首页|AAV衣壳亚基纯度分析中VP3变体峰的鉴定与机制研究

AAV衣壳亚基纯度分析中VP3变体峰的鉴定与机制研究

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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.
Characterization and Mechanism of VP3 Variant Peak in AAV Capsid Subunit Purity Analysis
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 prod-uct 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.

capillary electrophoresis sodium dodecyl sulfate techniquerecombinant adeno-associated viruspuritycapsidsubunit VP3 variant peak

陶翠平、夷佳、任浩然、周怡琳、孙浩行

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上海信致医药科技有限公司,上海 201100

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

2024

中国医药工业杂志
上海医药工业研究院,中国化学制药工业协会

中国医药工业杂志

CSTPCD
影响因子:0.487
ISSN:1001-8255
年,卷(期):2024.55(8)