CAJ | 학술논문

目的：构建结肠癌转移相关基因1（MACC1）RNAi慢病毒表达载体，并转染人乳腺癌细胞株MB-231，观察其最佳感染条件。方法设计并合成MACC1基因特异性的DNA寡核苷酸，连接到经AgeⅠ和EcoRⅠ双酶切线性化的pMAGic慢病毒质粒载体中，转化大肠埃希菌DH5a感受态细胞，筛选阳性菌落、扩增后提取质粒，进行DNA测序鉴定。用293FT 细胞包装产生慢病毒，感染MB-231细胞，选择感染效率高、感染复数（MOI）低的感染条件。结果 PCR与测序鉴定证实成功构建了 MACC1 RNAi的慢病毒载体，可以高效转染MB-231细胞，其最佳感染条件为MOI＝40。结论成功构建了MACC1 RNAi慢病毒表达载体，其可高效感染MB-231，为进一步研究靶向 MACC1 RNAi对乳腺癌细胞恶性生物学行为变化及基因治疗研究奠定了实验基础。
목적：구건결장암전이상관기인1（MACC1）RNAi만병독표체재체，병전염인유선암세포주MB-231，관찰기최가감염조건。방법설계병합성MACC1기인특이성적DNA과핵감산，련접도경AgeⅠ화EcoRⅠ쌍매절선성화적pMAGic만병독질립재체중，전화대장애희균DH5a감수태세포，사선양성균락、확증후제취질립，진행DNA측서감정。용293FT 세포포장산생만병독，감염MB-231세포，선택감염효솔고、감염복수（MOI）저적감염조건。결과 PCR여측서감정증실성공구건료 MACC1 RNAi적만병독재체，가이고효전염MB-231세포，기최가감염조건위MOI＝40。결론성공구건료MACC1 RNAi만병독표체재체，기가고효감염MB-231，위진일보연구파향 MACC1 RNAi대유선암세포악성생물학행위변화급기인치료연구전정료실험기출。
Objective To construct a lentiviral vector for RNA interference(RNAi)of MACC1 gene and to detect the best trans-fection condition by transfected into MB-231 cells .Methods The siRNA was designed and converted into cDNA of shRNA (small hair pin RNA) of siRNA for MACC1 gene .The cDNA was synthesized and inserted into pMAGic lentiviral plasmid vector which was linearized by enzyme Age Ⅰ and EcoRⅠ .The recombinant plasmid was transformed into competent E .coli DH5α cells .The positive recombinant colony was selected by ampicillin medium agar and identified by DNA sequencing .The recombinant lentivirus was packaged into mature lentivirus by 293FT cells and used to infect MB-231 cells .To detected the transfection condition of high efficiency of infection and low multiplicity of infection .Results PCR and sequencing verified that the recombinant lentivirus plasmid MACC1-shRNA was successfully constructed .The best transfection condition was MOI=40 by transfected into MB-231 .Conclu-sion The lentiviral RNAi expression vector targeting MACC 1 gene is successfully constructed and it can infect MB-231 cells effi-ciently ,which lays the experimental foundation for the research on the changes of malignant biological activity of tumor cell lines and gene therapy .