权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Characterization and specificity of newly defined protective antigens of Plasmodium berghei XAT

新定义的伯氏疟原虫 XAT 保护性抗原的表征和特异性

基本信息

批准号：
12670240
负责人：
KOBAYASHI Fumie
金额：
$ 2.18万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670240/
关键词：
malaria protective antigen vaccine Plasmodium berghei

项目摘要

Plasmodium berghei XAT is an irradiation induced attenuated variant of the lethal strain, P. berghei NK65. It has been suggested that Th1 type antibody, IgG2a, in hyperimmune sera suppressed the parasitemia, indicating that antibody-mediated immunity is important in P. berghei XAT infection. However, antigens which are involved in the protective immunity have not yet been identified. In the present study, we established hybridomas producing protective monoclonal antibodies against P. berghei XAT infection. The characterization, stage specificity and localization of the protective antigens were examined. Spleen cells from mice immunized with Plasmodium berghei XAT were fused with P3U1 myeloma cells. Seven hybridomas secreting monoclonal antibodies (mAb) which reacted strongly with P. berghei XAT were recloned. The clones were expanded in the form of ascites fluids. The isotypes of 7 mAbs were found to be IgMs (5 clones) and IgG1s (2 clones) and only mAbs identified as IgG1s were protective in passive transfer experiments. The other anti-P. berghei XAT mAbs were not protective on passive transfer. Protective mAbs showed high levels of antibody titer to P. berghei XAT and low levels to P. berghei NK65 in ELISA. The molecular weights of the recognized antigens are 150, 160 kDa as determined by Western blotting under reducing and nonreducing conditions. Indirect immunofluorescent antibody tests showed that the target antigens were synthesized only in the schizont stage. The detailed localization of the protective antigens was conferred by the immunoelectron microscopy. The antigens were localized in the parasitophorous vacuole space and in the clefts in the infected erythrocyte cytoplasm. To know the mechanisms by which the antigens recognized by these mAbs are involved in the protection would provide an important information for the development of a blood-stage malarial vaccine.

伯氏疟原虫XAT是伯氏疟原虫致死株NK65的辐射诱导减毒变异体。高免血清中Th1型抗体IgG2a可抑制原虫血症，提示抗体介导的免疫在伯氏肺吸虫XAT感染中起重要作用。然而，参与保护性免疫的抗原还没有被确定。在本研究中，我们建立了产生抗伯氏疟原虫XAT感染的保护性单抗的杂交瘤细胞。对保护性抗原的特性、阶段特异性和定位进行了检测。伯氏疟原虫XAT免疫小鼠的脾细胞与P3U1骨髓瘤细胞融合。重新克隆了7株分泌与伯氏疟原虫XAT强反应的单抗的杂交瘤细胞。克隆以腹水的形式进行扩增。在被动转移实验中，7株单抗的同工型分别为IgM(5个克隆)和IgG1s(2个克隆)，只有鉴定为IgG1s的单抗具有保护性。另一种是抗P.Berghei XAT单抗对被动转移无保护作用。保护性单抗对伯氏疟原虫XAT抗体效价高，对伯氏杆菌NK65抗体效价低。还原和非还原条件下的Western blotting测定识别抗原的相对分子质量分别为150、160 kDa。间接免疫荧光抗体检测显示，靶抗原仅在裂殖体阶段合成。免疫电子显微镜对保护性抗原进行了详细的定位。这些抗原定位于寄生虫的空泡空间和感染红细胞细胞质的裂隙中。了解这些单抗识别的抗原参与保护的机制将为血液期疟疾疫苗的开发提供重要信息。