The use of synthetic genes for the expression of ciliate proteins in heterologous systems
The use of synthetic genes for the expression of ciliate proteins in heterologous systems
复制标题
DOI：
10.1016/s0378-1119(02)00433-x
复制
发表时间：
2002-04-17
期刊：
影响因子：
3.5
通讯作者：
Clark, TG
中科院分区：
文献类型：
--
作者：
Lin, YK;Cheng, G;Clark, TG
常见的鱼类寄生虫，小瓜鱼，表达丰富的糖基化磷脂酰肌醇（GPI）锚定的膜蛋白，称为固定抗原，或i-抗原。这些蛋白质是宿主免疫应答的靶标，并且已被鉴定为重组亚单位疫苗开发的潜在候选物。然而，由于小瓜虫利用非标准遗传密码，相应基因产物的表达，无论是作为亚单位抗原在常规蛋白质表达系统中，或作为载体编码的抗原在DNA疫苗的情况下，是远非简单。为了克服这个问题，我们利用“装配聚合酶链反应”来制造两种基因的合成版本（命名为IAG 52 A [G5/CC]和IAG 52 B [G5/CC]），其编码与来自寄生虫株G5的52/55 kDa i抗原相似的抗原。这种方法可以消除不需要的终止密码子，并用癍点叉尾鮰的首选密码子替换基因的天然序列。为了确定合成的等位基因是否可以在使用标准遗传密码的细胞中表达，我们将IAG 52 A [G5/CC]引入到各种异源细胞类型中，并通过免疫荧光光学显微镜或Western印迹测试表达。当克隆到合适的启动子下游时，IAG 52 A [G5/CC]在大肠杆菌、哺乳动物COS-7细胞和癍点叉尾鮰中表达，在其中它引起抗原特异性免疫应答。有趣的是，COS-7细胞中相应基因产物的定位模式表明，虽然蛋白质正确折叠，但它不存在于细胞膜上，这表明GPI锚定添加所需的信号肽在纤毛虫和哺乳动物系统中不同。人工合成的等位基因的构建在开发针对小瓜虫的疫苗中具有实际效用，同时，为纤毛虫基因在异源系统中的表达提供了通用方法。(C)2002 Elsevier Science B. V.保留所有权利。
The common fish parasite, Ichthyophthirius multifiliis, expresses abundant glycosylated phosphatidylinositol (GPI)-anchored membrane proteins known as immobilization antigens, or i-antigens. These proteins are targets of the host immune response, and have been identified as potential candidates for recombinant subunit vaccine development. Nevertheless, because Ichthyophthirius utilizes a non-standard genetic code, expression of the corresponding gene products, either as subunit antigens in conventional protein expression systems, or as vector-encoded antigens in the case of DNA vaccines, is far from straightforward. To overcome this problem, we utilized `assembly polymerase chain reaction' to manufacture synthetic versions of two genes (designated IAG52A[G5/CC] and IAG52B[G5/CC]) encoding similar to52/55 kDa iantigens from parasite strain G5. This approach made it possible to eliminate unwanted stop codons and substitute the preferred codon usage of channel catfish for the native sequences of the genes. To determine whether the synthetic alleles could be expressed in cells that use the standard genetic code, we introduced IAG52A[G5/CC] into a variety of heterologous cell types and tested for expression either by immunofluorescence light microscopy or Western blotting. When cloned downstream of appropriate promoters, IAG52A[G5/CC] was expressed in Escherichia coli, mammalian COS-7 cells, and channel catfish where it elicited antigen-specific immune responses. Interestingly, the localization pattern of the corresponding gene product in COS-7 cells indicated that while the protein was correctly folded, it was not present on the cell membrane, suggesting that the signal peptides required for GPI-anchor addition differ in ciliate and mammalian systems. Construction of synthetic alleles should have practical utility in the development of vaccines against Ichthyophthirius, and at the same time, provide a general method for the expression of ciliate genes in heterologous systems. (C) 2002 Elsevier Science B.V. All rights reserved.