PAK4基因RNAi慢病毒载体的构建与鉴定

J4 ›› 2012, Vol. 38 ›› Issue (1): 11-14.

PAK4基因RNAi慢病毒载体的构建与鉴定

代炜¹,张红艳²|李彦姝²|李妍²|孙长伏¹

（1. 中国医科大学口腔医学院口腔颌面外科口腔颌面-头颈肿瘤外科|辽宁沈阳 110002；2. 中国医科大学细胞生物学教研室细胞生物学卫生部重点实验室|辽宁沈阳 110001）

收稿日期:2011-10-07 出版日期:2012-01-28 发布日期:2012-01-28
通讯作者: 孙长伏 E-mail:（Tel：024-22891026,E-mail:changfusun@hotmail.com)
作者简介:代炜（1978-）,男|辽宁省葫芦岛市人|主治医师|医学博士|主要从事肿瘤信号转导通路的研究。
基金资助:
国家自然科学基金资助课题（30871390， 30672331，30800415）

Construction and identification of lentiviral vector of RNA interference of PAK4 gene

DAI Wei¹,ZHANG Hong-yan²,LI Yan-shu²,LI Yan²,SUN Chang-fu¹

(1.Department of Oral Maxillofacial Surgery,Department of Oromaxillofacial-Head and Neck Tumor Surgery,School of Stomatology,China Medical University, Shenyang 110002,China;2. Key Laboratory of Cell Biology,Ministry of Health,Department of Cell Biology,Chinese Medical University,Shenyang 110001,China）

Received:2011-10-07 Online:2012-01-28 Published:2012-01-28

摘要/Abstract

摘要：

目的：构建PAK4基因RNAi慢病毒载体，并对其在涎腺腺样囊性癌细胞株SACC-83细胞中干扰效率进行鉴定。方法：针对PAK4基因RNAi有效靶序列，合成Oligo DNA，退火形成双链DNA，与经AgeⅠ和EcoRⅠ 酶切后的pGCsil-GFP载体连接产生shPAK4i-LV慢病毒载体，PCR筛选阳性克隆，测序鉴定。用shPAK4i-LV载体、pHelper1.0载体和pHelper 2.0质粒共转染包装细胞293T细胞，包装产生慢病毒，以293T细胞绿色荧光蛋白(GFP)的表达水平测定病毒滴度。结果：PCR扩增出插入片段，测序证实成功构建了PAK4-shRNA慢病毒载体shPAK4i-LV。包装并浓缩慢病毒，滴度为2E+9 TU•mL^-1。将病毒感染SACC-83细胞，real-time PCR检测PAK4的mRNA表达下调70%以上。结论：成功构建了shPAK4i-LV病毒载体并建立了SACC-83-shPAK4i细胞模型，为PAK4在肿瘤信号转导通路中的研究提供工作基础。

关键词: RNA干扰；PAK4基因；质粒构建；慢病毒；癌, 腺样囊性

Abstract:

To construct a lentiviral vector of RNA interference (RNAi) of PAK4 gene and to study the interference efficiency of PAK4 shRNA in SACC-83 cells in vivo.Methods The complementary oligo DNA was designed according to the effective sequence of siRNA targeting PAK4 gene and then synthesized and cloned into the pGCSIL-GFP vector.The obtained lentiviral vector containing PAK4i shRNA was named as shPAK4i-LV,and it was confirmed by PCR and sequencing.The HEK-293T cells were cotransfected with lentiviral vector shPAK4i-LV，pHelper1.0 and pHelper2.0.All virus stocks were produced by LipofectamineTM 2000 transfection.The titer of virus was tested according to the expression level of GFP.Results PCR and DNA sequencing demonstrated that the lentiviral vector shPAK4i-LV of PAK4 shRNA was constructed successfully.The titer of concentrated virus was 2E+9TU•mL^-1.The SACC-83 cell line was infected with shPAK4i-LV and the expression of PAK4 gene was inhibited about 70% which was confirmed by real-time PCR.Conclusion The shPAK4i-LV is constructed successfully and SACC-83-shPAK4i transient transfection cell model is established.It provides the basis for research on PAK4 signal transduction pathway in tumor.

Key words: RNA interference;PAK4 gene;plasmid construction;lentivirus;carcinoma,adenoid cystic

中图分类号:

R739.87

代炜|张红艳|李彦姝|李妍|孙长伏. PAK4基因RNAi慢病毒载体的构建与鉴定[J]. J4, 2012, 38(1): 11-14.

DAI Wei,ZHANG Hong-yan,LI Yan-shu,LI Yan,SUN Chang-fu. Construction and identification of lentiviral vector of RNA interference of PAK4 gene[J]. J4, 2012, 38(1): 11-14.

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