【摘要】 目的探讨用双嗜性逆转录病毒载体将外源基因导入日本血吸虫(Sj)细胞的生物学理论与实验依据。方法用生物信息学方法对双嗜性逆转录病毒rRam-1受体同源性分布、结构与功能作系统的分析与比较;利用携带外源E77.43基因的双嗜性逆转录病毒感染Sj童虫培养细胞,经PCR和RT-PCR检测感染细胞目的基因(E77.43)的整合与表达。结果根据生物信息学分析结果推断,Sj细胞膜上存在的SjCHGC09605和SjCHGC05362两种蛋白为非分泌性跨膜蛋白,可能具有细胞膜离子转运通道或受体蛋白的功能及双嗜性逆转录病毒感染的膜受体样作用,可能参与病毒对细胞的吸附和穿入过程;利用携带外源E77.43基因的双嗜性逆转录病毒感染Sj童虫培养细胞后,用PCR及RT-PCR检测到目的基因整合与表达,扩增的目的片段大小为330 bp,与理论值相符。结论用载有E77.43基因的双嗜性逆转录病毒感染Sj童虫细胞获得成功,推测SjCHGC09605和SjCHGC05362两种与rRam-1受体同源的蛋白可能是Sj感染过程中起作用的分子。研究结果为下一步用双嗜性逆转录病毒载体转导永生化基因到Sj细胞提供了生物学理论与实验依据。更多还原

【Abstract】 Objective To investigate the biological theory and experimental foundation for transduction of exogenous genes into cultured Schistosoma japonicum(Sj) cells using an amphotropic retrovirus.Methods The homologous distribution,structure,and function of receptors for an amphotropic retrovirus,Rattus norvegicus(rRam-1),were systematically analyzed and compared using bioinformatics methods.Afterwards,the cultured cells of 12-day-old Sj schistosomula infected with the amphotropic retrovirus containing exogenous gene E77.43 were identified with PCR and RT-PCR in order to integrate and express the target gene in virus-treated cells.Results Results of bioinformatics analysis suggested two possible non-secreted transmembrane proteins,SjCHGC09605 and SjCHGC05362,on the cytomembrane of Sj,with their possible roles as ion transportation or receptor proteins and membrane receptors for amphotropic retroviruses involving in the virus adsorption and penetration into the cells.Consequently,the integration and expression of the exogenous gene,E77.43,was detected in schistosomulum cells exposed to the viruses using PCR or RT-PCR analysis.Conclusion Schistosomulum cells were transduced successfully with the amphotropic retrovirus.Notably,two proteins,SjCHGC09605 and SjCHGC05362,that are homologous to the rRam-1 receptor may play an important role in the infection stage.Taken together,these results provide biological theory and experimental foundation for transferring an immortalized gene into Sj cells with an amphotropic retrovirus in future investigations.更多还原