从伪狂犬病病毒中删除Cre/LoxP介导的报告基因

Cre/LoxP Mediated Report Gene Deletion from Pseudorabies Virus

根据pEGFP-C1序列,设计并合成一对引物,通过PCR扩增出两端各含一个同向LoxP位点的GFP表达盒,克隆于转移载体pSKLR获得pSKLR-G-LoxP。通过经典同源重组方法,得到表达GFP的TK基因缺失伪狂犬病毒重组毒株S03109。该重组病毒在转染了pOG231(表达Cre重组酶)的293T细胞上连续传代,筛选得到重组病毒株S0419。荧光显微镜观察、Western blot及PCR检测结果表明,S03109感染细胞后持续表达GFP,而S0419不表达GFP。PCR证实S0419为含单个LoxP位点的TK基因缺失病毒,并且在细胞培养上遗传稳定,测序结果(GenBank登录号AY822465)表明,在Cre重组酶的作用下,两个同向LoxP序列之间的GFP表达盒被正确除去。对BALB/c小鼠的半数致死量(LD50)及免疫保护实验结果表明,S03109和S0419对BALB/c小鼠的LD50值均大于3×105PFU,免疫BALB/c小鼠,攻毒后平均存活率分别为67.5%与70%。以上结果表明,利用Cre/LoxP位点特异性重组系统,成功去除了插入重组伪狂犬病病毒基因组中的GFP报告基因。

A GFP expression cassette flanked by LoxP at two sites with the same orientation was amplified by PCR and inserted into the EcoRI site of pSKLR. Based on homologous recombination, the GFP expression recombinant virus S03109 was constructed using PrV RongA as a parental strain. After serial passage of S03109 on HEK-293T cell which was transfected with Cre encoding plasmid pOG231, the recombinant virus S0419 was obtained after plaque purification. Fluorescence microscope, Western blot analysis and PCR were employed to detect the expression of GFP. The results showed that S03109 infected-cells could express GFP but S0419 could not. PCR amplification and sequencing of S0419 TK gene indicated that the GFP expression cassette between two LoxP sites were deleted and only one LoxP site was left in TK locus. The sequencing result was submitted to GenBank （AY822465）. The LDs0 of S03109 and S0419 for BALB/c mice both exceeded 3 ×10^5 PFU. Using S03109 and S0419 to immunize BALB/c mice, the mean survival rates were 67.5 % and 70 % respectively after wild virus challenge. As a conclusion, the report gene GFP expression cassette was removed successfully from PrV genome by using Cre/LoxP recombinant system.