Genetically Engineered Pigs as Efficient Salivary Gland Bioreactors for Production of Therapeutically Valuable Human Nerve Growth Factor.
Genetically Engineered Pigs as Efficient Salivary Gland Bioreactors for Production of Therapeutically Valuable Human Nerve Growth Factor.
复制标题
基因工程猪作为高效唾液腺生物反应器生产具有治疗价值的人神经生长因子。
作者：
家畜唾液腺作为高效的生物反应器用于人类治疗性蛋白的生产具有巨大的潜力。神经生长因子（NGF）在动物唾液腺中天然表达，并已被批准用于人类临床治疗。本研究旨在利用转基因猪唾液腺作为生物反应器，高效合成人神经生长因子（hNGF）。通过克隆结合piggyBac转座子介导的基因转移产生hNGF-TG猪。这些hNGF-TG猪在它们的唾液腺中特异性表达hNGF蛋白，并将其以高水平分泌到唾液中。开发了手术和非手术方法以有效地从hNGF-TG猪收集唾液。成功地从收集的唾液中纯化了hNGF蛋白，并证实其具有生物活性。在另一个步骤中，通过与CRISPR/Cas9介导的同源重组组合的克隆来创建双转基因猪，其中内源性猪NGF（pNGF）基因被另一拷贝的hNGF转基因替换。这些双转基因猪表达hNGF而不表达pNGF，从而避免了纯化过程中hNGF与pNGF蛋白可能的“污染”。总之，TG猪唾液腺可以用作大规模合成功能性hNGF或其他有价值的蛋白质的稳健生物反应器。这种新的动物转基因方法将有利于人类健康和生物医学。
Farm animal salivary glands hold great potential as efficient bioreactors for production of human therapeutic proteins. Nerve growth factor (NGF) is naturally expressed in animal salivary glands and has been approved for human clinical treatment. This study aims to employ transgenic (TG) pig salivary gland as bioreactors for efficient synthesis of human NGF (hNGF). hNGF-TG pigs were generated by cloning in combination with piggyBac transposon-mediated gene transfer. These hNGF-TG pigs specifically expressed hNGF protein in their salivary glands and secreted it at high levels into saliva. Surgical and nonsurgical approaches were developed to efficiently collect saliva from hNGF-TG pigs. hNGF protein was successfully purified from collected saliva and was verified to be biologically active. In an additional step, the double-transgenic pigs, where the endogenous porcine NGF (pNGF) gene was replaced by another copy of hNGF transgene, were created by cloning combined with CRISPR/Cas9-mediated homologous recombination. These double-transgenic pigs expressed hNGF but not pNGF, thus avoiding possible “contamination” of hNGF with pNGF protein during purification. In conclusion, TG pig salivary glands can be used as robust bioreactors for a large-scale synthesis of functional hNGF or other valuable proteins. This new animal pharming method will benefit both human health and biomedicine.
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影响因子：
2.4
作者：
Amano O;Mizobe K;Bando Y;Sakiyama K
通讯作者：
Sakiyama K
影响因子：
1.8
作者：
Kurome, Mayuko;Ishikawa, Takayuki;Nagashima, Hiroshi
通讯作者：
Nagashima, Hiroshi
影响因子：
3.5
作者：
Coulibaly, S;Besenfelder, U;Brem, G
通讯作者：
Brem, G
DOI：
10.1073/pnas.74.6.2330
发表时间：
1977-01-01
影响因子：
11.1
作者：
MURPHY, RA;SAIDE, JD;YOUNG, M
通讯作者：
YOUNG, M
影响因子：
3.6
作者：
LAKSHMANAN, J;LANDEL, CP
通讯作者：
LANDEL, CP