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将完整的pAPN序列通过同源重组克隆入慢病毒表达载体pLVX-mCherry,并将该重组质粒pLVX-mCherry-pAPN与慢病毒包装质粒pLP1、pLP2、pLP-VSVG共转染293T细胞,在293T细胞中包装成含目的基因的重组慢病毒。将收获的重组病毒感染Vero细胞,经过嘌呤霉素筛选和细胞有限稀释法,筛选出表达pAPN的细胞。通过RT-PCR和间接免疫荧光试验(IFA)证明了所获细胞株能够稳定表达pAPN蛋白,命名为Vero-APN-B6。IFA试验和TCID50试验结果表明:与Vero细胞相比,Vero-APN-B6更能促进猪流行性腹泻病毒(PEDV)的增殖。利用该细胞株成功地从猪小肠组织中分离到了PEDV SC-MS株;经过鉴定该毒株为变异毒株。
The complete pAPN sequence was cloned into the lentiviral expression vector pLVX-mCherry by homologous recombination. The recombinant plasmid pLVX-mCherry-pAPN and the lentiviral packaging plasmid pLP1, pLP2 and pLP-VSVG were cotransfected into 293T cells. 293T cells Packaged in a recombinant lentivirus containing the gene of interest. The harvested recombinant virus was infected into Vero cells, and the pAPN-expressing cells were screened by puromycin selection and limiting cell dilution. The obtained cell lines stably expressed pAPN protein by RT-PCR and indirect immunofluorescence assay (IFA), and named Vero-APN-B6. IFA test and TCID50 test results showed that Vero-APN-B6 can promote the proliferation of porcine epidemic diarrhea virus (PEDV) more than Vero cells. PEDV SC-MS strain was successfully isolated from pig intestine by using this cell line. The strain was identified as a mutant strain.