论文部分内容阅读
目的 本课题利用含人类巨细胞病毒 (CMV)的高效启动子和增强子的pcDNA3质粒作为载体 ,在人宫颈癌细胞(Hela)中高效表达恶性疟原虫环子孢子蛋白 (CSP) ,观察其表达产物诱导BALB/c小鼠的免疫应答水平。方法 从我国恶性疟原虫海南FCC1/HN株基因组中特异扩增CSP基因 ;将其定向克隆到真核表达质粒pcDNA3的CMV启动子下游 ,构建重组质粒pcDNA3 PfCSP ,并导入Hela细胞 ;分离和纯化表达蛋白 ,并将其免疫接种BALB/c小鼠 ;通过ELISA、Dot ELISA、Westernblot分析、T淋巴细胞增殖试验、自然杀伤 (NK)细胞活性检测、T淋巴细胞亚群测定 ,观察其诱导BALB/c小鼠产生体液免疫和细胞免疫的应答水平。结果 免疫后的小鼠产生高效价的特异性抗体反应 ,ELISA检测抗体滴度达 1∶640 0 ;Westernblot结果在 38 3kDa相应位置处出现较清晰的显色条带。小鼠脾淋巴细胞转化试验结果显示 ,且表达蛋白抗原刺激免疫鼠和正常鼠测得OD值分别为 0 34± 0 0 4和 0 12± 0 0 3,两者之间差异显著 (P <0 0 1)。用MTT比色分析法检测小鼠NK细胞活性 ,免疫鼠NK细胞活性明显高于正常鼠 (P <0 0 5 )。用间接免疫荧光法测定免疫鼠淋巴细胞亚群 ,结果表明其CD4 + 、CD8+ T细胞数与正常鼠相比 ,均明显增多 ,经t检验差异显著 (P <0 0 5 )。结论
OBJECTIVE: To express Plasmodium falciparum circumsporozoite protein (CSP) highly efficiently in human cervical cancer cell (Hela) using pcDNA3 plasmid containing human cytomegalovirus (CMV) highly efficient promoter and enhancer as a vector and observe its expression The product induces an immune response level in BALB / c mice. Methods CSP gene was amplified from the genome of Hainan FCC1 / HN strain of Plasmodium falciparum in China and cloned into the downstream of CMV promoter of eukaryotic expression plasmid pcDNA3. The recombinant plasmid pcDNA3 PfCSP was constructed and introduced into Hela cells. The expression of CSP gene was isolated and purified BALB / c mice were immunized with BALB / c mice by ELISA, Dot ELISA, Western blot analysis, T lymphocyte proliferation assay, natural killer (NK) cell activity assay and T lymphocyte subsets assay. Mice produce humoral and cellular immune response levels. Results After immunization, the titer of antibody was high titer. The titer of antibody was 1:640 0 by ELISA. The clearer band was observed at the corresponding position of 38 3 kDa by Western blot. The results of mouse splenic lymphocyte transformation showed that the OD value of the spleen lymphocyte of mice immunized with the expressed protein antigen was 0 34 ± 0 0 4 and 0 12 ± 0 0 3, respectively (P <0 0 1). The activity of NK cells in mice was detected by MTT colorimetric assay. The activity of NK cells in immunized mice was significantly higher than that in normal mice (P <0.05). The results of indirect immunofluorescence assay showed that the number of CD4 + and CD8 + T cells in immunized mice was significantly higher than that in normal mice (P <0.05). in conclusion