摘要
目的探讨免疫系统补体成分Ficolin-A抗伯氏疟原虫(Plasmodium berghei)感染的效果。方法克隆扩增伯氏疟原虫裂殖子表面蛋白MSP119基因,构建pGEX-KG-MSP119质粒。将pGEX-KG-Ficolin-A质粒和pGEXKG-MSP119质粒分别转染至大肠埃希菌(E.coli)BL21,1 mmol/L异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,谷胱甘肽琼脂糖凝胶4B柱纯化重组蛋白,十二烷基硫酸钠-聚丙烯酰胺电泳(SDS-PAGE)和蛋白质印迹(Western blotting)鉴定蛋白表达情况。40只小鼠随机均分为5组,Ficolin-A蛋白组和MSP119蛋白组小鼠每次注射蛋白20μg,MSP119蛋白+Ficolin-A蛋白组小鼠每次注射2种蛋白各20μg,PBS对照组和GST对照组小鼠每次各注射PBS 200μl和GST 20μg,各组小鼠每2周免疫1次,共免疫4次。末次免疫后2周,各组小鼠腹腔注射感染伯氏疟原虫的红细胞300μl,分别于注射感染后第2、4、6、8和10天每组各取3只小鼠,尾静脉采血涂片,吉氏染色后,显微镜下计数感染的红细胞,计算疟原虫密度。感染疟原虫后第20天统计各组小鼠的存活数量。结果测序结果表明,pGEX-KG-Ficolin-A和pGEX-KG-MSP119质粒构建成功。SDS-PAGE和Western blotting结果表明,Ficolin-A融合蛋白的相对分子质量(Mr)约为69 000,MSP119融合蛋白约Mr41 000,均与预测值相符。动物实验结果显示,感染后第2、4、6、8和10天,MSP119蛋白+Ficolin-A蛋白组小鼠的疟原虫密度均略低于其他4组,至感染后第10天,疟原虫密度为(22.2±1.7)%,略低于MSP119蛋白组[(33.4±2.7)%]、Ficolin-A蛋白组[(36.2±3.1)%]、GST对照组[(43.8±4.8)%]和PBS对照组[(45.3±3.6)%],但差异均无统计学意义(P>0.05)。感染后第20天,PBS对照组8只小鼠均死亡,Ficolin-A蛋白组存活小鼠数量(3只)与GST对照组(2只)比较,差异无统计学意义(P>0.05);MSP119蛋白+Ficolin-A蛋白组存活小鼠数量(6只)显著高于GST对照组(P<0.05)。结论补体成分Ficolin-A对降低小鼠疟原虫密度效果不明显,联合MSP119使用可提高小鼠感染疟原虫后的生存机会。
Objective To evaluate the effect of Ficolin-A, a lectin complement against Plasmodium berghei in mice model. Methods The M 19 000 fragment of merozoite surface protein-1 of P. berghei （MSPI19） was cloned and then subcloned into the vector pGEX-KG. The recombinants of pGEX-KG-Ficolin-A and pGEX-KG-MSPll9 were transformed into Escherichia coil BL21, and followed by expression of the protein induced by 1 mmol/L IPTG. The fusion protein was purified by affinity chromatography using Glutathione Sepharose 4B, and then identified by SDS-PAGE and Western-blot- ting. Five mouse model groups were treated with PBS, GST, Ficolin-A, MSPI19, or Ficolin-A＋MSPllg, respectively. Each group had eight mice. Mice in Ficolin-A or MSP119 groups were injected with 20 p,g Ficolin-A or MSP119 protein each time, respectively. Mice in Ficolin-A＋MSP119 group were injected with 20 p^g Fieolin-A and 20 Ixg MSPII9 each time. Mice in control groups were injected with 200 Ixl PBS or 20 ug GST, respectively. All the mice received four immuniza- tions at 2-week intervals. Two weeks after the last immunization, all the mice were inoculated with 300 txl Plasmodium berghei-infected red blood ceils. On day 2, 4, 6, 8, and 10 post-infection, blood samples were collected from three mice of each group, and the Giemsa stained-blood films were microscopically examined. Density of malaria parasites was cal- culated. The survival rate was evaluated on day 20 post-infection. Results The recombinant vectors of pGEX-KG-Ficol-in-A and pGEX-KG-MSP119 were constructed. Purified fusion proteins, Ficolin-A-GST and MSP119-GST, were obtained. Western blotting analysis indicated that the relative molecular mass of fusion proteins Ficolin-A-GST and MSPIIg-GST was about M, 69 000 and Mr 41 000. Animal experiments showed that on day 10 after infection, the parasite density in Fi- colin-A＋MSP119 group [（22.2±1.7）%] was slightly lower than that of the groups MSP119 [（33.4±2.7）%1, Ficolin-A [（36.2± 3.1）%1, GST [（43.8±4.8）%1 and PBS[（45.3＋-3.6） %] , but the difference was not statistically significant （P〉0.05）. No mouse survived in PBS group on day 20 after infection. There was no significant difference in number of survival mice between Ficolin-A group （3 mice） and GST group （2 mice）. Six mice survived in Ficolin-A＋MSPI19 group, which was sig- nificantly more than that of GST group （P〈0.05）. Conduslon Ficolin-A cannot significantly suppress parasite density. However, Ficolin-A＋MSP119 can increase the survival rate of Plasmodium berghei-infected mice.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2014年第1期42-45,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
恩施州科技攻关计划课题~~
