Guo Hao, Gaowa Wudong, Zhao Hongzhe, Liu Chunyu, Hou Lina, Wen Yongjun, Wang Fengxue
Key Laboratory of Clinical diagnosis and treatment of Animal Diseases, Department of Agriculture and villages, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China.
Key Laboratory of Clinical diagnosis and treatment of Animal Diseases, Department of Agriculture and villages, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China.
Res Vet Sci. 2023 May;158:84-95. doi: 10.1016/j.rvsc.2023.03.010. Epub 2023 Mar 16.
Porcine Reproductive and Respiratory Syndrome (PRRS) threats the swine industry seriously. The spread of live vaccine virus leads to the emergence of recombinant virus, which brings biosafety problems. The replication-deficient virus as a vaccine candidate would avoid this problem. In the present study, the recombinant lentiviral plasmid pLV-EF1α-EGFP-2A-ORF4 was co-transfected with lentivirus in HEK293FT cells. The transfection mixture was harvested and transduced into Marc-145 to screen a cell line stably expressing the PRRSV ORF4 with puromycin. The cell line Marc-145-GP4 was confirmed with PCR, RT-PCR, IFA, and Western blotting using a monoclonal antibody against Glycoprotein 4 (GP4) of PRRSV. To obtain a replication-deficient PRRSV, Western blotting the recombinant plasmid pNM09-ΔORF4 was constructed by Overlap PCR and DNA recombinant technology with the pNM09 as a backbone plasmid. The pNM09-ΔORF4 was transfected into Marc-145-GP4 with electroporation after transcription in vitro. The replication-deficient virus was rescued on Marc-145-GP4 cells with trans-complementation of ORF4 gene and verified by RT-PCR and IFA. The results indicated that a cell line Marc-145-GP4 stably expressed PRRSV ORF4 was obtained. The recombinant GP4 was successfully expressed and obtained a monoclonal antibody Anti-A-GP4-70, which can specifically react with the virus. Finally, the replication-deficient virus rNM09-ΔORF4 can be rescued with low titer and could only reproduce on the Marc-145-GP4 cells. Unfortunately, the rNM09-ΔORF4 showed too low virus replication titer to determine it. This study lays the foundation for the rapid detection of PRRS and the functional study of GP4 and provides experience for replication-deficient PRRSV.
猪繁殖与呼吸综合征（PRRS）严重威胁着养猪业。活疫苗病毒的传播导致重组病毒的出现，带来了生物安全问题。复制缺陷型病毒作为候选疫苗可避免这一问题。在本研究中，重组慢病毒质粒pLV-EF1α-EGFP-2A-ORF4与慢病毒在HEK293FT细胞中共转染。收获转染混合物并转导至Marc-145细胞中，用嘌呤霉素筛选出稳定表达PRRSV ORF4的细胞系。使用针对PRRSV糖蛋白4（GP4）的单克隆抗体，通过PCR、RT-PCR、免疫荧光分析（IFA）和蛋白质免疫印迹法（Western blotting）对Marc-145-GP4细胞系进行了鉴定。为获得复制缺陷型PRRSV，以pNM09为骨架质粒，通过重叠PCR和DNA重组技术构建重组质粒pNM09-ΔORF4。体外转录后，通过电穿孔将pNM09-ΔORF4转染至Marc-145-GP4细胞中。在Marc-145-GP4细胞上通过ORF4基因的反式互补拯救出复制缺陷型病毒，并通过RT-PCR和IFA进行验证。结果表明，获得了稳定表达PRRSV ORF4的Marc-145-GP4细胞系。重组GP4成功表达，并获得了单克隆抗体Anti-A-GP4-70，该抗体可与病毒特异性反应。最后，可低滴度拯救出复制缺陷型病毒rNM09-ΔORF4，且其仅能在Marc-145-GP4细胞上繁殖。遗憾的是，rNM09-ΔORF4的病毒复制滴度过低，无法对其进行测定。本研究为PRRS的快速检测以及GP4的功能研究奠定了基础，并为复制缺陷型PRRSV提供了经验。
