羊口疮病毒ORFV118蛋白对山羊睾丸支持细胞周期、凋亡及免疫细胞因子的影响

doi:10.7668/hbnxb.20192480

摘要/Abstract

摘要：

旨在探究羊口疮病毒ORFV118蛋白对山羊睾丸支持细胞周期、凋亡以及诱导机体免疫应答相关的4种细胞因子(IL-1β、IL-6、IFN-γ和TNF-α)表达水平的影响。在ORFV118基因克隆基础上,成功构建真核表达载体pEGFP-ORFV118;将pEGFP-ORFV118转染山羊睾丸支持细胞,Western Blot 鉴定ORFV118蛋白表达的正确性;流式细胞仪检测ORFV118基因对细胞周期及凋亡的影响;RT-qPCR检测细胞周期相关基因CDK2和P21、凋亡相关基因Bax、Bcl-2、Caspase3、Caspase7、P53和BCL2L11的mRNA的表达水平,并利用RT-qPCR和ELISA方法检测免疫相关的细胞因子表达的变化。结果显示,成功扩增ORFV118基因,全长为309 bp。ORFV118蛋白的表达阻滞了细胞的DNA合成期(S期),促进了DNA合成后期(G2/M期)且下调基因CDK2的mRNA表达水平;可抑制细胞的凋亡作用,且下调促凋亡基因Caspase3、Caspase7、SOCS2的mRNA表达水平;对细胞因子IL-1β和IFN-γ呈不同程度的抑制作用,而对IL-6和TNF-α呈促进作用。

关键词: 山羊, 羊口疮病毒, ORFV118蛋白, 细胞周期, 细胞凋亡, 细胞因子

Abstract:

This aim of study was to explore the role of ORFV118 in regulating cell cycle,apoptosis and the immune-related cytokines(IL-1β,IL-6,IFN-γ and TNF-α)in goat testicular sertoli cells.We cloned the whole sequence of ORFV118 and constructed an expression vector of pEGFP-ORFV118.The expression of ORFV118 protein was then identified by using Western Blot method after the transfection of goat testicular sertoli cells with pEGFP-ORFV118.Following which,a flow cytometer equipment was used to measure the effect of ORFV118 expression on cell cycle and apoptosis of goat testicular sertoli cells,and also on regulating the expression of genes related to cell cycle,including CDK2 and P21,and genes relative to apottosis,including Bax,Bcl-2,Caspase3,Caspase7,P53 and BCL2L11.The expression of immune-related cytokines,including IL-1β,IL-6,IFN-γ and TNF-α,was detected by ELISA method and RT-qPCR after ORFV118 expression in goat testicular sertoli cells.The results showed that a total length of 309 bp ORFV118 gene sequence was cloned successfully.The cell's DNA synthesis phase(S phase)was inhibited and the late stage DNA synthesis(G2/M phase)was promoted by the expression of ORFV118,and the expression of CDK2 mRNA was decreased.The cell apoptosis blocked after ORFV118 expression,and the expression of Caspase3,Caspase7 and SOCS2 mRNA decreased.The immune-related cytokines,including IL-1β and IFN-γ,was inhibited in goat testis cells,while the expression of IL-6 and TNF-α were promoted.

Key words: Goat, Orf virus, ORFV118 protein, Cell cycle, Cell apoptosis, Cytokines

王宪军, 向华, 张旭梅, 任玉鹏, 朱江江. 羊口疮病毒ORFV118蛋白对山羊睾丸支持细胞周期、凋亡及免疫细胞因子的影响[J]. 华北农学报, 2022, 37(1): 222-231. doi: 10.7668/hbnxb.20192480.

WANG Xianjun, XIANG Hua, ZHANG Xumei, REN Yupeng, ZHU Jiangjiang. Effects of ORFV118 on Cell Cycle,Apoptosis and the Immune-related Cytokines in Goat Testicular Sertoli Cells[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 222-231. doi: 10.7668/hbnxb.20192480.

http://www.hbnxb.net/CN/Y2022/V37/I1/222

图/表 10

表1 细胞因子的引物序列

Tab.1 Primer sequences of cytokine

引物名称 Primer	引物序列(5'—3') Primer sequence(5'—3')	登录号 Accession No.	退火温度/℃ TM	扩增产物大小/bp PCR product length
IL-1β	S:TCACCAGCTCTACAACAAA A:AGGTCATCATCACGGAAG	KY038171.1	60	105
IL-6	S:GCTTCCAATCTGGGTTCA A:AGTTTCCTGATTTCCCTC	AB028635.1	60	114
IFN-γ	S:CAGAGCCAAATTGTCTCCTTC A:ATCCACCGGAATTTGAATCAG	AF020684.1	60	167
TNF-α	S:CCAGAGGGAAGAGCAGTCC A:GGCTACAACGTGGGCTACC	MH180383.1	60	111
GAPDH	S: GCAAGTTCCACGGCACAG A: TCAGCACCAGCATCACCC	XM_005680968.3	62	118

