High-level expression of Plasmodium vivax apical membrane antigen 1 (AMA-1) in Pichia pastoris:: Strong immunogenicity in Macaca mulatta immunized with P-vivax AMA-1 and adjuvant SBAS2
High-level expression of Plasmodium vivax apical membrane antigen 1 (AMA-1) in Pichia pastoris:: Strong immunogenicity in Macaca mulatta immunized with P-vivax AMA-1 and adjuvant SBAS2
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DOI：
10.1128/iai.67.1.43-49.1999
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发表时间：
1999-01-01
影响因子：
3.1
通讯作者：
Thomas, AW
中科院分区：
文献类型：
--
作者：
Kocken, CHM;Dubbeld, MA;Thomas, AW
顶膜抗原1(AMA-1)家族是一个很有前途的疟疾血液期疫苗候选家族，已在啮齿动物和非人类灵长类疟疾模型中诱导保护。正确的蛋白质构象似乎是诱导寄生虫抑制反应的关键，这些反应似乎主要是由抗体介导的。在这里，我们首次描述了间日疟原虫AMA-1(PV66/AMA-1)胞外区的高效分泌表达(超过50 mg/L)。为了防止非天然糖基化，还开发了一个保守突变的PV66/AMA-1基因(PV66 Delta Glyc)，该基因缺乏N-糖基化位点。PV66 Delta糖胞外域的表达产生了类似水平的非糖基化均一产物，并且很容易通过离子交换和凝胶过滤层析进行纯化。重组PV66 Delta Glyc(43-487)能与构象依赖的单抗反应，在SBAS2佐剂的作用下，毕赤酵母表达的PV66 Delta Glyc(43-487)对5只恒河猴具有高度的免疫原性，诱导免疫球蛋白G的酶联免疫吸附测定效价超过1：20万，这组猕猴在食蟹猴疟原虫感染后有较低的累积寄生虫量的趋势。
The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of the Plasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66 Delta glyc) lacking N-glycosylation sites was also developed. Expression of the PV66 Delta glyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66 Delta glyc(43-487) was reactive with conformation-dependent monoclonal antibodies, With the SBAS2 adjuvant, Pichia-expressed PV66 Delta glyc(43-487) was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000, This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.