摘要
目的:解整合素样金属蛋白酶10(disintegrin and metalloproteinase domain-containing protein 10,ADAM 10)是一种重要的膜锚定金属蛋白酶,参与多个重要生理过程,同某些癌症和神经退行性疾病有重要联系。拟通过毕赤酵母表达系统表达ADAM 10的金属蛋白酶结构域,探究ADAM 10对β-淀粉样蛋白(β-amyloid,Aβ42)的作用。方法:首先通过数据库检索的方式获得所需序列,将密码子进行酵母偏好性优化后,插入至pPIC9k分泌表达载体上。通过电转化、阳性筛选、多拷贝子筛选等过程后,用蛋白印迹(Western Blot)的检测方式筛选出能够表达重组蛋白并将其有效分泌至培养基中的阳性菌株。分别用Tricine-SDSPAGE电泳结合Western Blot和高效液相色谱-质谱联用(LC-MS/MS)的方法检验截短型ADAM 10(s ADAM10)对Aβ42的水解作用,并用硫黄素T(thioflavin T,ThT)荧光实验检验s ADAM 10对Aβ42聚集的影响。结果:通过毕赤酵母表达系统成功分泌表达了s ADAM 10,其分子量在35 kD左右,为已成功切除了前导肽的并在金属蛋白酶结构域中的糖基化位点上进行糖基化的成熟蛋白酶;重组蛋白能水解Aβ42,在一定程度上抑制Aβ42聚集,其被检测到的水解位点位于Aβ42多肽的中段。结论:s ADAM 10具有作为阿尔茨海默病(Alzheimer’s disease,AD)治疗药物的可能性。
Objective: To express the metalloproteinase domain of disintegrin and metalloproteinase domaincontaining protein 10( ADAM 10),a membrane-anchored metalloproteinase that takes important parts in many physiological processes and is related with some kinds of cancer and neurodegenerative diseases,by the Pichia expression system thus to explore the effect of ADAM 10 on β-amyloid( Aβ42). Methods: The sequence was first obtained by database search. After optimizing the codon for yeast preference,it was inserted into the p PIC9 k secreted expression vector. After electrotransformation,positive screening,multi-copy screening and other processes,some positive strains capable of expressing the recombinant protein and secreting it into the culture were found out by Western blot. The hydrolysis of Aβ42 by truncated ADAM 10( s ADAM 10) was examined by Tricine-SDS-PAGE electrophoresis combined with Western blot and by LC-MS/MS. The effect of s ADAM 10 on Aβ42’s aggregation was examined by Th T( Thioflavin T) fluorescence assay. Results: s ADAM 10 was expressed by the Pichia expression system and secreted into the culture successfully. According to the results,s ADAM 10 had a molecular weight of about 35 k D and should be a mature protease whose prodomain had been successfully removed with the successful glycosylation in the metalloproteinase domain;s ADAM 10 could hydrolyze Aβ42,which led to the inhibition on Aβ42’s aggregation in a certain degree;and the detected hydrolysis sites were located in the middle of the Aβ42.Conclusion: s ADAM 10 is a potential therapy for Alzheimer’s Disease( AD).
作者
王紫瑈
孙鑫
符馨月
李琪琪
张芳
陈豪
李前
赵宝全
孙曼霁
WANG Zi-rou;SUN Xin;FU Xin-yue;LI Qi-qi;ZHANG Fang;CHEN Hao;LI Qian;ZHAO Bao-quan;SUN Man-ji(Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)
出处
《中国新药杂志》
CAS
CSCD
北大核心
2020年第10期1103-1109,共7页
Chinese Journal of New Drugs
基金
国家“重大新药创制”科技重大专项资助项目(2012ZX09102301020)。
