[1]王海晶,杨和平,周向东.RNA干扰CA916798基因真核表达载体的构建与鉴定[J].第三军医大学学报,2008,30(01):23-26.
 WANG Hai-jing,YANG He-ping,ZHOU Xiang-dong.Construction and identification of CA916798 eukaryotic expression vector for RNA interference[J].J Third Mil Med Univ,2008,30(01):23-26.
点击复制

RNA干扰CA916798基因真核表达载体的构建与鉴定(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
30卷
期数:
2008年第01期
页码:
23-26
栏目:
论著
出版日期:
2008-01-15

文章信息/Info

Title:
Construction and identification of CA916798 eukaryotic expression vector for RNA interference
作者:
王海晶杨和平周向东
第三军医大学西南医院呼吸内科
Author(s):
WANG Hai-jingYANG He-pingZHOU Xiang-dong
Department of Respiratory Diseases,Southwest Hospital,Third Military Medical University
关键词:
CA916798RNA干扰真核表达载体构建转染
Keywords:
CA916798RNA interferenceeukaryotic expression vectorconstructiontransfection
分类号:
R394-33; R734.2
文献标志码:
A
摘要:
目的  构建CA916798基因RNA干扰(RNAi)的真核细胞表达载体。  方法  以CA916798为靶基因,以pGenSil-l质粒为载体,设计构建重组体,根据GenBank数据库提供的CA916798基因核苷酸序列,按照Tuschl设计原则,选择设计2条带发夹结构的核苷酸序列,克隆到空载体pGenSil-l中,转化DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析。用脂质体2 000将重组质粒转染A549/CDDP细胞并用G418筛选,MTT法测定药物敏感性并绘制细胞增殖曲线。  结果  构建针对CA916798的pRNAi-CA916798shRNA,经限制性内切酶酶切、PCR和DNA测序证实与设计完全一致;在荧光显微镜下观察到A549/CDDP细胞表达绿色荧光蛋白(GFP)证实重组质粒己转染入细胞并用G418剔除了未转染质粒的细胞,1.0 μg/ml顺铂作用后pRNAi-CA916798shRNA转染组细胞增殖受到明显抑制(P<0.01)。  结论  成功构建CA916798基因的shRNA表达载体,并筛选出转染了CA916798shRNA的A549/CDDP细胞。
Abstract:
Objective  To construct an eukaryotic expression vector encoding an shRNA targeting CA916798.   Methods  According to the CA916798 cDNA sequence in GenBank, 2 pairs of oligo nucleotides were designed and synthesized. After primer annealing, they were inserted into plasmid pGenSil-l to construct the shRNA eukaryotic expression vector. The recombinant plasmid were transformed into DH5α, and the positive strain were identified by enzyme digestion and sequence analysis. The recombinant vector were transfected into A549/CDDP cells with Lipofectamine 2000. Drug sensitivity and proliferation of the transfected cells were measured by MTT test.   Results  The pRNAi-CA916798 shRNA of recombinant plasmid was constructed successfully. The growth of A549/CDDP cells transfected by pRNAi-CA916798 shRNA was slowly than that of those with blank vector transfection after 1.0 μg/ml CDDP treatment (P<0.01).   Conclusion  The recombinant vector is established and transfected into A549/CDDP cells successfully,and A549/CDDP cell with CA916798 shRNA transfection are obtained.

参考文献/References:

王海晶, 杨和平, 周向东. RNA干扰CA916798基因真核表达载体的构建与鉴定[J].第三军医大学学报,2008,30(1):23-26.

