靶向人Trb3基因shRNA真核表达载体的构建及鉴定

作者:文浩杰;隋军;孙传政;蒋玉娥;曾木圣;李满枝;张晶;李科;李晓江;王虎 刊名:中国耳鼻咽喉头颈外科 上传者:韶晖

【摘要】目的构建并鉴定靶向果蝇同源蛋白3(tribbles-related protein 3,Trb3)基因的短发夹状RNA(short hairpin RNA,shRNA)真核表达载体,并检测其对靶基因的沉默效应。方法根据Genbank数据库中Trb3序列,设计、合成3对互补并特异性编码其shRNA序列的寡核苷酸,克隆到线性化的pSUPER.retro.puro质粒载体中,并对重组质粒进行酶切分析及测序鉴定;脂质体介导重组质粒和pSUPER.retro.puro Vector转染舌鳞状细胞癌Tca8113细胞株,RT-PCR和Western-Blot检测其对靶基因Trb3的表达影响。结果酶切及测序鉴定重组质粒pSUPER.puro-shTrb3序列正确;转染重组质粒后舌鳞状细胞癌Tca8113细胞中Trb3 mRNA和蛋白表达水平相对于空载体组明显下降,shRNA可以特异性沉默Trb3基因。结论成功构建了靶向人Trb3基因的shRNA真核表达载体,为进一步研究Trb3在舌鳞状细胞癌和其他头颈肿瘤中的作用奠定了基础。

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1*41, "Jifül , (1 650118; 2 510060) (tribbles-related protein 3, Trb3) (short hairpin RNA, shRNA) Trb3B51J, RT-PCR*0Western-BlotÉilJRXif RNA+ft; Construction and identification of the eukaryotic expression vector expressing short hairpin RNA targeting Human Trb3 Gene WEN Haojie', SUI Jun', SUN Chuanzheng', JANG Yu'e', ZENG Musheng2, L/ ManzhF, ZHANG Jing l, L/ Kef, L/ Xiaojiangl, WANG Hui (1 Department of Head and Heck Surgery, the Third Affiliated Hospital of Kunming Medical University, Head and Neck Cancer Research Center of Yunnan Province, Kunming, Yunnan, 650118, China; 2 State Key Laboratory of Oncology in South China, Department of Experimental (30960444) , (2009CD186) (08C0095) ( 303040966014 ) Email: wenhaojie1886@163.com iMiF.fr*: (Email: scz007@126.com) Research, Sun Yat-Sen University Cancer Center, Guangzhou, Guangdong, 510060, China) Corresponding author: SUN Chuanzheng (Email: scz007@126.com) [ABSTRACTI OBJECTIVE To construct and identify the short hairpin RNA (shRNA) eukaryotic expression vector for targeting human Trb3 gene and observe its effect on inhibiting expression of target gene. METHODS According to the GenBank database, three pairs of shRNA sequences targeting human Trb3 gene were designed and synthesized. The selected nucleotides were cloned in the linearized plasmid pSUPER.retro.vector respectively, and the shRNA recombinant plasmids were identified by DNA sequencing and restriction enzyme digestion. The recombinant plasmids were transfected into t

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