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目的:构建含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1- E7AS并鉴定,探讨腺相关病毒介导的反义E7基因技术用于治疗早期宫颈癌的可能性。方法:使用RT -PCR法扩增全长HPV16型E7基因,利用基因重组法将目的片段反向插入腺相关病毒骨架质粒pUF1并酶切鉴定。结果:RT PCR法扩增全长315bp的HPV16型E7基因,装入pGEM -Teasy载体进行测序,经NCBI数据库blast检索为100%的符合,经基因重组获得含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS,AccⅠ和KpnⅠ双酶切鉴定pUF1 E7AS。结论:含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS可成功构建。
Objective: To construct and identify the adeno-associated virus backbone plasmid pUF1-E7AS containing the antisense E7 gene of HPV16 and to explore the possibility of adeno-associated virus-mediated antisense E7 gene therapy for early cervical cancer. Methods: The full-length HPV16 E7 gene was amplified by RT-PCR and inserted into the adeno-associated virus backbone plasmid pUF1 by gene recombination. Results: The full-length 315bp HPV16 E7 gene was amplified by RT-PCR and inserted into pGEM-Teasy vector for sequencing. According to the blast of NCBI database, the E7 gene was sequenced. The adeno-associated E7 gene containing HPV16 antisense gene was obtained by gene recombination The virus backbone plasmid pUF1 E7AS, Acc Ⅰ and Kpn Ⅰ double digestion identified pUF1 E7AS. Conclusion: The adeno-associated virus backbone plasmid pUF1 E7AS containing HPV16 antisense E7 gene can be successfully constructed.