人Notch4基因RNAi慢病毒载体的构建及鉴定

Construction and identification of a lentiviral vector of RNA interference containing the human Notch4 gene

目的涎腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)是涎腺常见的恶性肿瘤,Notch4基因与SACC的发生、转移有关。文中构建人Notch4基因RNA干扰(RNA interference,RNAi)慢病毒载体并鉴定。方法针对人Notch4基因序列,按照RNAi序列设计原则,设计RNAi靶点序列,通过限制性内切酶MIu l和CIa l双酶切、T4 DNA连接酶连接,将Notch4插入慢病毒载体pLenOR-THM,构建pLenOR-THM-Notch4重组载体。质粒转化感受态细菌,筛选阳性克隆,经双酶切及测序鉴定正确后通过脂质体将慢病毒4质粒系统共转染293 T细胞,进行慢病毒包装并测定病毒滴度、观察感染效率。各组病毒载体转染ACC-M细胞后,用QRT-PCR和Western blot检测Notch4基因mRNA和蛋白的表达水平。结果成功构建慢病毒表达载体pLenOR-THM-Notch4,4质粒共转染293T细胞后可见大量绿色荧光。浓缩病毒后测定其滴度为6.2×108TU/ml。以复感染系数MOI(multiplicity of infection)为1感染293T细胞,感染效率在90%以上。结合QRT-PCR和Western blot检测结果,第2组慢病毒载体干扰效果最佳。结论成功构建并鉴定人Notch4 RNAi慢病毒表达载体。

Objective Salivary adenoid cystic carcinoma （SACC） is a common malignancy in salivary glands, and the Notch4 gene is involved in its development and metastasis. This study aimed to construct and identify a lentiviral vector of RNA inter- ference targeting human Notch4. Methods Based on the human Notch4 gene sequences, RNAi target sequences were designed in accordance with RNAi sequence design principles, and cloned into the lentiviral vector pLenOR-THM by restriction endonuclease Mlu 1 and CIa 1 double digestion and T4 DNA ligase ligation. After transformation into competent E. coli bacteria, the candidate clones were identified by DNA sequencing. The recombinant plasmid and the three packaging plasmids were eo-transfected into the human em- bryonic kidney 293T cells by lipofectamineTM 2000 to produce the lentiviral particles, and the viral titer was determined. The 293T cells were infected with the lentiviral particles obtained and the transfection efficiency was assessed under the fluorescent microscope. Then the lentiviral vector particles were transfected into ACC-M cells and the expression of Notch4 in the transfected cells was deter- mined by QRT-PCR and Western blot. Results The lentiviral RNAi vector pLenOR-THM-Notch4 for the Notch4 gene was construe- ted successfully. Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of the cells with the 4 plasmids of the lentiviral vector. The virus in the supernatant reached a titer of 6.2 x 10s TU/ml. The transfection effi- ciency of the collected virus exceeded 90% in the 293T cells with a multiplicity of infection of 1. The second lentiviral vector signifi- cantly inhibited the expression of Notch4 at both the mRNA and protein levels. Conclusion The lentiviral RNAi vector of Notch4 has been successfully constructed and identified.