耐辐射球菌PprI蛋白质异源高效表达及其抗放作用与机理的研究

Acute radiation injury(ARI) is a systemic disease caused by ionizing radiation from nuclear emergency such as nuclear reactor，tumour radiotherapy，spaceflight，nucear terror and A-bomb blast et al. The prevention and cure of ARI are secret and key research projects for the governments of various countries. The relevant reports are seldom found in literatures. Studies showed that Deinococcus radiodurans was extremely a radioresistant bacterium which might is due to its high-efficiency DNA repair system. PprI (inducer of pleiotropic proteins promoting DNA repair) is a general switch protein responsible extreme radioresistance of Deinococcus radiodurans. Recently，we have successfully constructed a novel recombinant eukaryotic expression plasmid i.e. pCMV-HA-pprI encoding pprI gene isolated from Deinococcus radiodurans R1. The PprI protain was successfully expressed by transfecting the pCMV-HA-pprI into different phyletic mammalian cells and confirmed by western blotting. The recombinant plasmid pCMV-HA-pprI was transfered into the mice muscle by in vivo electroporation，it showed significant effects on prevention and treatment of ARI, and had long biological half-life and few side effects. These finds have obtained the patent for invention in China. Based on the study above，we are going to make a further scientific researches on the method of high-efficiency expression and purification of PprI protein in eukaryotic cells，and its the radioprotective and therapeutic effects，mechanism，pharmacokinetics and biocompatibility etc on irradiated mammal and mammalian cells. There is no any reports on the scientific problem in the literatures at present. The objective of this study is to obtain a novel radioresistant proteinic drug that possesses Chinese intellectual property rights. The prokaryotic protein would fill in a gap in the fields of international radiation protection. This study should make our country can join the advanced ranks in the world in areas of the prevention and cure of radiation injury.

急性放射损伤是核反应堆、肿瘤放疗、航天航空、核恐怖袭击、中子弹、原子弹爆炸等核突发事件电离辐射引起的全身性疾病，其防治是近年各国政府高度机密重点投资的研究领域，文献报道极少。研究表明，耐辐射球菌对大剂量电离辐射具有极端抗性，这与该菌具有高效的DNA 损伤修复系统有关。其中，pprI基因是一种具有重要调控作用的开关基因。近年我们成功地构建了一种pprI原核基因的真核表达质粒，可在真核细胞和动物整体转染表达，对小鼠急性放射损伤具有显著的防治作用，已获国家发明专利。在此基础上，本项目拟深入研究PprI原核蛋白的真核细胞高效表达和纯化技术，研究其对细胞和哺乳动物放射损伤的防治作用、机理、药代药动学和生物相容性, 迄今未见国内外文献报道。本研究旨在使我国获得具有自主知识产权的创新型抗放蛋白新药，填补国际辐射防护领域尚无原核抗放蛋白的空白，使我国在放射损伤防治领域的研究跻身世界先进行列。

近年来，国内外各种辐射事故，例如核电站和肿瘤放疗事故不断发生，尤其是日益攀升的核恐怖、核袭击、核与辐射突发事件以及放射性“脏弹”、贫铀弹和中子弹的使用，其电离辐射可引起人体严重的急性放射损伤，死亡率极高。核安全受到世界各国的重视，而我国应对核突发事件的能力则极其薄弱。因此，本项目创造性地研制了两种新型有效的抗辐射损伤技术及其药物。. 1. 本项目发明构建了一种耐辐射球菌pprI基因重组真核载体，将该质粒通过活体电转染技术转入细胞，发现pprI基因可在哺乳细胞表达其原核蛋白，其对人离体细胞和哺乳动物致死性急性放射损伤具有非常显著的防护和救治作用。同时，初步阐明了pprI基因转染抗辐射作用的分子机理。pprI基因在国际上首次实现从原核生物用于真核生物的重大种系跨越。. 2. 本项目自行构建了一种与耐辐射球菌密码子不同的新的pprI基因序列，成功建立了一种可表达该基因的毕赤酵母重组工程菌株，研究了PprI蛋白高效表达和纯化的发酵工艺技术，在国际上首次获得大量纯化的PprI蛋白。研究发现，PprI蛋白注射对动物急性放射损伤具有非常显著的救治和防护作用，受照小鼠死亡率减少30%，并且无细胞和免疫毒性，具有良好生物相容性。同时，本项目深入研究了PprI蛋白抗辐射作用的多种分子机制。PprI蛋白的制备技术已申报美国专利并通过实质审查。该项成果填补了国际尚无原核蛋白辐射防护剂的空白，对急性放射损伤救治和防护具有重要应用价值。