基于CuAAC点击化学反应的PEG定点修饰提高T7噬菌体抗免疫清除能力的机制研究

Recently, antimicrobial resistance has become increasingly serious. Phage therapy on antimicrobial resistant bacteria has its unique advantage and broad application prospect. However, the rapid clearance in vivo prevents the development of phage therapy. Promoting the ability of anti-immune response of phage is the trend in development of phage therapy. PEGlytion can prolong the circulating half-life of phages but reduce their infectivity. We propose a hypothesis that site-specific PEGlytion on head protein would increase anti-immune response of phage and not reduce its infectivity. We plan to introduce site-specific mutation into T7 to keep only one amber codon at the 3’end of the gp10 head protein gene of T7 via phage genetic engineering. The synthesized non-natural amino acid, p-propargyloxy phenylalanine(pPa), will be inserted into head protein p10A site-specifically via synthetic biology by using tyrosyl-tRNA synthetase and tRNA_CUA of Methanococcus jannaschii. T7 will be site-specifically PEGlyted on the head via CuAAC reaction between the pPa and methoxypolyethylene glycol azide (mPEG-N3). We will analyze the change of anti-immune response ability of PEGlyted T7. The study will elucidate the mechanism of site-specific PEGlytion of head protein on the phage's anti-immune response ability and unravel the prolonging of the phage circulating half-time. This study can not only provide a new idea to increase phage's anti-immune response ability, but also promote the development of phage therapy, contribute to treatment of antimicrobial resistant bacteria infection and prevent the spread of the antimicrobial resistant bacteria.

细菌耐药性愈演愈烈，噬菌体治疗耐药细菌感染，因其独特于抗生素作用机制的优势，应用前景广阔。但在动物体内的快速清除严重制约了噬菌体治疗的发展，如何增强噬菌体抗免疫清除能力是噬菌体治疗研究的发展趋势和研究难点。已有研究表明PEG修饰噬菌体能延长其循环半衰期，但会降低感染能力。项目组提出对噬菌体头部定点PEG修饰能提高噬菌体抗免疫清除能力而不影响其感染力的假说。本项目拟对T7噬菌体进行遗传改造，利用詹氏甲烷球菌酪氨酰tRNA合成酶及tRNA_CUA在其头部蛋白定点插入对丙炔氧基-L-苯丙氨酸（pPa）；通过pPa与甲氧基聚乙二醇叠氮发生CuAAC点击化学反应，实现T7噬菌体头部蛋白定点PEG修饰，研究头部定点PEG修饰与噬菌体抗免疫清除能力之间的关系，验证其是否能有效延长噬菌体循环半衰期。本研究不仅可为提高噬菌体抗免疫清除能力提供新思路，也可为推动噬菌体治疗发展，治疗耐药细菌感染作出积极贡献。

细菌耐药性愈演愈烈，噬菌体治疗耐药细菌感染，因其独特于抗生素作用机制的优势，应用前景广阔。但在动物体内的快速清除严重制约了噬菌体治疗的发展，如何增强噬菌体抗免疫清除能力是噬菌体治疗研究的发展趋势和研究难点。聚乙二醇（polyethylene glycol，PEG）修饰技术广泛应用于多种蛋白质和多肽的化学修饰。 PEG修饰具有半衰期延长、免疫原性降低或消失、毒副作用减少以及物理、化学和生物稳定性增强等。有研究表明PEG修饰噬菌体能延长其循环半衰期，但会降低感染能力。因此，本项目组提出对噬菌体头部定点PEG修饰可能能提高噬菌体抗免疫清除能力而不影响其感染力。项目组首先合成了非天然氨基酸N-丙炔氧羰基-L-赖氨酸，并构建了MbPylRS/tRNACUA高效表达体系，为后期在噬菌体头部插入N-丙炔氧羰基-L-赖氨酸奠定了基础；通过基因组合成的方法去除T7Select基因组内所有琥珀密码子，在其头部蛋白gp10B的赭石密码前插入琥珀密码子，使其基因组只在头部蛋白末端携带琥珀密码子，获得突变体amber-T7Select；利用MbPylRS/tRNACUA高效表达体系识别琥珀密码子，在amber-T7Select头部蛋白C端插入N-丙炔氧羰基-L-赖氨酸，得到alkynyl-T7Select；通过CuAAA点击化学反应，用FITC-PEG-N3对噬菌体头部的炔基进行定点修饰，得到FITC-PEG-T7select，其感染能力与amber-T7select相比，无显著变化；最后用FITC-PEG-T7select及amber-T7select对SPF小鼠进行尾静脉注射后，不同时间点于另一条尾静脉取血，测小鼠血浆中剩余噬菌体滴度，结果表明FITC-PEG-T7select与amber-T7select相比，尾静脉注射12-24 h后，小鼠血液中FITC-PEG-T7select存留量为T7select WT的10-15倍。本研究不仅可为提高噬菌体抗免疫清除能力提供新思路，也可为推动噬菌体治疗发展，治疗耐药细菌感染作出积极贡献。