基于转基因猪唾液腺生物反应器技术的药用人神经生长因子制备研究

Use of transgenic animals as drug manufactories for production of human medicine holds great promise in the future. It will make animal husbandary become the upstream industry for the biomedicine industry. It also will be the feature and highlight of future innovation in farm animal industry, and has significant meaning for sustainable development of both animal husbandary and biomedicine industry. Development of proper bioreactors is the key for transgenic animals-based pharming. Currently our country's transgenic animal bioreactor technology still has the following problems. The success rate of generating transgenic animals is low, and the foreign gene is usually randomly integrated into host animal’s genome which frequently causes detrimental effects on host animal or expression silence of transgene. The bioactivity of expressed foreign protein is low and it is difficult to purify expressed foreign proteins from transgenic animals. To overcome these problems, this study aims to generate transgenic pigs specifically expressing valuable therapeutic human nerve growth factor (hNGF) in the salivary glands by CRISPR/Cas9 in combination with cloning technique, and purify biologically active hNGF from the saliva of transgenic pigs. This study not only provides an efficient and low cost approach for production of functional hNGF protein, but also establishes a new drug production method based on using transgenic pig’s salivary glands as bioreactors. This will promote application of drug-producing transgenic pigs to the biopharmaceutical industry in the future.

利用转基因动物生产药物具有非常可观的应用前景。它将拓宽畜牧业的生产领域，使畜牧业成为现代生物医药的上游产业，是未来畜牧科技创新的特色和亮点，对畜牧业及生物医药业的可持续发展均具有重要意义。实现转基因动物制药的关键是建立合适的转基因动物生物反应器技术。但目前我国转基因动物生物反应器技术还存在转基因动物制备的成功率较低，转基因随机整合导致表达沉默和毒性整合，所表达的外源蛋白活性低及分离纯化困难等问题。针对上述问题，本研究创新性地利用高效的CRISPR/Cas9系统结合体细胞克隆技术，建立唾液腺特异表达具有重要药用价值的人神经生长因子的转基因猪模型，从其唾液中纯化制备具有功能的人神经生长因子。探索一种高效、低成本制备高活性人神经生长因子的新方法，并建立一种基于转基因猪唾液腺生物反应器的药用蛋白制备新技术，以期为未来产药型转基因猪培育及其在生物医药领域的应用提供重要理论依据、技术支撑与实践基础。

转基因动物唾液腺作为一种新型生物反应器，对于药物蛋白的制备具有极大的潜在应用价值。而人神经生长因子（Human nerve growth factor, hNGF）是能够有效治疗许多人类神经元和非神经元疾病的重要药物蛋白。本研究的目的是利用转基因猪唾液腺作生物反应器生产hNGF。本研究将携带绿色荧光蛋白（Enhanced green fluorescent protein, EGFP）筛选标记基因的hNGF表达载体转染猪胎儿成纤维细胞，筛选获得转hNGF基因单细胞克隆团，将其作为供体细胞通过体细胞克隆法制备出4头原代转hNGF基因猪；EGFP表达观察，PCR和Southern blot分析都证明该4头原代转基因猪基因组整合了外源的EGFP和hNGF基因；酶联免疫吸附测定检测结果显示转基因猪唾液中分泌的hNGF浓度约达600-1200ng/mL；用含高浓度hNGF的转基因猪唾液处理培养的PC12细胞证明其具有促进类神经元细胞分化的活性。综上，本研究成功建立了一种利用转基因猪唾液腺作为生物反应器制备人类药物蛋白的新技术，并为制备hNGF蛋白提供了一种高效的新方法。