Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop.
Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop.
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DOI：
10.1016/j.vaccine.2004.01.069
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发表时间：
2004-10
期刊：
影响因子：
5.5
通讯作者：
J. Watson;V. Koya;S. Leppla;H. Daniell
中科院分区：
文献类型：
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作者：
J. Watson;V. Koya;S. Leppla;H. Daniell
疾病控制中心（CDC）将炭疽杆菌列为A类制剂，并估计每100 000名接触者遭受炭疽袭击的成本超过260亿美元。对炭疽疫苗纯度的担忧，需要多次注射，以及保护性抗原（PA）的有限供应，强调了对改进疫苗的迫切需要。因此，83 kDa免疫原性炭疽杆菌保护性抗原在转基因烟草叶绿体中表达。将PA基因（pag）与psbA调节信号沿着克隆到叶绿体载体中以增强翻译。通过PCR和Southern印迹分析证实了转基因的叶绿体整合。粗植物提取物含有高达2.5mg全长PA/g新鲜叶组织，这在储存的叶或粗提取物中显示出几个月的优异稳定性。在连续光照下，在成熟叶中观察到最大水平的表达。共表达的ORF 2伴侣蛋白从苏云金芽孢杆菌没有增加PA积累或诱导折叠成立方晶体的转基因叶绿体。胰蛋白酶，胰凝乳蛋白酶和弗林蛋白酶的蛋白水解裂解位点存在于PA中的保护在转基因叶绿体，因为只有全长PA 83没有任何降解产物。CHAPS和SDS洗涤剂以相同的效率提取PA，并且在可溶性级分中观察到PA。叶绿体衍生的PA在与致死因子（LF）结合时在裂解小鼠巨噬细胞中具有功能活性。粗叶提取物含有高达25μg功能性PA/ml。使用在温室中生长的实验性转基因品种，每株植物的PA平均产量为172毫克，每英亩可以生产4亿剂疫苗（不含污染物），使用田间商业品种，产量可以进一步提高18倍。
The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the 83kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts. The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts. Maximum levels of expression were observed in mature leaves under continuous illumination. Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF). Crude leaf extracts contained up to 25μg functional PA/ml. With an average yield of 172mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field.