非天然氨基酸pBpa在IL1β抗原表位的嵌入及其表征  

Insertion and characterization of unnatural amino acid p Bpa in epitope of IL1β

作　　者：李闰婷[1] 马韩轲 张丽萌[1,2] 聂晓宁 代冰雪 王林青 陈龙欣[1,2] LI Runting;MA Hanke;ZHANG Limeng;NIE Xiaoning;DAI Bingxue;WANG Linqing;CHEN Longxin(Molecular Biology Laboratory,Zhengzhou Normal University,Zhengzhou 450044,China;College of Life Sciences,Zhengzhou Normal University,Zhengzhou 450044,China)

机构地区：[1]郑州师范学院分子生物学实验室,郑州450044 [2]郑州师范学院生命科学学院,郑州450044

出　　处：《天津师范大学学报（自然科学版）》2022年第6期23-28,共6页Journal of Tianjin Normal University：Natural Science Edition

基　　金：国家级大学生创新创业训练计划资助项目(202012949006);国家自然科学基金资助项目(32071447);河南省自然科学基金资助项目(202300410509);河南省高等学校青年骨干教师培养计划(2018GGJS161);郑州师范学院大学生创新创业训练计划资助项目(DCY2020005)。

摘　　要：为了探究表位中嵌入光敏型非天然氨基酸的蛋白质与其特异性抗体的光交联反应及其产物,基于基因密码子扩展技术,原核表达嵌入携带功能基团的非天然氨基酸蛋白质IL1β-L67pBpa,通过Western blot方法验证该突变体蛋白及其抗体的光交联功能.结果表明,本研究成功构建了pET28a-IL1β-L67TAG突变体质粒用于原核表达,在蛋白质的指定位点嵌入非天然氨基酸pBpa,并得到了高效表达的IL1β-L67pBpa.365 nm紫外光照射后,该突变体中的非天然氨基酸pBpa光敏基团与抗体中的氨基酸在空间距离靠近时,pBpa嵌合体能与其表位抗体(康纳单克隆抗体)通过共价键的光交联形成新的复合物.本研究证实了将非天然氨基酸(活性基团)插入到蛋白质特定位点的可行性.In order to explore the photocrosslinking and product of the protein inserted with photosensitive unnatural amino acids(UAAs)cross-linking with its specific antibodies under long wavelength ultraviolet irradiation,the plasmid expressing IL1β-L67pBpa mutant with functional group was expressed by gene codon extension technology methods,and the photo-crosslinking function of the mutant protein against its antibody was verified by Western blot.The results show that the pET28a-IL1β-L67TAG mutant plasmid was successfully constructed to express the protein IL1β-L67pBpa with functional group.The UAAs could be inserted into the target site of the protein through prokaryotic expression,and the highly expressed IL1β-L67pBpa was obtained.When the space distance between the pBpa of the mutant and the amino acid in the antibody was close to each other,the chimeric pBpa could form a complex with its epitope antibody(canakinumab)through covalent bonds after irradiation with 365 nm ultraviolet light.The study verified that UAAs(active groups)can be inserted into the target sites of proteins.

关 键 词：非天然氨基酸 基因密码子扩展技术 抗体 蛋白质相互作用 光交联 

分 类 号：Q786[生物学—分子生物学] Q51