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的研究  

Effects of intracellular localization of proteins expressed by DNA vaccines on humoral immunity response upon DNA immunization

作　　者：杨军[1] 陈晓黎[1] 王一理[2] 司履生[2] 

机构地区：[1]西安交通大学医学院第二附属医院病理科,710004 [2]西安交通大学生命科学与技术学院癌症研究所,生物医学信息工程教育部重点实验室,710061

出　　处：《中国医药生物技术》2012年第4期270-275,共6页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(30300305);教育部"新世纪优秀人才支持计划"(NCET-10-0647);教育部博士点新教师基金(2007-0698106)

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-HPVl6L1融合蛋白和截短型EGFP-HPV16L1△NLS融合蛋白的重组pcDNA-EGFP-HPV16L1和pcDNA-EGFPHPV16L1△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细胞质内可见到绿色荧光: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,但重组pcDNA-EGFP-HPV16L1△NLS真核表达载体免疫组小鼠IgG抗体A_(450)值显著高于重组pcDNAEGFP-HPV16L1真核表达载体免疫组小鼠IgG抗体A_(450)值(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,定位于细胞质内的蛋白分子较定位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了参考。Objective To analyze whether different intracellular location of expressed proteins may affect humoral immunity response upon DNA immunization. Methods EGFP-HPV16 L1 and EGFP-HPV16L1ANLS fusion genes were amplified from pFB-EGFP-HPV16L1 and pFB-EGFP-HPV16L1ANLS plasmid, respectively, then were recombined into pcDNA3.1（＋） vector to generate recombinant pcDNA-EGFP-HPV16L1 and pcDNA -EGFP-HPV16L1 ANLS plasmid, respectively. The recombinant plasmids were transfected into CHO cells and fluorescences of EGFP tagged fusion proteins were observed under fluorescent microscope. The recombinant plasmids were subsequently injected into the tibial muscles to immunize BALB/c mices and titers of the antisera were determined using ELISA.Results The EGFP-HPV16 L1 and EGFP-HPV16L1ANLS were correctly inserted into pcDNA3.1 （＋） vectors. EGFP-HPV16 L1 fusion protein could be detected in the nucleus of CHO cells, while the EGFP-HPV16L1ANLS fusion protein was located in the cytoplasm of CHO cells. The two recombinant plasmids could induce humoral immunity response in BALB/c mices. After three times of immunization, the A4s0 values of antisera of pcDNA -EGFP-HPV 16L 1 ANLS plasmid immunized mices group were higher than that of recombinant pcDNA-EGFP-HPV 16L 1 immunized mices group （P 〈 0.001）. Conclusion Intracellular localization of the proteins expressed by DNA vaccines could affect humoral immunity response upon DNA immunization. Proteins localized in the cytoplasm could induce stronger humoral immunity response compared with proteins localized in the nucleus upon DNA immunization. The conclusion is very useful to design DNA vaccine to induce stronger humoral immunity response or to produce antibody upon DNA immunization.

关 键 词：抗体生成 免疫疗法 核定位信号 基因免疫 

分 类 号：R392.1[医药卫生—免疫学]