人乳头瘤病毒58型E6E7融合蛋白的原核表达、纯化及鉴定

摘要

Objective: To construct pET-42a( + )-HPV58EdE7 prokaryotic expression plasmid and to express and purify human papillomavirus type 58(HPV58) E6E7 fusion protein. Methods: The HPV58 E6E7 fusion gene were amplified by PCR and cloned into pET-42a( + ). The recombinant plasmids were successfully introduced into E.co-li BL21 and were induced by IPTG. SDS-PAGE and Western blot analysis were used to detect the fusion protein. Results: The recombinant plasmid of pET-42a( + )-HPV58E6E7 was identified and confirmed with enzyme digestion and sequencing. HPV58E6E7 fusion protein was expressed and purified sucessfully. SDS-PAGE and Western blot showed that the fusion protein was correct and effective. Conclusion: pET-42a (+) -HPV58E6E7 prokaryotic expression plasmid was constructed successfully. High-level expression of HPV58E6E7 fusion protein was achieved and purified. The fusion protein can be used as antigen of immunological test to HPV58 therapeutic vaccine.%目的:构建pET-42a(+)-HPV58E6E7原核表达质粒,诱导表达人乳头瘤病毒(HPV)58型E6E7融合蛋白.方法:采用PCR方法扩增出HPV58 E6E7融合基因的全长序列,利用DNA重组技术将其定向插入原核表达载体pET-42a(+)中,构建pET-42a( +)-HPV58E6E7原核表达质粒,用限制性内切酶酶切和核酸序列检测对重组质粒进行鉴定；将其转入宿主菌大肠杆菌BL21进行诱导以表达HPV58E6E7融合蛋白,并用谷胱甘肽琼脂糖树脂纯化回收HPV58E6E7融合蛋白,用SDS-PAGE及Western印迹鉴定表达蛋白的相对分子质量及抗原性.结果:PCR、限制性内切酶酶切和核酸序列检测证实重组质粒中插入的目的基因大小、方向正确；HPV58E6E7融合蛋白得到高效原核表达及纯化,表达蛋白的分子大小正确,抗原性良好.结论:pET-42a(+)-HPV58E6E7原核表达质粒构建成功,HPV58E6E7融合蛋白得到高效表达及有效纯化,为检测HPV58型治疗性疫苗的免疫效果提供了抗原.