人类microRNA-1246慢病毒抑制载体的构建以及鉴定

Construction and Identifacation of a Lentiviral Vector for MicroRNA-1246 of Human Inhibition

目的:构建microRNA-1246慢病毒抑制载。方法针对microRNA-1246成熟体的反义核苷酸片段,应用基因重组技术将目的基因片段克隆到GV280慢病毒载体中,通过酶切、PCR、DNA测序验证重组克隆的成功构建,将GV280-mir-1246down、Helper1.0、Helper2.0共转染293T细胞获得重组慢病毒,浓缩提纯病毒液并检测病毒滴度,用病毒液感染siha细胞,通过检测绿色荧光蛋白(GFP)验证GV280-mir-1246down在siha细胞的表达情况,用嘌呤霉素筛选GV280-mir-1246down稳转细胞株。用细胞计数的方法检测细胞的生长率。结果①对菌落进行PCR鉴定筛选出构建正确的慢病毒载体,DNA测序证实插入的基因序列正确;②GV280-mir-1246down、Helper1.0、Helper2.0共转染293T细胞产生重组慢病毒;③目的mir-246down被慢病毒成功导入siha细胞中,同时达到稳定表达,转到率几乎达100%,荧光显微镜下能直接观察到GFP;④细胞计数结果显示在siha细胞中microRNA-1246的下调细胞的生长速度减弱。结论成功构建microRNA-1246慢病毒抑制载体,建立siha细胞mir-1246-down的稳转株,microRNA-1246在siha细胞中下调的siha细胞的增殖能力下降.这为研究microRNA-1246在宫颈鳞癌siha细胞的作用及其机制提供了可靠的基础。

Objective To construct and identify a lentiviral vector for microRNA-1246 inhibiton.Methods The gene sequences of microRNA-1246 Antisenseolignuclleotides were chosen,the antisense sequences of microRNA-1246 were designed and linked into linearized GV280 vector.The recombinants were screened and identified by colony PCR and DNA sequencing,The GV280-mir-246down,Helper1.0、Helper2.0 were mixed in suitable proportion and then transfected into the cell of 293T to get recombinant lentiviral vector.The concentrated and purified of virus bulk were detected the drops of it,indeed the virus was used to infect the carcinoma cell of Cervical squamous of siha.The GV280-mir-1246down,s expression in the siha cell tested by testing the GFP.Results①The corrected of recombination lentiviral vector was screened from the bacterial which was anthenticated by PCR,DNA testing proved that the inserted gene sequence was correct.②The cells of 293T which was transfected GV280-mir-1246down、Helper1、0 and Helper2.0 produced recombinant lentiviral vector.③The target gene sequences were successfully inserted into siha cell by GV280 lentiviral vector,the recombinant lentiviruses which carring mir-246down could infect and deliver mir-246down and GFP genes to siha cells.The infection efficiency was almost 100%.Conclusion The lentiviral microRNA inhibitor vector has been successfully constructed,which provides a stable tool for further study of therole of microRNA-1246 on cervical cancer.