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目的 探讨优化密码对人乳头瘤病毒 6b型 (HPV 6b)基因表达及免疫原性的影响 ,为治疗性DNA疫苗的研究奠定基础。方法 设计合成含优化密码及pRB结合位点突变的HPV 6bE7(hu mE7)全长基因。测序验证无误后 ,定向克隆于pcDNA3的KpnⅠ和EcoRⅠ位点 ,成功构建真核表达质粒pcDNA3 hu mE7。体外转染COS 1细胞 ,免疫荧光检测其E7蛋白表达。C57BL 6小鼠胫前肌内接种裸DNA ,观察其诱导的细胞免疫反应。结果 pcDNA3 hu mE7在COS 1细胞获得明显表达 ,表达产物主要位于细胞核中。DNA免疫结果显示 ,与含有野生型E7基因的表达质粒pcDNA3 wtE7比较 ,pcDNA3 hu mE7免疫小鼠脾细胞培养上清中IFN γ产生明显升高 ,CD8+ 和CD4 + 淋巴细胞活性增强。结论 优化密码能促进HPV 6bE7蛋白表达及DNA疫苗诱导的细胞免疫反应。
Objective To investigate the effect of optimized codons on the gene expression and immunogenicity of human papillomavirus type 6b (HPV 6b), and lay a foundation for the study of therapeutic DNA vaccine. Methods The full-length HPV 6bE7 (hu mE7) gene with optimized coding and pRB binding sites was designed and synthesized. After verified by sequencing, the recombinant plasmid pcDNA3-hu mE7 was successfully cloned into KpnⅠand EcoRI sites of pcDNA3. COS 1 cells were transfected in vitro, and the expression of E7 protein was detected by immunofluorescence. C57BL 6 mice were inoculated with naked DNA into the anterior tibial muscle to observe the induced cellular immune response. Results The pcDNA3 hu mE7 was expressed in COS 1 cells and the expressed product was mainly located in the nucleus. The results of DNA immunization showed that IFNγ production in splenocyte culture supernatant of mice immunized with pcDNA3 hu mE7 was significantly increased and the activity of CD8 + and CD4 + lymphocytes was enhanced compared with the pcDNA3 wtE7 expression vector containing wild type E7 gene. Conclusion The optimized password can promote HPV 6bE7 protein expression and DNA vaccine-induced cellular immune response.