摘要
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 approaches and the possible mechanisms of the papillomavirus （PV） L 1 gene expression and the immune responses of DNA vaccinations. Methods Recombinant eukaryotic expression vectors containing codon optimized BPVL1-GFP （HL1-GFP） fusion gene and wild type BPVL1-GFP （WL1-GFP） fusion gene transfected HaCAT cells alone or cotransfected with pSVtRNA^ser separately. The transient expressions of the fusion proteins were examined by fluorescence microscopy and Western blot method. Message RNA expression of different L1 genes was measured by semi-quantitative RT-PCR. For in vivo studies, some female BALB/c mice were immunized intramuscularly with wild-type and codon modified L1 DNA vaccine individually, meanwhile, some other female BALB/c mice were immunized with the combination of pSVtRNA^ser and L1 DNA vaccine. Results Fluorescence microscopy and Western blot results showed that HL1-GFP gene readily expressed in HaCAT cells, while WL1-GFP gene failed to express. Semi-quantitative RT-PCR suggested that there was no significant difference in mRNA expression levels between HL1 and WL1 genes. Cells cotransfected with pSVtRNA^ser and WL1-GFP gene produced higher levels of L1 protein than those transfected with WL1 gene only. In vivo study results indicated that mice immunized with HL1 DNA vaccination elicited significantly higher levels of Ll-specific serum IgG than those with WL1 immunization. The mice immunized with tRNA^ser and WL1 gene produced higher IgG antibody level than those with WL1 immunization only. Condusion Codon usage optimization can significantly enhance PV L1 protein expression in mammalian cells and the humoral immune response of DNA vaccine, and supplementation of exogenous tRNA can also promote wild-type L1 protein expression in mammalian cells and the humoral immune response.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第6期516-520,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金(30271193
30470086)
山东省自然科学基金(Y2002004)
