快速纯化重组腺联病毒的受体结合捕捉方法

摘要【目的】建立用于重组腺联病毒(AAV)纯化的受体结合捕捉方法。【方法】将AAV受体的多囊肾病(PKD)结构域1和2与类弹性蛋白多肽(ELP)在重组大肠杆菌中进行融合表达,利用相变循环(ITC)纯化ELP-PKD融合蛋白;分别用昆虫和AAV-293细胞制备rAAV-GFP,与ELP-PKD融合蛋白共孵育后进行ITC,从沉淀复合物提取病毒DNA进行PCR检测;在优化条件下利用ELP-PKD蛋白结合捕捉rAAV-GFP,利用电子显微镜观察、免疫转印和细胞感染试验进行rAAV鉴定。【结果】ELP-PKD融合蛋白获得正确、可溶性表达,ITC纯化的蛋白纯度大于90%;ELP-PKD蛋白能特异结合rAAV-GFP,结合具有p H、温度和时间依赖性,受体结合捕捉方法可在1h内完成,从两种细胞纯化rAAV-GFP的回收率分别为58%和56%;rAAV-GFP洗脱具有p H和温度依赖性,洗脱rAAV-GFP的回收率分别为46%和44%;纯化rAAV-GFP具有AAV的典型形态和结构蛋白。【结论】建立的ELP-PKD结合捕捉法可用于不同细胞源rAAV的快速纯化。 [Objective]To establish a receptor-binding capture method for recombinant adeno-associated virus(rAAV)purification.[Methods]We expressed polycystic kidney disease(PKD)domains 1 and 2 of AAV receptor in E.coli as an elastin-like polypeptide(ELP)fusion protein.We purified the fusion protein by inverse transition cycling(ITC).We generated two versions of rAAV-GFP and incubated them with ELP-PKD protein.We recovered the protein-bound rAAV-GFP by ITC and extracted the viral DNA for PCR analysis.We optimized the conditions for rAAV-GFP purification and identified the purified rAAV by electron microscopy and Western blotting.[Results]ELP-PKD fusion protein was correctly expressed as a soluble protein which was purified to more than 90%purity.We demonstrated the specific affinity of ELP-PKD fusion protein for rAAV-GFP binding.We purified rAAV-GFP with 58%recovery from insect cells or 56%recovery from AAV-293 cells.After elution,we obtained final rAAV-GFP recovery rates of 46%and 44%from the two cell types,respectively.We demonstrated that the purified rAAV-GFP had the typical morphology and structural proteins of AAV.[Conclusion]We established ELP-PKD-binding capture method for quick purification of rAAV from different cell types.

作者陈裴裴刘晓明卢会鹏周晓慧张鑫宇夏晓莉孙怀昌 Peipei Chen;Xiaoming Liu;Huipeng Lu;Xiaohui Zhou;Xinyu Zhang;Xiaoli Xia;Huaichang Sun(Jiangsu Co-Innovation Center for Prevention and Control of important Animal Infectious Diseases and Zoonoses,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,Jiangsu Province,China;Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals,Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,Jiangsu Province,China)

机构地区扬州大学兽医学院江苏农牧科技职业技术学院

出处《微生物学报》 CAS CSCD 北大核心 2021年第3期621-630,共10页 Acta Microbiologica Sinica

基金 Supported by the National Key R&D Program of China(2018YFC0840404-3) by the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。

关键词腺联病毒受体 ELP融合表达受体结合捕捉重组AAV纯化 adeno-associated virus receptor ELP fusion expression receptor-binding capture recombinant AAV purification

分类号 R73 [医药卫生—肿瘤]