Department of International Health, University of Copenhagen, Copenhagen K, Denmark.
Malar J. 2010 Nov 15;9:325. doi: 10.1186/1475-2875-9-325.
The PFD1235w Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE) adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed.
The recombinant proteins were run on SDS-PAGE and Western blots for quantification and size estimation. Insect cell and E. coli-produced recombinant proteins were coupled to a bead-based Luminex assay to measure the plasma antibody reactivity of 180 samples collected from Tanzanian individuals. The recombinant proteins used for immunization of rats and antisera were also tested by flow cytometry for their ability to surface label 3D7PFD1235w-IE.
All seven pAcGP67A constructs were successfully expressed as recombinant protein in baculovirus-infected insect cells and subsequently produced to a purity of 60-97% and a yield of 2-15 mg/L. By comparison, only three of seven pET101/D-TOPO constructs expressed in the E. coli system could be produced at all with purity and yield ranging from 3-95% and 6-11 mg/L. All seven insect cell, but only two of the E. coli produced proteins induced antibodies reactive with native PFD1235w expressed on 3D7PFD1235w-IE. The recombinant proteins were recognized in an age- and transmission intensity-dependent manner by antibodies from 180 Tanzanian individuals in a bead-based Luminex assay.
The baculovirus based insect cell system was distinctly superior to the E. coli expression system in producing a larger number of different recombinant PFD1235w protein domains and these were significantly easier to purify at a useful yield. However, proteins produced in both systems were able to induce antibodies in rats, which can recognize the native PFD1235w on the surface of IE.
PFD1235w 恶性疟原虫红细胞膜蛋白 1（PfEMP1）抗原与儿童重症疟疾相关，可在黏附于 ICAM1 的感染红细胞（IE）表面表达。然而，这种 PfEMP1 的精确三维结构及其表面暴露的表位尚不清楚。本研究采用昆虫细胞和大肠埃希菌（E. coli）系统表达由 pfd1235w 变体基因编码的单域和双域蛋白。评估了所得重组蛋白的产量和纯度及其诱导大鼠抗体的能力，这些抗体与 3D7PFD1235w-IE 上表达的天然 PFD1235w PfEMP1 抗原发生反应。还分析了它们对来自先前感染坦桑尼亚的供体的人抗疟抗体的识别。
SDS-PAGE 和 Western blot 用于定量和大小估计分析重组蛋白。将昆虫细胞和大肠埃希菌产生的重组蛋白偶联到基于珠子的 Luminex 测定法中，以测量来自 180 名坦桑尼亚个体的血浆抗体反应性。还通过流式细胞术测试了用于免疫大鼠和抗血清的重组蛋白，以检测它们在 3D7PFD1235w-IE 上表面标记的能力。
7 个 pAcGP67A 构建体均成功在杆状病毒感染的昆虫细胞中作为重组蛋白表达，随后纯度达到 60-97%，产量为 2-15mg/L。相比之下，7 个 pET101/D-TOPO 构建体中只有 3 个可在大肠埃希菌系统中表达，其纯度和产量范围分别为 3-95%和 6-11mg/L。7 个昆虫细胞产生的所有蛋白，而非 2 个大肠埃希菌产生的蛋白，均可诱导与 3D7PFD1235w-IE 上表达的天然 PFD1235w 发生反应的抗体。重组蛋白在基于珠子的 Luminex 测定法中以年龄和传播强度依赖的方式被来自 180 名坦桑尼亚个体的抗体识别。
与大肠埃希菌表达系统相比，基于杆状病毒的昆虫细胞系统在生产大量不同的重组 PFD1235w 蛋白结构域方面明显具有优势，且这些蛋白更容易以有用的产量进行纯化。然而，两种系统产生的蛋白均能在大鼠中诱导产生可识别 IE 表面天然 PFD1235w 的抗体。
