国际生物医学工程杂志
國際生物醫學工程雜誌
국제생물의학공정잡지
INTERNATIONAL JOURNAL OF BIOMEDICAL ENGINEERING
2013年
1期
1-4,8
,共5页
李陪%董智雄%朱长军%刘海燕
李陪%董智雄%硃長軍%劉海燕
리배%동지웅%주장군%류해연
RNA干扰%esiRNA文库%KIF4A
RNA榦擾%esiRNA文庫%KIF4A
RNA간우%esiRNA문고%KIF4A
RNA interference%esiRNA library%KIF4A
目的 建立KIF4A核糖核酸内切酶Ⅲ酶切制备的小分子干扰RNA(esiRNA)文库,并探讨这种新型siRNA制备方法的优势.方法 制备502 bp的KIF4A基因组序列作为转录模板；体外转录获得双链RNA；用核糖核酸内切酶ⅢGST融合蛋白(GST-RNaseⅢ)酶切双链RNA,制备KIF4A esiRNA文库；应用KIF4A esiRNA和化学合成的KIF4A siRNA转染胃癌细胞株SGC-7901,应用Q-RT PCR和WesternBlot分别检测KIF4A的mRNA和蛋白水平.结果 利用GST-RNaseⅢ成功制备了KIF4A esiRNA文库,该文库可有效抑制SGC-7901细胞内的KIF4A表达,且抑制效果明显优于化学合成的KIF4A siRNA.结论 用生物学方法制备的KIF4A esiRNA文库可有效抑制KIF4A在细胞内的表达,且抑制效果优于化学合成的siRNA.
目的 建立KIF4A覈糖覈痠內切酶Ⅲ酶切製備的小分子榦擾RNA(esiRNA)文庫,併探討這種新型siRNA製備方法的優勢.方法 製備502 bp的KIF4A基因組序列作為轉錄模闆；體外轉錄穫得雙鏈RNA；用覈糖覈痠內切酶ⅢGST融閤蛋白(GST-RNaseⅢ)酶切雙鏈RNA,製備KIF4A esiRNA文庫；應用KIF4A esiRNA和化學閤成的KIF4A siRNA轉染胃癌細胞株SGC-7901,應用Q-RT PCR和WesternBlot分彆檢測KIF4A的mRNA和蛋白水平.結果 利用GST-RNaseⅢ成功製備瞭KIF4A esiRNA文庫,該文庫可有效抑製SGC-7901細胞內的KIF4A錶達,且抑製效果明顯優于化學閤成的KIF4A siRNA.結論 用生物學方法製備的KIF4A esiRNA文庫可有效抑製KIF4A在細胞內的錶達,且抑製效果優于化學閤成的siRNA.
목적 건립KIF4A핵당핵산내절매Ⅲ매절제비적소분자간우RNA(esiRNA)문고,병탐토저충신형siRNA제비방법적우세.방법 제비502 bp적KIF4A기인조서렬작위전록모판；체외전록획득쌍련RNA；용핵당핵산내절매ⅢGST융합단백(GST-RNaseⅢ)매절쌍련RNA,제비KIF4A esiRNA문고；응용KIF4A esiRNA화화학합성적KIF4A siRNA전염위암세포주SGC-7901,응용Q-RT PCR화WesternBlot분별검측KIF4A적mRNA화단백수평.결과 이용GST-RNaseⅢ성공제비료KIF4A esiRNA문고,해문고가유효억제SGC-7901세포내적KIF4A표체,차억제효과명현우우화학합성적KIF4A siRNA.결론 용생물학방법제비적KIF4A esiRNA문고가유효억제KIF4A재세포내적표체,차억제효과우우화학합성적siRNA.
Objective To establish KIF4A 3'UTR esiRNA library and analyze the advantage of method in studying the function of KIF4A in SGC-7901 cells.Methods The GST-fusion protein of Escherichia coli endoribonuclease Ⅲ (GST-RNase Ⅲ) was used to digest the double strand RNA (dsRNA),which was transcribed in vitro from a 502bp template of KIF4A genome (T7-KIF4A 3'UTR).The KIF4A esiRNA library generated from the above method and chemically synthesized KIF4A siRNA were then used to transfect SGC-7901 cells at 10 nmol/L and 20 nmol/L.Real-time quantitative PCR and Western Blot were used to detect the mRNA and protein level of KIF4A,respectively.Results The KIF4A esiRNA library was effectively established from dsRNA digestion using GST-RNase Ⅲ.KIF4A expression was significantly reduced in SGC-7901 cells transfected with KIF4A esiRNA or siRNA.In addition,the inhibitory effect of KIF4A esiRNA was more effective than that of chemically synthesized siRNA.Conclusion KIF4A esiRNA library which was obtained using biological method can more effectively inhibited the expression of KIF4A more effectively in SGC-7901 cells than chemically synthesized siRNA.Therefore,esiRNA library can be used as a new and more effective method in studying the function of KIF4A in SGC-7901 cells.
