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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

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【作者】 李疆覃海德刘鹏张经纬冯炳健冯启胜陈丽珍潘志刚黄丽惜张如华余杏娟曾益新

【Author】 LI Jiang*, QIN Hai-de, LIU Peng, ZHANG Jing-wei, FENG Bing-jian, FENG Qi-sheng, CHEN Li-zhen, PAN Zhi-gang, HUANG Li-xi, ZHANG Ru-hua, YU Xing-juan, ZENG Yi-xin. *Research Department of Sun Yat-Sen University Cancer Center, Guangzhou 510060, China

【机构】 中山大学肿瘤医院实验研究部深圳太太药业集团中山大学肿瘤医院实验研究部 510060广州510060广州510060广州

【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。

【Abstract】 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】 SARS病毒spike基因DNA疫苗
【Key words】 SARS-CoVspike geneDNA vaccine
【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 )
  • 【文献出处】 中华微生物学和免疫学杂志 ,Chinese Journal of Microbiology and Immunology , 编辑部邮箱 ,2005年04期
  • 【分类号】R392
  • 【下载频次】172
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