恶性疟原虫海南FCC1/HN株CSP抗CSP抗原基因在Hela细胞中表达产物的免疫应答研究(英文)

Immune responses to the expressed products of the CSP antigen gene of Plasmodi um falciparum southern China isolate FCC1/HN in Hela cell

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 )。

Objective To construct a eukaryotic expression system with pcDNA3-PfCSP/Hela for the Circumsporozoite protein (CSP) gene of Plasmodium falciparum (P. falciparum), to observe the immune responses in BALB/c mice induced by the expressed proteins. Methods The recombinant plasmid pcDNA3-PfCSP was transformed into the Hela cell line. The expressed protein was isolated and analyzed by using SDS-PAGE and used for immunization of BALB/c mice by subcutaneous, intravenous, and intraperitoneal adminstration. Enzyme-linked immunosorbent assay(ELISA), Dot-ELISA, Western blot, T lymphocyte proliferation test, natural killer cell(NKC) activity assay, and CD4+ and CD8+ T cell detection were used for observation of humoral and cellular immune responses. Results Immune sera strongly reacted with the expressed protein, antibody titer was up to 1∶6400 as detected by ELISA. Western blot analysis revealed a specific band at 38.3?Kda. When the spleen cells of normal and immunized BALB/c mice were specifically stimulated with expressed protein, the optical densities were 0.12±0.03 and 0.34±0.04, respectively. The latter were significantly higher than the former (P<0.01). We used the MTT colorimetric assay to measure NKC activity of mice spleen. The results showed that the NKC activity of immunized BALB/c mice was remarkably higher than that of the controls (P<0.05). CD4+ and CD8+ T cells were detected by using monoclonal antibody immunofluorescence methods. The results showed that the percentage of CD4+ and CD8+ T cells of immunized group were significantly higher than that of control group (P<0.05).Conclusions The humoral and cell-mediated immune responses and elevated NKC activity to products made with a eukaryotic expression system could be specifically detected in BALB/c mice. These findings indicate that the expressed protein could enhance the immune function in mice.