大鼠免疫球蛋白样转录物3过表达腺病毒载体的构建及鉴定

Construction and Identification of Recombinant Adenovirus Vector which Overexpresses Rat Immunoglobulin- like Transcript 3

目的构建免疫球蛋白样转录物3(immunoglobulin-like transcript 3,ILT3)过表达腺病毒载体,以期进一步研究ILT3基因功能及其在心血管疾病中的作用。方法针对大鼠ILT3设计引物,通过PCR扩增获取目的基因,以琼脂糖凝胶电泳鉴定,行胶回收与PCR产物纯化。使用限制性内切酶Bam HⅠ与AgeⅠ对载体质粒以及纯化后的PCR产物进行双酶切,将酶切后的PCR产物与载体连接,连接产物加入至感受态细胞培养后挑取菌落进行PCR鉴定、测序、比对及质粒抽提。将ILT3重组质粒与腺病毒辅助包装质粒共转染至HEK293细胞,获取病毒粗提液并进行扩增、纯化,终点稀释法进行病毒效价检测,在荧光显微镜下观察腺病毒转染效率。结果 PCR与测序鉴定表明GV314-ILT3重组质粒构建成功。终点稀释法测得病毒效价为1×109PFU/ml,荧光显微镜下观察显示病毒转染效率较高。结论携带ILT3基因的腺病毒载体构建成功,获取的腺病毒具有较高的效价与转染效率。

Objective To construct recombinant adenovirus vector which overexpresses immunoglobulin - like transcript 3 （ILT3） in order to further study the function of ILT3 and its effect in cardiovascular diseases. Methods The primers of rat ILT3 were designed, and the target gene were amplified by PCR and identified by agarose gel electrophoresis. The gel was recovered and PCR products were purified. Digested plasmid vector and PCR products using restriction endonuelease BamHI and ageI were connected and added to competent cells, and the cultured colonies were identified by PCR and sequencing. ILT3 recombinant plasmid and adenovirus packaging plasmid were co - transfeeted into HEK293 cells, and the obtained adenovirus were amplified and purified. Virus titer were detected using the end- point dilution assay, and the transfection efficiency was observed under the fluorescence microscope. Results PCR and sequencing analysis showed that the recombinant plasmid of GV314 - ILT3 was successfully constructed. The virus titer was 1 × 109pFU/ml and the transfection efficiency was high under fluorescence microscope. Conclusion The adenovirus vector which overexpresses ILT3 was constructed successfully, and the obtained adenovirus exhibited high titer and transfection efficiency.