细胞与分子免疫学杂志
細胞與分子免疫學雜誌
세포여분자면역학잡지
2009年
10期
866-869
,共4页
白文栋%张瑞%孟艳玲%曹云新%王涛%赵晶%杨安钢
白文棟%張瑞%孟豔玲%曹雲新%王濤%趙晶%楊安鋼
백문동%장서%맹염령%조운신%왕도%조정%양안강
Runx3%腺病毒%恶性脑胶质瘤细胞
Runx3%腺病毒%噁性腦膠質瘤細胞
Runx3%선병독%악성뇌효질류세포
Runx3%adenovirus%malignant glioma cells
目的:制备含有融合Flag标签的Runx3基因的复制缺陷型重组腺病毒,感染神经胶质细胞瘤U251,观察外源Runx3在细胞中的表达及亚细胞定位.方法:用PCR的方法扩增Runx3基因,并将Flag标签蛋白的编码基因与RUNX3基因进行融合,构建腺病毒穿梭载体pShuttle-CMV-Runx3,经Kpn Ⅰ/Xho Ⅰ双酶切鉴定并测序.利用电转化方法将经Pme Ⅰ线性化的pShuttle-CMV-Runx3穿梭载体导入BJ5183重组细菌,获取重组腺病毒质粒Ad-Runx3,再将经Pac Ⅰ线性化的Ad-Runx3重组病毒骨架质粒转染293A包装细胞,包装并扩增病毒.利用该病毒感染神经胶质细胞瘤U251,用免疫印迹法观察外源Runx3在细胞中的表达,用间接免疫荧光法观察其在细胞内的定位.结果:构建并包装表达Runx3蛋白的重组腺病毒,用重组腺病毒感染U251细胞后,经免疫印迹和间接免疫荧光法检测,可见外源导入的Runx3蛋白在细胞核内的特异性定位.结论:成功制备了含有融合Flag标签的转录因子Runx3基因的重组腺病毒,感染U251细胞,在细胞中观察到该分子表达后定位于细胞核中,为研究Runx3在神经胶质瘤发生中的作用奠定了实验基础.
目的:製備含有融閤Flag標籤的Runx3基因的複製缺陷型重組腺病毒,感染神經膠質細胞瘤U251,觀察外源Runx3在細胞中的錶達及亞細胞定位.方法:用PCR的方法擴增Runx3基因,併將Flag標籤蛋白的編碼基因與RUNX3基因進行融閤,構建腺病毒穿梭載體pShuttle-CMV-Runx3,經Kpn Ⅰ/Xho Ⅰ雙酶切鑒定併測序.利用電轉化方法將經Pme Ⅰ線性化的pShuttle-CMV-Runx3穿梭載體導入BJ5183重組細菌,穫取重組腺病毒質粒Ad-Runx3,再將經Pac Ⅰ線性化的Ad-Runx3重組病毒骨架質粒轉染293A包裝細胞,包裝併擴增病毒.利用該病毒感染神經膠質細胞瘤U251,用免疫印跡法觀察外源Runx3在細胞中的錶達,用間接免疫熒光法觀察其在細胞內的定位.結果:構建併包裝錶達Runx3蛋白的重組腺病毒,用重組腺病毒感染U251細胞後,經免疫印跡和間接免疫熒光法檢測,可見外源導入的Runx3蛋白在細胞覈內的特異性定位.結論:成功製備瞭含有融閤Flag標籤的轉錄因子Runx3基因的重組腺病毒,感染U251細胞,在細胞中觀察到該分子錶達後定位于細胞覈中,為研究Runx3在神經膠質瘤髮生中的作用奠定瞭實驗基礎.
목적:제비함유융합Flag표첨적Runx3기인적복제결함형중조선병독,감염신경효질세포류U251,관찰외원Runx3재세포중적표체급아세포정위.방법:용PCR적방법확증Runx3기인,병장Flag표첨단백적편마기인여RUNX3기인진행융합,구건선병독천사재체pShuttle-CMV-Runx3,경Kpn Ⅰ/Xho Ⅰ쌍매절감정병측서.이용전전화방법장경Pme Ⅰ선성화적pShuttle-CMV-Runx3천사재체도입BJ5183중조세균,획취중조선병독질립Ad-Runx3,재장경Pac Ⅰ선성화적Ad-Runx3중조병독골가질립전염293A포장세포,포장병확증병독.이용해병독감염신경효질세포류U251,용면역인적법관찰외원Runx3재세포중적표체,용간접면역형광법관찰기재세포내적정위.결과:구건병포장표체Runx3단백적중조선병독,용중조선병독감염U251세포후,경면역인적화간접면역형광법검측,가견외원도입적Runx3단백재세포핵내적특이성정위.결론:성공제비료함유융합Flag표첨적전록인자Runx3기인적중조선병독,감염U251세포,재세포중관찰도해분자표체후정위우세포핵중,위연구Runx3재신경효질류발생중적작용전정료실험기출.
AIM: To construct the replicative defecient adenovirus Ad-Runx3 expressing Runx3, and to express it in U251 malignant glioblastoma cells. METHODS: The runx3 gene with a flag tag was amplified by PCR using pCMV5-AML2 as a template, and was confirmed by DNA sequencing. The adenovirus shuttle vector pShuttle-CMVRunx3 was constructed by introducing runx3 DNA fragment into the sites of Kpn Ⅰ and Xho Ⅰ of pShuttle-CMV vector. This recombinant plasmid was linearized by Pmel and electronically transfected into BJ5183 cells to get the recombinant adenovirus vector Ad-Runx3. The recombinant adenovirus expressing Runx3 was infected into U251 malignant glioblastoma cells. The expression of exogenous Runx3 was observed by immonoblotting and its localization was detected by immunostaining using anti-Flag tag antibody. RESULTS: The recombinant adenovirus expressing Runx3 with a Flag tag was constructed and infected into U251 glioblastoma cells. The exogenous Runx3 protein was detected only in the nuclei. CONCLUSION: The recombinant adenovirus expressing Runx3 with a Flag tag is constructed successfully, and the Runx3 protein expressed in the nuclei of infected cells. The study laid a foundation for further research of the function of Runx3 in gliocarcinogenesis.
