SHP-1基因重组腺病毒的构建与表达

目的 构建并鉴定人蛋白酪氨酸磷酸酶基因(SHP-1)重组腺病毒Ad-SHP1.方法 将人SHP-1 cDNA克隆于穿梭质粒pAdTrack-CMV,PmeⅠ线性化后,与腺病毒骨架质粒pAdEasy-1共转化BJ5183细菌,获得带有SHP-1基因的重组腺病毒质粒pAd-SHP1,经PacⅠ线性化后通过Lipofectamine2000转染HEK293包装细胞,观察细胞内绿色荧光蛋白表达情况.收获重组腺病毒,经PCR和Western Blotting鉴定后,扩增并纯化重组腺病毒,测定病毒滴度.结果 经测序、酶切电泳和感染后蛋白表达检测均表明成功构建重组腺病毒Ad-SHP1;病毒滴度为1.58×1010 pfu/mL.结论 成功构建带有人SHP-1 cDNA的重组腺病毒,为进一步研究结外鼻型NK/T细胞淋巴瘤发生机制及肿瘤的基因治疗奠定了基础.

Construction and Expression of Recombinant Adenovirus Carrying Human SHP-1 Gene

OBJECTIVE: To construct and identify recombinant adenovirus Ad-SHP1 carrying human SH2 domain-containing protein tyrosine phosphatase (SHP-1). METHODS: Human SHP-1 cDNA from healthy volunteers' peripheral blood lymphocyte was cloned into the shuttle plasmid pAdTrack-CMV by standard procedure. The plasmid pAdTrackCMV-SHP1 was selected and then linearized by Pme I, followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183. Recombinant adenovirus Ad-SHP1 were obtained after packaged in HEK293 cells and the green fluorescence in HEK293 cells was observed. Recombinant adenovirus was confirmed with PCR and the expressed SHP-1 protein was verified with Western Blotting. Ad-SHP1 was multiplied and purified. The titer of virus was measured with fluorescent counter. Results The results of sequencing, restriction endounclease digestion and Western Blotting indicated that Ad-SHP1 was successfully constructed. The titer of Ad-SHP1 reached 1.58 x 10(10) pfu/mL. CONCLUSION: Recombinant adenovirus Ad-SHP1 had been successfully constructed. It could express SHP-1 protein stably and effectively and this will be very helpful for the further study of the generation of extranodal NK/T cell lymphoma and the corresponding therapy.