相似文献/References:

[1]范婷婷,唐良萏.Nek2基因沉默对卵巢癌SKOV3细胞侵袭能力的影响[J].第三军医大学学报,2012,34(15):1514.
 Fan Tingting,Tang Liangdan.Silencing Nek2 via RNAi suppresses invasiveness in ovarian cancer SKOV3 cells[J].J Third Mil Med Univ,2012,34(01):1514.
[2]梁佳,林力,寿铸,等.鼻咽癌畸胎瘤细胞源性生长因子表达及siRNA对HNE-1细胞株增殖的抑制[J].第三军医大学学报,2012,34(15):1572.
 Liang Jia,Lin Li,Shou Zhu,et al.Expression of PCDGF in nasopharyngeal carcinoma and its silencing in cell proliferation[J].J Third Mil Med Univ,2012,34(01):1572.
[3]刘丹,尹东,汤蕾,等.LPS预处理对心肌细胞缺氧/复氧损伤的保护作用[J].第三军医大学学报,2012,34(17):1707.
 Liu Dan,Yin Dong,Tang Lei,et al.Protective effect of lipopolysaccharide pretreatment on cardiomyocytes anoxia/reoxygenation injury[J].J Third Mil Med Univ,2012,34(01):1707.
[4]赵亮,高蕊,刘菁,等.异位表达FOXO4对胃癌细胞凋亡的影响[J].第三军医大学学报,2015,37(18):1830.
 Zhao Liang,Gao Rui,Liu Jing,et al.Effects of ectopic expression of Forkhead box O4 on cell apoptosis in human gastric cancer cells[J].J Third Mil Med Univ,2015,37(01):1830.
[5]黄士隋,史良会,黄广岩.RNA干扰下调LAT1表达对胃腺癌SGC-7901细胞增殖、侵袭、转移及细胞周期的影响[J].第三军医大学学报,2013,35(04):320.
 Huang Shisui,Shi Lianghui,Huang Guangyan.Effect of RNA interference targeting LAT1 on proliferation, migration and invasion of SGC7901 cells[J].J Third Mil Med Univ,2013,35(01):320.
[6]党微旗,唐浩,曹红,等.可调控STAT3干扰载体抑制BIU-87细胞侵袭的体外研究[J].第三军医大学学报,2013,35(05):400.
 Dang Weiqi,Tang Hao,Cao Hong,et al.Effect of CRE-dependent RNA interference targeting STAT3 on invasion and migration in human bladder cancer BIU-87 cells[J].J Third Mil Med Univ,2013,35(01):400.
[7]张彦,郑晓东,唐鹏,等.Sphk1基因对人乳腺癌MCF-7细胞增殖、凋亡和迁移能力的影响[J].第三军医大学学报,2012,34(21):2141.
 Zhang Yan,Zheng Xiaodong,Tang Peng,et al.Sphk1 interference suppresses proliferation, apoptosis and migration in human MCF-7 breast cancer cells[J].J Third Mil Med Univ,2012,34(01):2141.
[8]王勇,唐川,兰曦,等.沉默CD133基因对CD133+肝癌干细胞放射敏感性的影响[J].第三军医大学学报,2012,34(23):2373.
 Wang Yong,Tang Chuan,Lan Xi,et al.RNAi targeting CD133 enhances radiosensitivity in CD133 positive liver cancer stem cells[J].J Third Mil Med Univ,2012,34(01):2373.
[9]祝冰晶,王宇亮,罗虎,等.CA916798基因通过PI3K/AKT通路参与肺癌顺铂耐药[J].第三军医大学学报,2013,35(07):618.
 Zhu Bingjing,Wang Yuliang,Luo Hu,et al.CA916798 gene is involved in cisplatin resistance in human lung cancer through PI3K/AKT pathway[J].J Third Mil Med Univ,2013,35(01):618.
[10]赵亮,李静,魏强,等.人PGI基因siRNA慢病毒质粒的构建及对白血病细胞增殖的影响[J].第三军医大学学报,2013,35(01):20.
 Zhao Liang,Li Jing,Wei Qiang,et al.Construction of a lentiviral vector expressing human phosphoglucose isomerase gene siRNA and its influence on leukemia cell proliferation[J].J Third Mil Med Univ,2013,35(01):20.

更新日期/Last Update: 2008-05-05