补体成分Ficolin-A抗小鼠感染伯氏疟原虫的研究  

Study on Ficolin-A against Infection of Plasmodium berghei in Mouse Model

作　　者：向田 刘杲 戴万案 李宗清 陈凡[2] 

机构地区：[1]湖北省恩施州中心医院,恩施445000 [2]湖北大学生命科学院.武汉430062

出　　处：《中国寄生虫学与寄生虫病杂志》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~~

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.

关 键 词：Ficolin-A 伯氏疟原虫 裂殖子表面蛋白1羧基端Mr19 000片段 

分 类 号：R382.319[医药卫生—医学寄生虫学]