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人乳头瘤病毒16型转化作用在宫颈癌发生中可能起重要作用,其转化基因主要位于基因组早期区域E6、E7开放阅读框架内。为了进一步研究HPV16转化作用,我们以Pstl+ECoRI酶解HPV16DNA,以pUC-19为载体,大肠杆菌JM103细胞为宿主菌,经两次定向次级克隆,组建了质粒pEP-8。经限制性核酸內切酶和Southem转印杂交证实,其插入片段为-1.43Kb带有E6、E7 ORF的DNA片段。在E6区上游序列还含有两个TATA盒,1个Cat盒和1个细胞特异性增强子。该质粒的组建为检测HPV感染细胞中mRNA转录提供了特异性的早期基因探针,同时为进一步研究HPV转化作用及转化蛋白打下了基础。
Human papillomavirus type 16 transformation may play an important role in the carcinogenesis of cervical cancer. The transgene is mainly located in the E6, E7 open reading frame in the early genome. In order to further study the transformation of HPV16, HPV16 DNA was digested with Pstl + ECoRI. The plasmid pEP-8 was constructed by pUC-19 vector and Escherichia coli JM103 as host cells. The restriction endonuclease and Southem blot hybridization confirmed that the inserted fragment was -1.43Kb DNA fragment with E6, E7 ORF. The upstream sequence in the E6 region also contains two TATA boxes, a Cat box and a cell-specific enhancer. This plasmid was constructed to detect early gene probes specific for mRNA transcripts in HPV-infected cells, and laid the foundation for further study of HPV transformation and transformation of proteins.