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目的探讨促进乳头状瘤病毒(PV)晚期基因L1的蛋白表达和DNA疫苗免疫效应的途径及可能的机制。方法分别将含人源化优化密码BPVL1-GFP融合基因(HL1-GFP)和野生型BPVL1-GFP融合基因(WL1-GFP)的真核表达质粒体外单独转染或与tRNAser真核表达质粒共转染人角质形成细胞HaCAT,荧光显微镜及Western blot方法观察L1-GFP融合蛋白的瞬时表达情况。半定量RT-PCR分析不同PV L1基因mRNA的表达。BALB／c小鼠股四头肌内分别接种野生型、优化密码L1 DNA疫苗,同时将L1 DNA疫苗与tRNAser真核表达质粒混合后接种小鼠,观察诱导产生的体液免疫反应。结果野生型WL1-GFP融合基因在HaCAT细胞中未能有效表达,优化密码HL1-GFP基因在HaCAT细胞中的蛋白表达水平明显增强;但两者在mRNA水平差异无统计学意义。WL1-GFP与tRNAser真核表达质粒共转染细胞中L1蛋白的表达较单独转染WL1-GFP基因时增强。动物DNA免疫结果表明,优化密码HL1 DNA疫苗接种可使小鼠特异性L1中和抗体水平明显升高;而野生型WL1 DNA疫苗接种组小鼠仅产生低滴度的L1抗体,WL1 DNA疫苗与tRNAser真核表达质粒混合接种使小鼠的L1抗体水平增加。结论优化密码能显著促进乳头状瘤病毒晚期基因L1在哺乳动物细胞中的表达和DNA疫苗的免疫反应;补充外源tRNAser基因能够提高野生型L1基因的蛋白表达和DNA疫苗诱导的体液免疫应答。
Objective To explore the pathway and possible mechanism of promoting the expression of L1 gene and DNA vaccine in late stage of papilloma virus (PV). Methods Eukaryotic expression plasmids containing humanized optimized BPVL1-GFP fusion gene (HL1-GFP) and wild-type BPVL1-GFP fusion gene (WL1-GFP) were transfected in vitro or co-transfected with tRNAser eukaryotic expression plasmid Human keratinocytes were stained with HaCAT. The transient expression of L1-GFP fusion protein was observed by fluorescence microscopy and Western blot. Semi-quantitative RT-PCR analysis of different PV L1 gene mRNA expression. BALB / c mice were intramuscularly inoculated with wild-type, optimized L1 code DNA vaccine, at the same time L1 DNA vaccine and tRNAser eukaryotic expression plasmid were mixed inoculated mice to observe the induced humoral immune response. Results The wild-type WL1-GFP fusion gene was not expressed efficiently in HaCAT cells. The protein expression level of HL1-GFP gene was significantly increased in HaCAT cells. However, there was no significant difference between the two in mRNA level. The expression of L1 protein co-transfected with WL1-GFP and tRNAser eukaryotic expression plasmids was stronger than that of WL1-GFP transfection alone. Animal DNA immunization results showed that the optimized password of HL1 DNA vaccination can significantly increase the level of mouse specific L1 neutralizing antibody; while wild-type WL1 DNA vaccinated mice only produced low titer of L1 antibody, WL1 DNA vaccine and Mixed vaccination with tRNAser eukaryotic expression plasmids raised L1 antibody levels in mice. Conclusion The optimized codon usage can significantly promote the expression of L1 gene in mammalian cells and the immune response of DNA vaccine. Supplementation of exogenous tRNAser gene can increase the expression of wild-type L1 gene and DNA vaccine-induced humoral immune response.