腺病毒伴随病毒表达载体表达人乳头瘤病毒18型E6E7基因构建及其转化作用的鉴定

目的：探讨人乳头瘤病毒（HPV）的E6E7基因在细胞恶性转化中所起的作用。方法：将人乳头瘤病毒（HPV）的E6E7基因克隆至腺病毒伴随病毒表达载体中，通过包装的重组病毒感染，将E6E7基因导入并整合到永生293细胞的基因组中。结果：本研究成功地构建了HPV18 E6E7 AAV病毒并感染了永生293细胞，PCR／Southern杂交分析表明E6E7基因在转化细胞293TL中确有表达，转化细胞293TC和293TL具有明显的转化表型，和亲本293细胞相比，生长速度快，接触抑制消失，集落形成率提高20倍，且集落明显增大，形成时间短。结论：成功地构建了HPV18 E6E7 AAV病毒，HPV18 E6E7基因引起永生化人上皮细胞293的恶性转化。此病毒可用于感染正常上皮细胞，研究其致癌机制。

Construction of human papilloma virus type 18 E6E7 genes in adeno-associated virus expression vector and checking its activity for malignant transformation

Objective To construct human papillomavirus type 18 (HPV18 E6E7) adeno associated virus (AAV) for studying the role of HPV E6E7 in the development of human cancer. Methods HPV18 E6E7 genes were inserted into adeno associated virus expression vector and then infected 293 cell line.The expression of HPV18 E6E7 genes were confirmed by using RT PCR/Southern blot assay. Results There was HPV18 E6E7 genes in the malignantly transformed cell line. The 293TL cells compared with the parent cells transformed cells grew more rapidly,lost their contact inhibition and formed more and large colonies in soft agar.Conclusion HPV18 E6E7 AAV was successfully constructed and could induce malignant transformation. HPV18 E6E7 AAV can be use for studying the immortalization and malignant transformation of human normal epithelial cells.