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目的构建mCD99L2(mouse CD99 antigen-like2)基因RNA干扰慢病毒表达载体,检测其对293FT细胞的感染效率。方法应用基因工程技术,首先设计并合成4对siRNA序列,退火、酶切并连接于慢病毒表达载体SD1259,构建携带针对目的基因mCD99L2的siRNA慢病毒穿梭质粒表达载体(其中包括1条阴性对照序列);然后使用慢病毒包装质粒混合物和构建好的慢病毒穿梭质粒共转染293FT细胞,转染48h后收集上清离心过滤,浓缩病毒,利用绿色荧光蛋白作为报告基因,对病毒滴度和感染效率进行检测。结果构建3个携带针对目的基因mCD99L2的siRNA慢病毒穿梭质粒表达载体和1个阴性对照质粒,经测序鉴定正确;共转染293FT细胞包装病毒并浓缩后滴度达1×107/m1,适合感染目的细胞。结论应用基因工程技术成功构建了mCD99L2基因RNA干扰慢病毒表达载体,为进一步构建类人霍奇金淋巴瘤可视化细胞模型及动物模型奠定了基础。
Objective To construct a RNA interference lentiviral vector expressing mCD99L2 gene and test its infective efficiency on 293FT cells. Methods Four pairs of siRNA sequences were designed and synthesized by annealing, digestion and ligated into the lentiviral vector SD1259. The vector containing siRNA targeting lentiviral vector mCD99L2 (including one negative control sequence ). Then 293FT cells were co-transfected with the lentivirus packaging plasmid mixture and the constructed lentiviral shuttle plasmid. After 48h transfection, the supernatant was collected and centrifuged for filtration. The virus was concentrated, and the green fluorescent protein Efficiency testing. Results Three siRNA lentiviral shuttle plasmids carrying siRNA targeting mCD99L2 and one negative control plasmid were constructed and correctly identified by sequencing. The 293FT cells were co-transfected with virus and the titer was 1 × 107 / ml, which was suitable for infection The purpose of cells. Conclusion The RNAi lentiviral vector of mCD99L2 gene was successfully constructed by genetic engineering technique, which laid the foundation for further construction of human Hodgkin ’s lymphoma visualization cell model and animal model.