中华耳鼻咽喉科杂志
中華耳鼻嚥喉科雜誌
중화이비인후과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY
2001年
1期
47-50
,共4页
张必成%余鹰%邱元正%钱骏%周鸣%李忠花%张小慧%向娟娟%朱诗国%李桂源
張必成%餘鷹%邱元正%錢駿%週鳴%李忠花%張小慧%嚮娟娟%硃詩國%李桂源
장필성%여응%구원정%전준%주명%리충화%장소혜%향연연%주시국%리계원
鼻咽%DNA,重组%基因文库%遗传学技术
鼻嚥%DNA,重組%基因文庫%遺傳學技術
비인%DNA,중조%기인문고%유전학기술
目的采用SMART (switching mechanism at 5′ end of RNA transcript)技术，快速构建了高质量的成人鼻咽上皮组织定向cDNA文库。方法从成人正常鼻咽上皮组织分离总RNA，利用经修饰的oligo（dT）引物（含sfi IB酶切位点）合成cDNA第一链，同时根据真核生物mRNA 5′端帽子结构特点，利用SMART核苷酸（含sfi IA酶切位点）作为cDNA第二链在mRNA 5′端延伸出去的模板，进而以此序列为引物利用LD-PCR（Long-distance-PCR）合成双链cDNA，双链cDNA经sfi I(IA & IB)酶切和过柱分级分离后，克隆入经sfi I酶切的λTripIEX2载体后经体外包装而成cDNA文库。结果获得1.5×106个重组子，重组率达100%。文库扩增后，滴度达3.8×109 pfu/ml，插入cDNA平均长度为1.5 kb。结论构建的成人鼻咽cDNA 文库具有良好的质量，该cDNA文库为进一步筛选、克隆鼻咽癌抑瘤基因及鼻咽组织特异性表达基因奠定了基础。
目的採用SMART (switching mechanism at 5′ end of RNA transcript)技術，快速構建瞭高質量的成人鼻嚥上皮組織定嚮cDNA文庫。方法從成人正常鼻嚥上皮組織分離總RNA，利用經脩飾的oligo（dT）引物（含sfi IB酶切位點）閤成cDNA第一鏈，同時根據真覈生物mRNA 5′耑帽子結構特點，利用SMART覈苷痠（含sfi IA酶切位點）作為cDNA第二鏈在mRNA 5′耑延伸齣去的模闆，進而以此序列為引物利用LD-PCR（Long-distance-PCR）閤成雙鏈cDNA，雙鏈cDNA經sfi I(IA & IB)酶切和過柱分級分離後，剋隆入經sfi I酶切的λTripIEX2載體後經體外包裝而成cDNA文庫。結果穫得1.5×106箇重組子，重組率達100%。文庫擴增後，滴度達3.8×109 pfu/ml，插入cDNA平均長度為1.5 kb。結論構建的成人鼻嚥cDNA 文庫具有良好的質量，該cDNA文庫為進一步篩選、剋隆鼻嚥癌抑瘤基因及鼻嚥組織特異性錶達基因奠定瞭基礎。
목적채용SMART (switching mechanism at 5′ end of RNA transcript)기술，쾌속구건료고질량적성인비인상피조직정향cDNA문고。방법종성인정상비인상피조직분리총RNA，이용경수식적oligo（dT）인물（함sfi IB매절위점）합성cDNA제일련，동시근거진핵생물mRNA 5′단모자결구특점，이용SMART핵감산（함sfi IA매절위점）작위cDNA제이련재mRNA 5′단연신출거적모판，진이이차서렬위인물이용LD-PCR（Long-distance-PCR）합성쌍련cDNA，쌍련cDNA경sfi I(IA & IB)매절화과주분급분리후，극륭입경sfi I매절적λTripIEX2재체후경체외포장이성cDNA문고。결과획득1.5×106개중조자，중조솔체100%。문고확증후，적도체3.8×109 pfu/ml，삽입cDNA평균장도위1.5 kb。결론구건적성인비인cDNA 문고구유량호적질량，해cDNA문고위진일보사선、극륭비인암억류기인급비인조직특이성표체기인전정료기출。
Objective To construct a directional cDNA library from human adult nasopharynx by SMART (switching mechanism at 5′ end of RNA transcript) technique. Methods The total RNA was separated from human adult nasopharynx epithelial fissue and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer(contained sfi IB site) while the SMART oligonudeotide(contained sfi IA site) was utilized as a template so that the frist-strand cDNA could be extended over the 5′end of mRNA. The double-strand cDNA was amplified by LD-PCR(long-distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction enzyme.After cDNA size fractionation through CHROMA SPIN column,the double-strand cDNA was ligated into the sfi I-digested λTripIEx2 vector and then the recombinant DNA was packaged in vitro. Results The unamplified human adult nasopharynx cDNA library consists of 1.5×106 independent clones in which the percentage of recombinant clones is about 100%.The titer of the amplified cDNA library is 3.8×109 pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. Conclusion These results shows that the human adult nasopharynx cDNA library has an excellent quality and lays solid foundation for screening and cloning new tumor suppressor genes of nasopharyngeal carcinoma(NPC) and tissue-specific genes of human nasopharynx.
