AAV衣壳亚基纯度分析中VP3变体峰的鉴定与机制研究  

Characterization and Mechanism of VP3 Variant Peak in AAV Capsid Subunit Purity Analysis

作　　者：陶翠平 夷佳 任浩然 周怡琳 孙浩行 TAO Cuiping;YI Jia;REN Haoran;ZHOU Yilin;SUN Haohang(Shanghai Belief-Delivery BioMed Co.,Ltd.,Shanghai 201100)

机构地区：[1]上海信致医药科技有限公司,上海201100

出　　处：《中国医药工业杂志》2024年第8期1148-1155,共8页Chinese Journal of Pharmaceuticals

摘　　要：该研究旨在探究采用十二烷基硫酸钠-毛细管凝胶电泳(CE-SDS)技术分析腺相关病毒(AAV)衣壳亚基纯度时,VP3亚基峰前端产生未知信号峰的原因。先采用PCR法对重组腺相关病毒(rAAV)衣壳蛋白VP3亚基碱基序列的第2位起始密码子进行位点突变,获得突变后的rAAV,再通过CE-SDS技术鉴定该位点突变与该未知信号峰的关联性,结合LC-MS对未知峰的相对分子质量进行检测和肽段分析。结果表明,VP3亚基碱基序列中如含有2个起始密码子(ATG),则可能从第2个起始密码子开始翻译,表达产物为亚基VP3变体(VP3'),即对应CE-SDS技术检测到的未知信号峰。在对AAV衣壳蛋白进行亚基纯度分析时应将其作为AAV衣壳亚基峰。This study aimed to investigate the cause of an unidentified peak that appeared ahead of the VP3 subunit peak during the purity analysis of adeno-associated virus(AAV)capsid subunits by capillary electrophoresis sodium dodecyl sulfate(CE-SDS)technique.Initially,a point mutation was introduced to the second start codon within the base sequence of the recombinant adeno-associated virus(rAAV)capsid protein’s VP3 subunit via PCR to generate a mutant rAAV.Subsequently,the relationship between this mutation and the unidentified signal peak was elucidated using CE-SDS technique.Additionally,the relative molecular weight and peptide analysis of the unknown peak was detected by LC-MS.The results indicated that if the VP3 subunit’s base sequence contain two initiation codons(ATG),the translation might start from the second initiation codon.And the expression product was a variant of the VP3 subunit(VP3'),which aligned with the unidentified signal peak observed in CE-SDS analysis.Consequently,this unidentified peak should be regarded as a AAV capsid subunit peak during the purity assessment of AAV capsid proteins.

关 键 词：十二烷基硫酸钠-毛细管凝胶电泳技术 重组腺相关病毒 纯度 衣壳 亚基VP3变体峰 

分 类 号：TQ937[化学工程] R917[医药卫生—药物分析学]