CAJ | 학술논문

目的利用抑制性消减杂交技术构建太空诱变宫颈癌细胞的消减文库,为进一步从基因表达水平研究空间特殊环境影响肿瘤细胞生物学行为改变的分子机制奠定基础.方法采用Super SMART和SSH技术,以太空诱变的宫颈癌48A9细胞株作为Tester,地面正常对照组宫颈癌细胞株作为Driver,分离太空诱变的宫颈癌48A9细胞中差异表达的基因片段;连接T载体,转化宿主菌,构建太空诱变的宫颈癌48A9细胞cDNA抑制性消减文库.结果经蓝白筛选后随机挑取300个阳性克隆,以接头1和2R内侧序列为引物进行菌液PCR扩增鉴定阳性克隆,结果显示其中288个克隆有插入片段,阳性率为96%,大小分布在0.2～1.0 kb间.结论本实验利用抑制性消减杂交技术成功的构建了高质量的太空诱变宫颈癌细胞消减文库,为进一步从基因水平阐明太空特殊环境对肿瘤细胞生物学行为改变的分子机制奠定了基础.
목적 이용억제성소감잡교기술구건태공유변궁경암세포적소감문고,위진일보종기인표체수평연구공간특수배경영향종류세포생물학행위개변적분자궤제전정기출.방법 채용Super SMART화SSH기술,이태공유변적궁경암48A9세포주작위Tester,지면정상대조조궁경암세포주작위Driver,분리태공유변적궁경암48A9세포중차이표체적기인편단;련접T재체,전화숙주균,구건태공유변적궁경암48A9세포cDNA억제성소감문고.결과 경람백사선후수궤도취300개양성극륭,이접두1화2R내측서렬위인물진행균액PCR확증감정양성극륭,결과현시기중288개극륭유삽입편단,양성솔위96%,대소분포재0.2～1.0 kb간.결론 본실험이용억제성소감잡교기술성공적구건료고질량적태공유변궁경암세포소감문고,위진일보종기인수평천명태공특수배경대종류세포생물학행위개변적분자궤제전정료기출.
Objectives Construct a subtractive library of Caski cell line induced by exposing to the space environment by suppression subtractive hybridization and pave the way to explain the molecular mechanisms of the changes at the gene level. Methods Super SMART cDNA synthesis and suppression subtractive hybridization (SSH) were performed to isolate differentially expressed cDNA fragments from strains subclonal 48A9 cell line. cDNA from the 48A9 cell line were used as " tester" , and the other from the control Caski cell line as "driver". Subtractive products were directly inserted into T/A cloning vector, and then transformed into host bacteria to set up a subtractive cDNA library of specially or highly expressed genes in strains subclonal 48A9 cell line. Results mRNA were directly extracted and purified with good quality. Double strand cDNA were reverse transcripted integratedly, and then cut by Rsa I into even length short segments. Liga-tion was identified as high effective. After two hybridizations, a subtractive library of differentially expressed genes in strains subclonal 48A9 cell line was successfully constructed by SSH. Conclusion SSH is an effective approach to isolate differentially expressed genes.