蛋白表达的亚细胞定位影响基因免疫诱导的体液免疫应答水平
首发时间:2011-11-24
摘要:目的 研究表达产物的不同亚细胞定位对基因免疫诱导的体液免疫应答水平的影响,为基因疫苗的设计提供参考。方法 利用分子克隆技术,分别构建能表达不同细胞定位的EGFP-HPV16L1融合蛋白和截短型EGFP-HPV16L1△NLS融合蛋白的重组pcDNA-EGFP-HPV16L1和pcDNA-EGFP-HPV16L1△NLS真核表达载体;通过转染CHO细胞,并在倒置荧光显微镜下观察表达产物的细胞定位;用重组pcDNA载体免疫BALB/c小鼠;EGFP作为抗原,ELISA法检测血清抗体吸光度。结果 1、重组pcDNA-EGFP-HPV16L1转染的CHO细胞胞核内可见到绿色荧光,pcDNA-EGFP-HPV16L1△NLS真核表达载体转染的CHO细胞胞浆内可见到绿色荧光;2、两种不同的重组pcDNA载体均可诱导BALB/c小鼠体液免疫反应,且重组pcDNA-EGFP-HPV16L1△NLS真核表达载体免疫组小鼠抗体A450吸光度显著高于且重组pcDNA-EGFP-HPV16L1真核表达载体免疫组小鼠抗体A450吸光度(P<0.001)。结论 表达产物的不同细胞定位可影响基因免疫诱发的体液免疫应答水平,表达于细胞浆的蛋白分子较表达于细胞核的蛋白分子能诱导机体更强的体液免疫反应,这为以增强体液免疫反应为目的的基因疫苗的设计提供参考依据。
关键词: 关基因免疫 体液免疫 核定位信号 HPV 16L1蛋白
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The intracellular localization of the proteins expressed by the DNA vaccine can affect the humour immunity responsion level in DNA immunization
Abstract:Objective Analyses the different location in cell affectting the humour immunity responsion level using DNA immunization. Methods after EGFP-HPV16 L1 and EGFP-HPV16L1△NLS fusion genes were amplified from pFB-EGFP-HPV16L1 and pFB-EGFP-HPV16L1△NLS plasmid, they were recombined into pcDNA3.1(+) vector respectively to generate the recombinant pcDNA-EGFP-HPV16L1 and pcDNA -EGFP-HPV16L1△NLS plasmids. The recombinant plasmids were used to transform CHO cells, The fluorescence of EGFP tagged fusion protein was observed under fluorescent microscope. And then, the recombinant plasmids were njected into the tibial musle to immunize BALB/c mices. The titers of these antisera were determined using ELISA. Results the EGFP-HPV16 L1 and EGFP-HPV16L1△NLS were correctly inserted into pcDNA3.1 (+) vectors. EGFP-HPV16 L1 fusion proteins could be detected in the the nuclear of CHO cells, while the EGFP-HPV16L1△NLS fusion proteins location in the cytoplasm of CHO cells. The two recombinant plasmids could induce the humour immunity responsion in BALB/c mices. After three times immunization, the A450 value of antisera of pcDNA -EGFP-HPV16L1△NLS plasmids immunized mices group is higher than it of the recombinant pcDNA-EGFP-HPV16L1the immunized mices group (P<0.001). Conclusion The intracellular localization of the proteins expressed by the DNA vaccine could affect the humour immunity responsion level in DNA immunization. The proteins localized in cytoplasm could induce stronger humour immunity responsion level and the proteins localized in nuclear in DNA immunization. expressed by the DNA vaccine. The conclusion is very useful to design the DNA vaccine to induce strong humour immunity responsion level or to produce antibody through DNA immunization.
Keywords: DNA immunization humour immunity responsionl human papillomavirus 16 l1 nuclear location signal (NLS)
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