SARS冠状病毒spike基因DNA疫苗的初步研究  

Immunogenicity of DNA vaccine that express spike gene fragment of SARS coronavirus

作　　者：李疆[1] 覃海德[1] 刘鹏[1] 张经纬 冯炳健[1] 冯启胜[1] 陈丽珍[1] 潘志刚[1] 黄丽惜[1] 张如华[1] 余杏娟[1] 曾益新[1] 

机构地区：[1]中山大学肿瘤医院实验研究部,广州510060 [2]深圳太太药业集团

出　　处：《中华微生物学和免疫学杂志》2005年第4期297-301,共5页Chinese Journal of Microbiology and Immunology

基　　金：973计划SARS专项项目 (2 0 0 3CB5 14 10 7) ;广东省防治非典科技攻关项目 (2 0 0 3Z3 0 45 1) ;横向联合课题 (深圳太太药业集团 )

摘　　要：目的　构建SARS病毒spike基因片段真核表达质粒DNA疫苗;检测spike基因片段的免疫原性;筛选SARS病毒疫苗构建的理想靶抗原。方法　采用RT PCR从SARS冠状病毒北京0 1(BJ0 1)株cDNA获取了spike基因cDNA的全长D12、编码N末端氨基酸的cDNA片段D14和编码C末端氨基酸的cDNA片段D2 3;构建重组真核表达质粒pcDNA3 /D12、pcDNA3 D14、pcDNA3 D2 /3;用3种重组质粒和pcDNA3空质粒载体(对照)DNA皮下注射免疫Wistar大鼠;ELISA检测免疫后大鼠血清S蛋白特异性抗体IgG的产生;同位素51 Cr实验检测特异性CTL应答;ELISPOT检测单细胞水平IFN -γ分泌。结果　pcDNA3 /D2 3疫苗免疫组大鼠血清有S蛋白阳性抗体IgG产生、抗体滴度>10 0 0 ;脾淋巴细胞可诱导特异的CTL应答和IFN γ分泌,pcDNA3 /D2 3疫苗组与其它组之间统计分析P <0 .0 5。结论SARS冠状病毒S蛋白的C末端具有较强的免疫原性,是疫苗构建的理想候选靶抗原,本研究结果为SARS病毒的疫苗研究提供了资料。Objective To construct the SARS coronavirus spike gene DNA vaccines and to find the immunogenicity of spike gene. Methods We collected two DNA fragments coding the N-terminal(D14, 1-663AA) and C-terminal(D23, 634-1255AA) of the Spike protein and the whole spike gene(D12, 1-1255AA) from the SARS virus (Beijing 01 strain) cDNA by RT-PCR and recombinated the DNA fragments with plasmid pcDNA3. The naked DNA plasmid containing the S gene and the two fragments of the N-terminal and C-terminal of the S gene and the empty vector pcDNA3 as control were used to immunize Wistar rats intramuscularly. The humoral antibody IgG responsed to S gene was detected in the serum of vaccination rats in 3 rd, 5 th, 6 th and 7 th weeks after the first dosage of immunization. The cell-mediated immune(CMI) response of vaccines was detected by the MHC class-Ⅰ limited CD8+ virus-specific cytotoxic T lymphocytes(CTLs) with 51Cr release experiments and the cytokine level of IFN-γ with the ELISPOT. Results D23 detected vaccinated animals had antiboby at 5 th week and the titer raised by 1000-fold. Other experiment and control groups didn′t produce antibody. The group of D23 was found to induce stronger specific CTL activity than other groups (P<0.05, between D23 and control group). No specific CTL was found in the groups of D14, D12 and control group and the spleenocytes of group of D23 released more IFN-γ than that of spleenocytes from other groups. Conclusion These results identified that the C-terminal of spike gene was with more preferential antigenicity expression for vaccine.

关 键 词：spike基因 SARS冠状病毒S蛋白 PCDNA3 步研究 SARS病毒 Wistar大鼠 cDNA片段 质粒DNA疫苗 ELISA检测 ELISPOT 抗体IGG CTL应答 IFN-γ 基因cDNA RT-PCR 真核表达质粒 基因片段 免疫原性 疫苗构建 大鼠血清 单细胞水平 脾淋巴细胞 

分 类 号：R392[医药卫生—免疫学]