journal of Radiological Medicine and Protection
2010年
3期
274-278
,共5页
王芹%李进%宋力%刘强%岳井银%穆传杰%唐卫生%樊飞跃
王芹%李進%宋力%劉彊%嶽井銀%穆傳傑%唐衛生%樊飛躍
왕근%리진%송력%류강%악정은%목전걸%당위생%번비약
IRM-2小鼠%cDNA文库%SMART技术
IRM-2小鼠%cDNA文庫%SMART技術
IRM-2소서%cDNA문고%SMART기술
IRM-2 mice%cDNA library%SMART techique
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.
目的 分離和鑒定IRM-2小鼠輻射抗性相關基因.方法 採用SMART技術構建IRM-2小鼠全長cDNA文庫,提取雄性IRM-2小鼠脾髒總RNA,以此為模闆,通過逆轉錄酶PowerScript反轉錄閤成第1鏈cDNA,通過長距離PCR閤成併擴增雙鏈cDNA.該擴增產物經純化、Sfi I酶切、去除小于500 bp片段後,將cDNA連接到sfi I消化過的PDNR-LIB質粒載體中,用電轉化法將重組質粒轉化到大腸桿菌DH5α,得到IRM-2小鼠cDNA原始文庫併用PCR法對文庫的質量進行鑒定.隨機從cDNA文庫挑選130箇暘性剋隆進行測序,與GenBank基因庫進行同源性比較.結果 構建的cDNA文庫的容量為2.25×106箇剋隆,重組率達95%,平均插入片段長度約1.2 kb,全長基因比例為55%.從cDNA文庫挑選的暘性剋隆中有21條EST序列與小鼠已知基因不同源,在GenBank的EST數據庫的註冊號為DW474856～DW474876.結論 成功構建瞭IRM-2小鼠全長cDNA文庫,21條EST提示IRM-2小鼠體內可能存在輻射抗性相關基因,為進一步分離和鑒定輻射抗性相關基因奠定瞭基礎.
목적 분리화감정IRM-2소서복사항성상관기인.방법 채용SMART기술구건IRM-2소서전장cDNA문고,제취웅성IRM-2소서비장총RNA,이차위모판,통과역전록매PowerScript반전록합성제1련cDNA,통과장거리PCR합성병확증쌍련cDNA.해확증산물경순화、Sfi I매절、거제소우500 bp편단후,장cDNA련접도sfi I소화과적PDNR-LIB질립재체중,용전전화법장중조질립전화도대장간균DH5α,득도IRM-2소서cDNA원시문고병용PCR법대문고적질량진행감정.수궤종cDNA문고도선130개양성극륭진행측서,여GenBank기인고진행동원성비교.결과 구건적cDNA문고적용량위2.25×106개극륭,중조솔체95%,평균삽입편단장도약1.2 kb,전장기인비례위55%.종cDNA문고도선적양성극륭중유21조EST서렬여소서이지기인불동원,재GenBank적EST수거고적주책호위DW474856～DW474876.결론 성공구건료IRM-2소서전장cDNA문고,21조EST제시IRM-2소서체내가능존재복사항성상관기인,위진일보분리화감정복사항성상관기인전정료기출.
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.
