Construction and identification of CA916798 eukaryotic expression vector for RNA interference

王海晶; 杨和平; 周向东

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Construction and identification of CA916798 eukaryotic expression vector for RNA interference

更新时间：2026-01-09

- Construction and identification of CA916798 eukaryotic expression vector for RNA interference
- Journal of Army Medical University Vol. 30, Issue 1, Pages: 23-26(2008)
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  CLC： R394-33;R734.2
- Published：2008
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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].陆军军医大学学报(原第三军医大学学报),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 Amry Med Univ (J Third Mil Med Univ),2008,30(01):23-26.
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].陆军军医大学学报(原第三军医大学学报),2008,30(01):23-26. DOI：

　WANG Hai-jing,YANG He-ping,ZHOU Xiang-dong.Construction and identification of CA916798 eukaryotic expression vector for RNA interference[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(01):23-26. DOI：

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(P0.01)。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 (P0.01). Conclusion The recombinant vector is established and transfected into A549/CDDP cells successfully，and A549/CDDP cell with CA916798 shRNA transfection are obtained.

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