表达Cre重组酶的真核细胞系的建立及在重组伪狂犬病病毒研究中的应用

Construction of eukaryotic cell lines expressing the site-specific recombinase Cre and application in recombination of Pseudorabies virus

根据Cre基因序列设计并合成1对引物,PCR扩增出Cre基因编码区,克隆于pIREShyg载体,得到重组质粒pIREShyg-Cre,转染HEK-293A细胞后经400μg.mL-1 Hygromycin B的筛选,将其中1个阳性克隆命名为293A-Cre。利用伪狂犬病病毒(Pseudorabies virus,PRV)S03109株(带有gfp报告基因表达盒及loxP位点)感染293A-Cre细胞,荧光显微镜观察、PCR及Western blot检测gfp基因的表达。结果表明,S03109感染293A-Cre二代后loxP位点间的gfp表达盒被删除,获得重组病毒S0419。将S0419感染已转染pBlulox的293A-Cre细胞,在RK13细胞上筛选得到表达LacZ的重组病毒S06293。S06293在293A-Cre细胞上传代2次,X-Gal原位染色表明LacZ的表达消失。测序结果证实,在Cre重组酶作用下,2个同向loxP序列之间的外源基因序列被正确除去。以上结果表明,本研究构建的稳定表达Cre重组酶的293A-Cre细胞系可以用于在包含有loxP位点的PRV基因组中外源基因的快速删除或整合。

A pair of primer was designed according to Cre recombinase gene sequence. Cre coding sequence were obtained by PCR and then cloned into bicistronic vector plREShyg. The resulting recombinant vector plREShyg-Cre was transfected into HEK-293A cells using Lipofectamine 2000. After selection with 400 μg · mL^-1 hygromicin B, one positive clone was denominated as 293A- Cre. 293A-Cre cells were infected by Pseudorabies virus （PRV） S03109 strain which containing a green fluorescent protein （GFP） expression cassette flanked by two loxP sites with the same orientation in tk locus. Fluorescence microscope, PCR and Western blot analysis were employed to detect the expression of gfp. The results showed that after two generation serial passages of PRV S03109 strain on 293A-Cre cells the gfp expression cassette was deleted from S03109 genome and the resulted virus was named S0419. After transfection of 293A-Cre with pBlulox and then infection with S0419, the recombinant virus S06293 which expresses LacZ gene was obtained by plaque purification on RK13 cells. After two generation serial passages of S06293 on 293A-Cre, sequences between two loxP sites were deleted completely. PCR amplification and sequencing of the second generation progeny virus tk gene confirmed that pBlulox sequences between two loxP sites were deleted and only one loxP site was left in PRV tk locus. In conclusion, 293A-Cre cell lines expressing the site-specific Cre recombinase was constructed, and it could be used to manipulate foreign gene deletion and integration from P. virus containing loxP site.