【摘要】 目的 :通过人工改变逆转录病毒多聚腺苷酸化信号 ,检测病毒转录能否产生病毒 宿主融合转录产物及其是否具有转化细胞的能力 ,探讨病毒致癌的机制。方法 :用分子生物学技术人工定点突变鼠逆转录病毒多聚腺苷酸化信号 ,使之产生多聚腺苷酸缺陷性病毒 ;用该病毒转染大鼠REF 1细胞 ,并用G418筛选抗性细胞。通过Northernblot方法检查通读RNA的表达 ;通过细胞形态和软琼脂集落形成实验检测细胞的转化。结果 :多聚腺苷酸化信号缺陷病毒PS可在CRIP包装细胞形成病毒 ;用该病毒感染大鼠REF 1细胞 ,可捕获下游宿主细胞序列 ;多聚腺苷酸化信号缺陷病毒感染的REF 1混合细胞中的部分细胞可使REF 1细胞集聚能力增强 ,在软琼脂中以集落式生长。结论 :多聚腺苷酸信号缺陷病毒感染REF 1细胞后可通过表达病毒 宿主通读RNA激活下游宿主序列使细胞发生转化 ,这可能是病毒致癌的重要机制更多还原

【Abstract】 Objective:To check if mutated polyadenylation signal retroviruses can produce viral host readthrough transcripts (Rth) and have the ability to transform normal REF 1 cells. Methods: The polyadenylation signal of wild type retrovirus vector was mutated by PCR site directed mutagensis to produce polyadenylation signal deficient retroviruses. The REF 1 cells were infected by the viruses and selected by G418. Viral host readthrough RNAs were checked by Northern blot. The cell growth and soft agar assay were run to test the transformed cells. Results: Northern blot analysis of viral RNA from infected pools and individual G418 resistant clones demonstrated that mutation of consensus LTR polyadenylation signals generated Rth viral RNA in the infected REF 1 cells. Rth transcripts were approximately between 0.1 10 kb longer than the parental viral genomes. Phenotypic analysis results showed that the Ref 1 cells infected with polyadenylation signal mutant viruses tended to grow in cluster manner. Pools of REF 1 cells infected with mutant viruses were able to form colonies in soft agar with a higher efficiency than control or uninfected cells. Conclusion: Host readthrough transcripts generated by polyadenylation signal mutant viruses may contribute to transformation phenotypes. The mutant vectors and the method we have described may be useful as tools to trap and identify genes involved in retroviral insertion mediated transformation.更多还原