含恶性疟原虫Pf70基因片段表达载体DNA的免疫学特性  

Immune characteristics of plasmid DNA containing Plasmodium falciparum Pf70 gene fragment

作　　者：叶凯[1] 李其岚[2] 徐洪[1] Kironde Fred Alexander S 马辉文[1] 

机构地区：[1]武汉大学药学院 [2]武汉大学生命科学学院 [3]CRID Projects,Department of Biochemistry,Medical Schoolof Makerere University

出　　处：《中华微生物学和免疫学杂志》2003年第5期359-363,共5页Chinese Journal of Microbiology and Immunology

基　　金：联合国工业发展组织 /世界银行 /世界卫生组织资助项目 (TDR/RPCB/2 0 0 0 )

摘　　要：目的　研究恶性疟原虫Pf70DNA片段作为DNA疫苗候选抗原基因的可能性。方法　应用PCR方法从含有恶性疟原虫Pf70基因DNA片段的质粒pGEX Pf70中扩增出包含Pf70DNA片段的长度为 95 7bp的PCR产物。将其插入带有乙肝表面抗原基因的真核表达载体pCMV S中 ,构建出重组质粒pCMV S Pf70。用提纯的重组质粒pCMV S Pf70作为DNA免疫制剂 ,以质粒pCMV SDNA和经过谷胱甘肽亲和层析法纯化的融合蛋白GST Pf70作为对照 ,免疫昆明种小白鼠。每隔 14d加强免疫1次 ,加强免疫 2次后第 7天采小鼠全血 ,用流式细胞仪检测CD8+ T淋巴细胞和CD4 + T淋巴细胞的水平 ,以检测体液免疫和细胞免疫状况。结果　接种了重组质粒pCMV S Pf70小鼠CD8+ T淋巴细胞的绝对量增加 ,其百分比含量增加了 39% ;CD4 + T淋巴细胞的相对含量保持恒定 ,绝对量稍有增加。用ELISA法分别检测经重组质粒pCMV S Pf70免疫的小鼠和经融合蛋白GST Pf70免疫的小鼠血清中抗Pf70蛋白质的抗体滴度 ,发现前者抗体滴度约为 1∶80 0 ,远远低于后者的滴度 (1∶5 0 0 0 )。研究结果证明 ,重组质粒pCMV S Pf70DNA可以诱导小鼠产生很强的细胞免疫和相对于蛋白质免疫而言较弱的体液免疫 ;Pf70蛋白质可以诱导小鼠产生很强的体液免疫。Objective To study the feasibility of Plasmodium falciparum Pf70 gene fragment as a component of DNA vaccine against the parasite infection. Methods A 957bp Pf70 gene fragment was amplified by PCR using pGEX Pf70 as template containing the gene fragment. The PCR product was inserted into an eucaryotic expression vector, pCMV S, in the same reading frame with HBsAg gene in the vector to form a recombinant plasmid named pCMV S Pf70. Kunming mice were immunized with either purified plasmid pCMV S Pf70, or GST Pf70 fusion protein purified using glutathione affinity chromatography. The immunizations were boosted twice at an interval of 14 days. Whole blood was collected from mice orbit on the day 7 after last boost and subjected to the assay of flow cytometry to determine the levels of CD4 + and CD8 + T lymphocytes. Results Compared with no immunized mice, it was found that the number and the percentage of CD8 + T lymphocyte were obviously increased, whereas the percentage of CD4 + T lymphocyte was keeping unchanged and the number of this kind of T lymphocyte was slightly increased in the mice immunized with plasmid DNA. The humoral immune responses of the immunized mice with either plasmid pCMV S Pf70 or fusion protein GST Pf70 were determined with ELISA. The result indicated that the titer of antibodies in the sera from DNA immunized mice reached to 1∶800 and that was much lower than what in the sera from the mice immunized with GST Pf70 fusion protein (1∶5?000). Conclusion Pf70 gene fragment may be a good candidate for P.falciiparum blood stage multiple DNA vaccine. [

关 键 词：疟疾 恶性疟原虫 Pf70基因 DNA疫苗 

分 类 号：R392[医药卫生—免疫学]