应用CRISPR/Cas9构建稳定表达CPV结构蛋白VP2的HEK293T细胞系

Establishment of HEK293T cell lines stably expressing canine parvovirus structural protein VP2 by CRISPR/Cas9

为了构建犬细小病毒(canine parvovirus,CPV)结构蛋白VP2稳定表达的HEK293T细胞系,以2017年新分离的CPV-WH株为材料,扩增其VP2基因,并进行进化树分析。根据安全插入位点AAVS1位点序列设计sgRNA,并构建Cas9及sgRNA的表达载体PX335-U6-sgRNA-Cas9,同时构建含有VP2的特异性同源序列的片段HM-puro-eGFP-VP2-HA,将两者共转染HEK293T细胞后,通过嘌呤霉素筛选稳定表达VP2的HEK293T细胞株,并通过测序确定VP2正确插入。进化树分析显示CPV-WH的VP2存在S557N、T570K 2个氨基酸突变,该氨基酸位点可能与病毒免疫逃逸有关。通过荧光观察和免疫印迹确定了稳定表达细胞系中VP2的表达。本研究利用CRISPR/Cas9技术成功构建了表达CPV结构蛋白VP2的HEK293T细胞,为后期制备CPV样颗粒提供细胞模型。

In order to construct HEK293 T stable cell lines expressing canine parvovirus structural protein VP2,the VP2 gene of canine parvovirus(CPV-WH strain) newly isolated in 2017 was amplified and analyzed by evolutionary tree.SgRNA was designed according to the sequence of AAVS1 locus,and expression vectors PX335-U6-sgRNA-Cas9 were constructed.Meanwhile,the specific Homologous Fragment HM-puro-eGFP-VP2-HA containing VP2 was constructed.After co-transfection of the above two plasmids into HEK293 T cells,HEK293 T stable cell lines expressing VP2 were screened by purinomycin,and the correct insertion of VP2 was determined by sequencing.Evolution tree analysis showed that there were two amino acid mutations(S557 N and T570 K) in VP2,which might be related to virus immune escape.The stable expression of VP2 in the cell lines was determined by observing the fluorescent label protein and immunoblotting.In this study,HEK293 T stable cell lines expressing canine parvovirus structural protein VP2 was successfully constructed by CRISPR/Cas9,which provides a cell model for the preparation of canine parvovirus-like particles.