Purification, characterization, and immunogenicity of a disulfide cross-linked Plasmodium vivax vaccine candidate antigen, merozoite surface protein 1, expressed in Escherichia coli
Purification, characterization, and immunogenicity of a disulfide cross-linked Plasmodium vivax vaccine candidate antigen, merozoite surface protein 1, expressed in Escherichia coli
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DOI：
10.1128/iai.69.9.5464-5470.2001
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发表时间：
2001-09-01
影响因子：
3.1
通讯作者：
Lanar, DE
中科院分区：
文献类型：
--
作者：
Dutta, S;Ware, LA;Lanar, DE
间日疟原虫裂殖子表面蛋白1（MSP-1）42-kDa片段（PvMSP-1 p42）是一种有前途的针对疟原虫血液期的疫苗候选抗原。我们已经开发了一种生产这种疫苗靶点的方法，同时考虑到它在人类志愿者中的用途。谷胱甘肽和硫氧还蛋白还原酶基因突变的大肠杆菌新菌株Origami（DE 3）产生的可溶性PvMSP-1 p42比传统的大肠杆菌多60%。coliBL 21（DE 3）菌株。重组PvMSP-1 p42纯化到大于或等于99%的纯度与一个快速的两步方案，设计容易放大。最终产物具有低内毒素含量，并且在其冻干形式下是稳定的。发现PvMSP-1 p42具有预测的一级和三级结构，并且如预测的那样由含有一个游离半胱氨酸的单个构象异构体组成。该产物被针对间日疟原虫MSP-1的构象单克隆抗体识别。用两种小鼠品系和佐剂Montanide ISA 51和Montanide ISA 720进行PvMSP-1 p42的免疫原性研究。发现两种制剂均诱导高水平的免疫球蛋白G1（IgG 1）、IgG 2b和IgG 2a抗体沿着低水平的IgG 3。来自所有PvMSP-1 p42免疫组中动物的淋巴细胞在用PvMSP-1 p42刺激后显示增殖反应;在培养上清液中检测到细胞因子白细胞介素2（IL-2）、γ干扰素、IL-4和IL-10。这些结果表明，PvMSP-1 p42与两种佐剂组合在小鼠中引发细胞和体液应答。
The Plasmodium vivax merozoite surface protein 1 (MSP-1) 42-kDa fragment (PvMSP-1 p42) is a promising vaccine candidate antigen against the blood stage of the malarial parasite. We have developed a process for the production of this vaccine target, keeping in mind its use in human volunteers. A novel strain, Origami (DE3), of Escherichia coli with mutations in the glutathione and thioredoxin reductase genes yielded 60% more soluble PvMSP-1 p42 than the conventional E. coli BL21(DE3) strain. Recombinant PvMSP-1 p42 was purified to greater than or equal to 99% purity with a rapid two-step protocol designed for easy scaling up. The final product had a low endotoxin content and was stable in its lyophilized form. PvMSP-1 p42 was found to have the predicted primary and tertiary structures and consisted of a single conformer containing one free cysteine, as predicted. The product was recognized by conformational monoclonal antibodies against P. vivax MSP-1. Immunogenicity studies of PvMSP-1 p42 were carried out with two strains of mice and the adjuvants Montanide ISA51 and Montanide ISA720. Both formulations were found to induce high levels of immunoglobulin G1 (IgG1), IgG2b, and IgG2a antibodies along with low levels of IgG3. Lymphocytes from animals in all the PvMSP-1 p42-immunized groups showed proliferative responses upon stimulation with PvMSP-1 p42; the cytokines interleukin 2 (IL-2), gamma interferon, IL-4, and IL-10 were detected in the culture supernatants. These results indicate that PvMSP-1 p42 in combination with both of the adjuvants elicited cellular and humoral responses in mice.