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[目的]研究HPV16E7蛋白的生物学特性及其在宫颈癌新疗法中的应用。[方法]以临床确诊的HPV16感染患者子宫颈细胞DNA为模板,采用PCR方法克隆HPV16E7蛋白编码基因,经BamHⅠ和EcoRⅠ双酶切构建pET28a(+)原核表达载体,IPTG诱导蛋白表达,Ni2+-MAC层析柱纯化,SDS-PAGE电泳,Westernblot鉴定目的蛋白。无菌分离宫颈癌患者的外周血单个核细胞,与E7蛋白共培养24h,以瘤内注入途径作用人宫颈癌的裸鼠荷瘤模型。[结果]成功构建了pET28a-E7原核表达载体,表达了E7蛋白。与空白组和阳性对照相比,蛋白刺激组可以明显抑制肿瘤的生长,并可使荷瘤模型的瘤体缩小。[结论]本实验为宫颈癌的临床治疗提供新的思路。
[Objective] To study the biological characteristics of HPV16E7 protein and its application in new therapy of cervical cancer. [Methods] The HPV16E7 protein coding gene was cloned by PCR from the clinically diagnosed cervical intraepithelial neoplasia (HPV16) infected patients. The prokaryotic expression vector pET28a (+) was constructed by digestion with BamH Ⅰ and EcoR Ⅰ. IPTG induced the expression of Ni2 + -MAC Purification by chromatography, SDS-PAGE electrophoresis, Western blotting to identify the target protein. Peripheral blood mononuclear cells from patients with cervical cancer were aseptically separated and co-cultured with E7 protein for 24 hours. The tumor-bearing model of human cervical carcinoma was established by intratumoral injection. [Result] The prokaryotic expression vector pET28a-E7 was successfully constructed and E7 protein was expressed. Compared with the blank group and the positive control group, the protein-stimulated group could significantly inhibit the growth of the tumor and reduce the tumor size of the tumor-bearing model. [Conclusion] This experiment provides a new idea for the clinical treatment of cervical cancer.