稳定表达IL-10的PK-15细胞的构建及其在PCV2增殖中的应用

doi:10.16431/j.cnki.1671-7236.2022.03.031

Abstract

Abstract: 【Objective】 This experiment was conducted to investigate the effects of interleukin-10 (IL-10) on the replication of Porcine circovirus type 2 (PCV2), screen high infectious PCV2 cell lines, and improve the virus titer of PCV2, and lay a foundation for the subsequent vaccine research and development and the study of the role of IL-10 in PCV2 infection.【Method】 Porcine IL-10 gene was amplified by PCR, the target gene was linked to lentivirus expression vector (pCDH-CMV-MCS-EF1-GFP+Puro), and the recombinant plasmid pCDH-CMV-IL-10 was obtained.It was co-transfected with package plasmids psPAX2 and pMD2.G into 293T cells for lentivirus packaging.PK-15 cells were infected with the collected lentivirus supernatant and screened by puromycin to obtain PK-15-IL-10 cell lines.The control cells were named PK-15-pCDH and PK-15, respectively.After PCV2 was infecting PK-15-IL-10, PK-15-pCDH and PK-15 cell lines, cell fluid was collected at 24, 48 and 72 h, respectively, and cell viability was detected by CCK-8.The expression of IL-10 gene and the replication of PCV2 were detected by Real-time quantitative PCR and Western blotting.Indirect immunofluorescence assay (IFA) was used to observe the replication of PCV2 in cells and determine the virus titer of PCV2 (TCID₅₀).【Result】 The recombinant plasmid pCDH-CMV-IL-10 was successfully constructed.293T cells were co-transfected with the packaged plasmids psPAX2 and pMD2.G, and the cell status was the best and the fluorescence was the strongest at 48 h.PK-15 cells were co-transfected with lentivirus supernatant for 48 and 72 h, respectively.The fluorescence of pCDH-CMV-IL-10 group was the strongest, and they were cultured in complete medium with puromycin concentration of 2.5 μg/mL to obtain stable cell lines with green fluorescence.Real-time quantitative PCR and Western blotting results showed that the expression of IL-10 gene in pCDH-IL-10 cell lines was significantly higher than that in control group PK-15-pCDH and PK-15.The copy number of PCV2 was increased by four times, and its replication capacity was enhanced.After the virus was diluted and passed for 3 generations, PCV2 in PK-15-IL-10 cells was extremely significantly higher than that in PK-15 cells (P<0.01).Cell proliferation assay showed that overexpression of porcine IL-10 gene had no significant effect on cell viability.IFA results showed that fluorescence in PK-15-IL-10 cells was stronger than that in PK-15 cells.TCID₅₀ of PCV2 in PK-15-IL-10 cells was extremely significantly higher than that of PK-15 cells at 48 h after infection (P<0.01).【Conclusion】 The lentiviral expression vector of pCDH-CMV-IL-10 was successfully constructed and used to infect PK-15 cells.After further culture, PK-15-IL-10 cell line expressing IL-10 was screened, and infection with PCV2 could promote the replication of PCV2 in PK-15 cells.These results provided reference for later vaccine research, and laid a foundation for further study on the effect of IL-10 on the replication of PCV2 in PK-15 cells.

Key words: Porcine cyclovirus type 2(PCV2); interleukin-10(IL-10); immunosuppression; lentiviral expression vector; stable-expressing cell lines

CLC Number:

S852.65⁺9.2

ZHANG Jinghan, LI Zhilan, HAN Diangang, HE Shuai, LIU Jinhua, LYU Nianci, ZOU Fengcai, CHAI Jun, ZHANG Yifang. Construction of PK-15 Cells Stably Expressing IL-10 and Its Application in PCV2 Proliferation[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 1085-1095.

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Construction of PK-15 Cells Stably Expressing IL-10 and Its Application in PCV2 Proliferation

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