基于light-oxygen-voltage(LOV)结构域光敏剂的细胞毒性研究  

Cytotoxicity Studies of Light-oxygen-voltage(LOV)Domain Photosensitizers

作　　者：许爽 万奔 沙娜 赵开弘[1] XU Shuang;WAN Ben;SHA Na;ZHAO Kai-Hong(National Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070)

机构地区：[1]华中农业大学农业微生物资源发掘与利用实验室,武汉430070

出　　处：《生物化学与生物物理进展》2025年第2期487-500,共14页Progress In Biochemistry and Biophysics

摘　　要：目的光敏剂被特定波长的光激发后,产生的活性氧类能破坏细胞组织,介导细胞死亡,对微生物感染、肿瘤等相关疾病的治疗具有重要意义。方法基于粳稻类向光素1B(Oryza sativa japonica phototropin-1B-like)的LOV(lightoxygen-voltage)结构域,设计得到光敏剂LovPSO2及其突变体LovPRO2。在445 nm、70μmol·m^(-2)·s^(-1)蓝光照射下,每隔2 min测量LovPSO2和LovPRO2的单线态氧产量,持续10 min,每隔1 min测量其超氧阴离子产量,持续5 min,并研究温度、光照对其稳定性的影响,最后将其转入E.coli BL21(DE3)和HeLa细胞中表达并分析光毒性效果。结果在445 nm、70μmol·m^(-2)·s^(-1)蓝光照射下,LovPSO2是一种能产生大量单线态氧的Ⅱ型光敏剂(ΦΔ=0.61),LovPRO2是一种能够同时产生单线态氧和超氧阴离子的光敏剂。蛋白质稳定性分析结果表明,LovPSO2和LovPRO2具有较好的温度稳定性,其中LovPRO2的光稳定性更好。蛋白质的光毒性分析结果表明,445 nm、30 mW/cm^(2)蓝光照射30 min后,LovPSO2和LovPRO2对E.coli BL21(DE3)菌株有较好的光毒性,致死率高达90%。结论LovPSO2和LovPRO2可作为抗菌光敏剂,在食品和医疗等方面均有较为广阔的应用前景。Objective At present,the most commonly used photosensitizers in photodynamic therapy are still chemical photosensitizers,such as porphyrin and methylene blue,in order to specifically target cellular tissues,and thus poison cells,chemical photosensitizers need to use antibody conjugation or a transgenically encoded tag with affinity for the modified photosensitizing ligand,e.g.FlAsH,ReAsh or Halo Tag.Gene-encoded photosensitizers can directly poison cells by targeting specific cell compartments or organelles.However,currently developed gene-encoded photosensitizers have low reactive oxygen species production and low cytotoxicity,so it is necessary to continue to develop and obtain photosensitizers with higher reactive oxygen species production for the treatment of microbial infections and tumors.Methods In this study,we developed a photosensitizer LovPSO2 based on the light-oxygen-voltage(LOV)structural domain of phototropin-1B-like from Oryza sativa japonica.LovPSO2 was expressed in E.coli BL21(DE3)and purified to obtain protein samples,the purified protein samples were added 3μmol/L singlet oxygen probe of SOSG and 5μmol/L superoxide anion probe of DHE after fixed to A445=0.063±0.003,respectively,then measured every 2 min of singlet oxygen production for 10 min and every 1 min of superoxide anion production for 5 min under blue light irradiation at 445 nm,70μmol·m^(-2)·s^(-1).Results The results showed that LovPSO2 could produce a large amount of singlet oxygen under blue light irradiation at 445 nm,70μmol·m^(-2)·s^(-1),and its singlet oxygen quantum yield was 0.61,but its superoxide anion yield was low,so in order to improve the superoxide anion yield of LovPSO2,a mutant with a relatively high superoxide anion yield was obtained by further development and design on its basis LovPRO2.The stability of proteins is crucial for research in drug development and drug delivery,among others.Temperature and light are the key factors affecting the production of reactive oxygen species(ROS)by photosensitive protei

关 键 词：光敏剂 活性氧类 单线态氧 超氧阴离子 

分 类 号：Q63[生物学—生物物理学]