快速纯化重组腺联病毒的受体结合捕捉方法

【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。

Quick purification of recombinant adeno-associated viruses with the receptor-binding capture

[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.