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

摘要/Abstract

摘要： 【目的】研究白细胞介素-10（IL-10）对猪圆环病毒2型（Porcine circovirus type 2，PCV2）复制的影响，筛选PCV2高感染性细胞系和提高PCV2病毒滴度，为后续疫苗的研发及IL-10在PCV2感染中的作用研究提供参考。【方法】利用PCR技术扩增猪IL-10基因，将目的基因与慢病毒表达载体（pCDH-CMV-MCS-EF1-GFP+Puro）进行连接，获得重组质粒pCDH-CMV-IL-10，将其与包装质粒psPAX2和pMD2.G共转染293T细胞进行慢病毒包装。用收集的慢病毒液感染PK-15细胞，经嘌呤霉素筛选后得到细胞株PK-15-IL-10，对照组细胞分别命名为PK-15-pCDH和PK-15。PCV2感染PK-15-IL-10、PK-15-pCDH和PK-15细胞株后，在24、48和72 h分别收集细胞液，利用CCK-8检测细胞活力。利用实时荧光定量PCR和Western blotting检测IL-10基因的表达水平和PCV2的复制情况；利用间接免疫荧光试验（IFA）观察PCV2在细胞中的复制情况及测定PCV2的病毒滴度（TCID₅₀）。【结果】试验成功构建了重组质粒pCDH-CMV-IL-10，将其与包装质粒psPAX2和pMD2.G共转染293T细胞后，48 h时细胞状态最好，荧光最强。分别收集共转染48和72 h的慢病毒液上清感染PK-15细胞，pCDH-CMV-IL-10组的荧光最强，将其在嘌呤霉素浓度为2.5 μg/mL的完全培养基中继续培养，获得仍有绿色荧光的稳转细胞株。实时荧光定量PCR和Western blotting检测发现，IL-10基因在pCDH-IL-10细胞株中的表达量明显高于对照组PK-15-pCDH和PK-15，PCV2的拷贝数增加了4倍，复制能力增强，且将病毒稀释连续传3代后，PK-15-IL-10细胞中的PCV2极显著高于PK-15细胞（P<0.01）。细胞增殖试验表明，猪IL-10基因在细胞中过表达对细胞活力无明显影响；IFA结果表明，PK-15-IL-10细胞中的荧光比PK-15细胞更强，PCV2在PK-15-IL-10细胞中的TCID₅₀在感染后48 h极显著高于PK-15细胞（P<0.01）。【结论】本研究成功构建了pCDH-CMV-IL-10的慢病毒表达载体，并利用其感染PK-15细胞，继续培养后筛选出过表达IL-10的PK-15-IL-10细胞株，用PCV2感染该细胞株能促进PCV2在PK-15细胞中的复制。本试验结果为后期疫苗研究提供了参考，为进一步研究IL-10对PCV2在PK-15细胞中复制的影响奠定了基础。

关键词: 猪圆环病毒2型(PCV2); 白细胞介素-10(IL-10); 免疫抑制; 慢病毒表达载体; 稳定表达细胞系

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

中图分类号:

S852.65⁺9.2

张敬寒, 李枝兰, 韩佃刚, 何帅, 刘金华, 吕念词, 邹丰才, 柴俊, 张以芳. 稳定表达IL-10的PK-15细胞的构建及其在PCV2增殖中的应用[J]. 中国畜牧兽医, 2022, 49(3): 1085-1095.

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.

