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目的:构建携带增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)和胶质细胞生长因子2(glialgrowthfactor2,GGF2)的双基因真核表达载体pIRES2EGFPGGF2,研究GGF2在大鼠脊髓内的表达。方法:采用RTPCR方法从大鼠胎脑组织总RNA中扩增出GGF2全序列cDNA,利用脑炎心肌炎病毒的内部核糖进入位点(internalribosomeentrysite,IRES),构建GGF2与EGFP双基因真核表达载体pIRES2EGFPGGF2。采用基因注射法将阳离子脂质体Transfectam和pIRES2EGFPGGF2基因混合后转染至大鼠胸段脊髓组织中,观察GGF2在大鼠脊髓中的表达。结果:成功构建pIRES2EGFPGGF2质粒,在转染的局部脊髓内观察到GGF2mRNA表达显著增加。荧光显微镜下观察发现,注射局部灰质和白质神经元内均有较多绿色荧光蛋白表达。结论:阳离子脂质体能介导EGFP和GGF2双基因载体在脊髓内同时表达,EGFP可作为报告基因观察GGF2在脊髓内的表达。
OBJECTIVE: To construct the eukaryotic expression vector pIRES2EGFPGGF2 carrying enhanced green fluorescent protein (EGFP) and glial growth factor 2 (GGF2), and to study the expression of GGF2 in rat spinal cord. Methods: The full-length cDNA of GGF2 was amplified by RT-PCR from rat fetal brain tissue. The eukaryotic expression vector pIRES2EGFPGGF2 was constructed by internal ribosome entry site (IRES) of encephalitis virus. . The cationic liposomes Transfectam and pIRES2EGFPGGF2 were mixed by gene injection and then transfected into thoracic spinal cord tissue of rats to observe the expression of GGF2 in rat spinal cord. RESULTS: Plasmid pIRES2EGFPGGF2 was successfully constructed and a significant increase in GGF2 mRNA expression was observed in the transfected local spinal cord. Fluorescence microscopy showed that more green fluorescent protein was expressed in the local gray matter and white matter neurons. CONCLUSION: Cationic liposomes can mediate the simultaneous expression of EGFP and GGF2 double gene vectors in the spinal cord. EGFP can be used as a reporter gene to observe the expression of GGF2 in the spinal cord.