Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts.
Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts.
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DOI：
10.1006/jmbi.2001.4921
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发表时间：
2001-08-31
影响因子：
5.6
通讯作者：
Wiebe, PO
中科院分区：
文献类型：
--
作者：
Daniell, H;Lee, SB;Panchal, T;Wiebe, PO
关键词：
产肠毒素大肠杆菌（LTB）的B亚基和霍乱弧菌的霍乱毒素（CT B）是候选疫苗抗原。将未修饰的CTB编码序列整合到叶绿体基因组中（每个细胞高达10，000个拷贝），导致高达4.1%的总可溶性烟草叶蛋白作为功能性寡聚体积累（比通过核基因组表达的未修饰的LTB基因的表达水平高410倍）。然而，报道的表达水平低估了转基因叶绿体中CTB的实际积累，这是由于未煮沸样品中寡聚形式的聚集与纯化细菌抗原观察到的聚集相似。PCR和Southern杂交分析证实了CTB基因稳定整合到叶绿体基因组中。蛋白质印迹分析表明，叶绿体合成的CTB组装成寡聚体，并与纯化的天然CTB抗原相同。此外，结合试验证实，叶绿体合成的CTB结合肠膜GM 1-神经节苷脂受体，表明正确的折叠和二硫键的CTB五聚体内的转基因叶绿体形成。与发育不良的核转基因植物相反，当CTB在叶绿体中组成型表达时，叶绿体转基因植物在形态上与未转化的植物没有区别。通过PCR和Southern印迹分析证实，导入的基因在后代中稳定遗传。在转基因叶绿体中有效的跨粘膜载体分子和递送系统（如CTB）的增加的产生使得基于植物的口服疫苗和需要口服施用的具有CTB的融合蛋白在商业上是可行的。外源基因在转基因质体中的成功表达和无标记叶绿体转化技术的可用性预示着在转基因植物的可食用部分中开发疫苗的良好前景。此外，由于许多蛋白质的四级结构是必不可少的，它们的功能，这项调查表明，其他外来的多聚体蛋白在转基因叶绿体中正确表达和组装的潜力。
The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.
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影响因子：
3.7
作者：
Gómez, N;Carrillo, C;Escribano, JM
通讯作者：
Escribano, JM
影响因子：
56.9
作者：
HAQ, TA;MASON, HS;ARNTZEN, CJ
通讯作者：
ARNTZEN, CJ
影响因子：
4
作者：
BENDICH, AJ
通讯作者：
BENDICH, AJ
DOI：
10.1038/nbt1293-1574
发表时间：
1993-12-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
LEBENS, M;JOHANSSON, S;HOLMGREN, J
通讯作者：
HOLMGREN, J
影响因子：
3
作者：
Arakawa, T;Chong, DKX;Langridge, WHR
通讯作者：
Langridge, WHR