参考文献

[1] MUTTHI P, THEERAWATANASIRIKUL S, ROYTRAKUL S, et al.Interferon gamma induces cellular protein alteration and increases replication of Porcine circovirus type 2 in PK-15 cells[J].Archives of Virology, 2018, 163(11):2947-2957.
[2] SEGALES J.Porcine circovirus type 2 (PCV2) infections:Clinical signs, pathology and laboratory diagnosis[J].Virus Research, 2012, 164(1-2):10-19.
[3] XU T, ZHANG Y H, TIAN R B, et al.Prevalence and genetic analysis of Porcine circovirus type 2 (PCV2) and type 3 (PCV3) between 2018 and 2020 in central China[J].Infection Genetics and Evolution, 2021, 94:105016.
[4] CAO J, LIN C, WANG H, et al.Circovirus transport proceeds via direct interaction of the cytoplasmic dynein IC1 subunit with the viral capsid protein[J].Journal of Virology, 2015, 89(5):2777-2791.
[5] KARUPPANNAN A K, OPRIESSNIG T.Porcine circovirus type 2 (PCV2) vaccines in the context of current molecular epidemiology[J].Viruses, 2017, 9(5):99.
[6] WU X, WANG X, SHI T, et al.Porcine circovirus type 2 Rep enhances IL-10 production in macrophages via activation of p38-MAPK pathway[J].Viruses, 2019, 11(12):1141.
[7] HEDRICH C M, BREAM J H.Cell type-specific regulation of IL-10 expression in inflammation and disease[J].Immunologic Research, 2010, 47(1-3):185-206.
[8] FERREIRA C M, BARBOSA A M, BARREIRA-SILVA P, et al.Early IL-10 promotes vasculature-associated CD4⁺T cells unable to control Mycobacterium tuberculosis infection[J].JCI Insight, 2021, 23:e150060.
[9] PATTISON M J, MACKENZIE K F, ARTHUR J S.Inhibition of JAKs in macrophages increases lipopolysaccharide-induced cytokine production by blocking IL-10-mediated feedback[J].The Journal of Immunology, 2012, 189(6):2784-2792.
[10] 杜谦.猪圆环病毒2型对猪肺泡巨噬细胞IL-10/IL-12 p40表达的影响及调控机制[D].杨凌:西北农林科技大学, 2016. DU Q.Effect of Porcine cyclovirus type 2 on IL-10/IL-12 p40 expression and regulatory mechanism of pig alveolar macrophages[D].Yangling:Northwest A&F University, 2016.(in Chinese)
[11] DOSTER A R, SUBRAMANIAM S, YHEE J Y, et al.Distribution and characterization of IL-10-secreting cells in lymphoid tissues of PCV2-infected pigs[J].Journal of Veterinary Science, 2010, 11(3):177-183.
[12] DU Q, ZHANG H, HE M, et al.Interleukin-10 promotes Porcine circovirus type 2 persistent infection in mice and aggravates the tissue lesions by suppression of T cell infiltration[J].Frontiers in Microbiology, 2019, 10:2050.
[13] HAN J, ZHANG S, ZHANG Y, et al.Porcine circovirus type 2 increases interleukin-1beta and interleukin-10 production via the MyD88-NF-kappa B signaling pathway in porcine alveolar macrophages in vitro[J].Journal of Veterinary Science, 2017, 18(2):183-191.
[14] ZHANG X, ZHAO Y, MA C, et al.PK15 cell line stably overexpressing IL2 enhances PCV2 replication[J].Virus Genes, 2021, 57(1):111-116.
[15] QIAN G, LIU D, HU J, et al.Ochratoxin A-induced autophagy in vitro and in vivo promotes Porcine circovirus type 2 replication[J].Cell Death & Disease, 2017, 8(6):e2909.
[16] YANG X, CHEN F, CAO Y, et al.Comparative analysis of different methods to enhance Porcine circovirus 2 replication[J].Journal of Virological Methods, 2013, 187(2):368-371.
[17] VENEGAS-VARGAS C, TAYLOR L P, FOSS D L, et al.Cellular and humoral immunity following vaccination with two different PCV2 vaccines (containing PCV2a or PCV2a/PCV2b) and challenge with virulent PCV2d[J].Vaccine, 2021, 39(39):5615-5625.
[18] SACHDEVA G, D'COSTA J, CHO J E, et al.Chimeric HIV-1 and HIV-2 lentiviral vectors with added safety insurance[J].Journal of Medical Virology, 2007, 79(2):118-126.
[19] MA T, QUYANG T, QUYANG H, et al.Porcine circovirus 2 proliferation can be enhanced by stably expressing porcine IL-2 gene in PK-15 cell[J].Virus Research, 2017, 227:143-149.
[20] MENG X J.Porcine circovirus type 2 (PCV2):Pathogenesis and interaction with the immune system[J].Annual Review of Animal Biosciences, 2013, 1:43-64.
[21] YANG K, DONG L, DUAN Z, et al.Expression profile of long non-coding RNAs in porcine lymphnode response to Porcine circovirus type 2 infection[J].Microbial Pathogenesis, 2021, 158:105118.
[22] DARWICH L, RESENDES A, BALASCH M, et al.Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with Porcine circovirus type 2 (PCV2)[J].Research in Veterinary Science, 2008, 84(2):194-198.
[23] FOSSUM C, FUXLER L, KRUSE R, et al.Cytokine induction by immunostimulatory DNA in porcine PBMC is impaired by a hairpin forming sequence motif from the genome of Porcine circovirus type 2 (PCV2)[J].Veterinary Immunology and Immunopathology, 2011, 139(2-4):156-166.
[24] WANG L X, TALEBIAN F, LIU J Q, et al.IL-10 contributes to the suppressive function of tumour-associated myeloid cells and enhances myeloid cell accumulation in tumours[J].Scandinavian Journal of Immunology, 2012, 75(3):273-281.
[25] ROJAS J M, AVIA M, SEVILLA N.IL-10:A multifunctional cytokine in viral infections[J].Journal of Immunology Research, 2017, 2017:6104054.