Pir-b基因慢病毒载体的构建和表达

本研究构建pir—b基N慢病毒载体并检测其在293T细胞中的表达。从小鼠的mRNA中钓取pir—b基因的开放阅读框，与测序载体连接，经过测序鉴定以后，构建含有pir—b基N的慢病毒穿梭质粒，与包装质粒一起共转染293T细胞，收获上清病毒浓缩纯化后转染293T细胞，用Western blot检测PIR—B蛋白的表达。同时以含有egfp基因的慢病毒作为转染效率的对照，结果表明：含有pir—b开放阅读框的慢病毒穿梭质粒构建成功，序列测定的结果与预期完全一致，含有pir-b的慢病毒载体包装成功。Western blot的结果证实外源PIB—B蛋白在293T细胞中正常表达。结论：本研究成功构建了含有pir—b基因的慢病毒载体，转染293T细胞以后正常表达PIR—B蛋白，这为以后深入研究pir—b在免疫调节中的作用奠定了坚实的基础，

Construction and Expression of pir-b Gene Lentiviral Vector

The purpose of this research was to construct a lentiviral vector containing pir-b gene, and to detect the expression of pir-b gene in 293T ceils. The open reading frame （ORF） of pir-b gene from the mRNA of mouse was cloned, then inserted into a sequencing vector. The pir-b gene was subcloned into the transfer plasmid of the lentivirus system, which was transfected together with the packaging plasmids into 293T cells by Lipofectin 2000, thereafter, the supernatant was collected and concentrated to transfect 293T cells. Western blot was used to detect the expression of the exogenous PIR-B after 293T cells were infected by the virus, while the lentivirus harboring egfp gene was packaged as the control group. The result indicated that the ORF of the pir-b gene was successfully cloned, the sequence of which was consistent to that was expected and the PIR-B protein could be expressed in 293T cells normally. It is concluded that the lentiviral vector containing pir-b gene was constructed succesfuly, which would contribute to illustrating the important role of pir-b gene in the immunological regulation.