表2 细胞周期及凋亡相关基因的引物序列

Tab.2 Primers sequences of cell cycle and apoptosis-related genes

目的基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	登录号 Accession No.	退火温度/℃ TM	产物长度/bp Products length
BaX	S:CGCATTGGAGATGAATTGGACAGTAAC	KJ782303.1	60	128
	A:CAGTTGAAGTTGCCGTCGGAAAAC
Bcl-2	S:TGTGGATGACCGAGTACCTGAACC	KJ782304.1	60	127
	A:AGAGACAGCCAGGAGAAATCAAACAG
Caspase3	S:ACGCATATTCTACAGCACCTGGTTAC	JN986829.1	60	82
	A:ATCTCACAAAGAGCCTGGATGAACC
Caspase7	S:GGAACAGATGGCAAGACAGCAATAAAG	XM_018041622.1	60	101
	A:GCCTGAATGAAGAAGAGTTTGGGTTTC
BCL2L11	S:AACCTTCCGATGTAAGTTCTGAGTGTG	NM_001075310.1	60	117
	A:TGTCTTGCCGCTCTGTCTGTAAAG
P53	S:AGTGTGGTGGTGCCCTATGAGTC	X81704.1	60	137
	A:GAGTCTTCCAGTGTGATGATGGTGAG
SOCS2	S:TTCGCATTCAGATTACCTACT	XM_018047627.1	60	192
	A:CCTGTCCGCTTATCCTTG
PARP1	S:TCGCATATCAGCAAGTTACCCAAGG	XM_027564618.1	60	102
	A:CACCTCCACACCATCCACAGTAATAC
CDK2	S:GCACTCACTGGCATTCCTCTTCC	NM_001285608.1	60	140
	A:ATGGACCCATCTGCGTTGATAAGC
P21	S:GGTAGAGTTGGCTTTATGGGACACAG	NM_176645.3	60	114
	A:TCAGGGCTATCAATGGAGAAACACATC
UXT	S:GCAAGTGGATTTGGGCTGTAAC	JN711468.1	60	180
	A:ATGGAGTCCTTGGTGAGGTTGT

图1 ORFV118基因的扩增及克隆鉴定
A.ORFV118基因PCR扩增; B.质粒pMD19T-ORFV118的PCR扩增。M.2 000 bp DNA ladder;P.阳性对照;N.阴性对照;1.ORFV118基因。图4同。

Fig.1 Amplification,cloning and identification of ORFV118 gene
A.ORFV118 gene amplified by PCR;B.Recombinant plasmid pMD19T-ORFV118 amplified by PCR.M.2 000 bp DNA ladder;P.Positive product;N.Negative product;1.ORFV118 gene.The same as Fig.4.

图2 ORFV118蛋白的生物信息学分析
A.ORFV118蛋白的疏水性分析;B.ORFV118蛋白的信号肽预测;C.ORFV118蛋白的跨膜结构域分析;D.ORFV118蛋白的保守结构域预测;E.ORFV118蛋白的二级结构预测;F.ORFV118蛋白的三级结构预测;G.ORFV118蛋白的糖基化位点预测;H.ORFV118蛋白的磷酸化位点预测;I.ORFV118蛋白的亚细胞定位分析。

Fig.2 Bioinformatics analysis of ORFV118 protein
A.Hydrophobicity analysis of ORFV118 protein;B.Signal peptide prediction of ORFV118 protein;C.Transmembrane domain analysis of ORFV118 protein;D.Conserved domain prediction of ORFV118 protein;E.Secondary structure prediction of ORFV118 protein;F.Tertiary structure prediction of ORFV118 protein;G.Glycosylation site prediction of ORFV118 protein;H.Phosphorylation site prediction of ORFV118 protein;I.Predicted subcellular localization of ORFV118 protein.

图3 pEGFP-ORFV118的双酶切鉴定
M.5000 DNA Ladder;1.pEGFP-ORFV118的Hind Ⅲ、BamHⅠ双酶切鉴定。

Fig.3 Enzyme identification results of pEGFP-ORFV118
M.5000 DNA Ladder;1.pEGFP-ORFV118 digested by Hind Ⅲ and BamHⅠ.

图4 RT-PCR检测pEGFP-ORFV118重组质粒的mRNA表达分析

Fig.4 mRNA analysis of pEGFP-ORFV118

图5 ORFV118蛋白表达的Western Blot鉴定

Fig.5 Western Blot identification of ORFV118 protein

图6 ORFV118蛋白对IL-1β、IL-6、IFN-γ和TNF-α表达的影响
不同小写字母表示处理较对照在0.05水平上差异显著;不同大写字母表示处理较对照组在0.01水平上差异极显著。图7—8同。

Fig.6 Effect of ORFV118 on IL-1β,IL-6,IFN-γ and TNF-α expression
Different lowercase letters indicate significant difference at the level of 0.05 between the treatment and the control; Different capital letters indicate extremely significant difference at the level of 0.01 between the treatment and the control.The same as Fig.7—8.

图7 ORF118蛋白转染对山羊睾丸支持细胞周期的影响
A.pEGFP-N1对山羊睾丸支持细胞周期的影响; B.pEGFP-ORFV118对山羊睾丸支持细胞周期的影响;C.不同组别细胞周期结果; D.pEGFP-ORFV118对凋亡相关基因表达的影响。

Fig.7 Effect of ORF118 protein transfection on cell cycle of goat testicular sertoli cells
A.Effect of pEGFP-N1 on cell cycle of goat primary testis;B.Effect of pEGFP-ORFV118 on cell cycle of goat primary testis; C.Cell cycle results of different groups;D.Effect of pEGFP-ORFV118 on the expression of cell cycle-related genes.

图8 ORF118蛋白转染对山羊睾丸支持细胞凋亡的影响
A.pEGFP-N1对山羊睾丸支持细胞凋亡的影响; B.pEGFP-ORFV118对山羊睾丸支持细胞凋亡的影响;C.RT-qPCR检测凋亡相关基因的影响。

Fig.8 Effect of ORF118 protein transfection on cell apoptosis of goat testicular sertoli cells
A.Effect of pEGFP-N1 on apoptosis of goat testicular sertoli cells;B.Effect of pEGFP-ORFV118 on apoptosis of goat testicular sertoli cells C.Effect of RT-qPCR detect apoptosis-related genes.

参考文献 35

[1]	Muhsen M,Protschka M,Schneider L E,Müller U,Köhler G,Magin T M,Büttner M,Alber G,Siegemund S.Orf virus(ORFV)infection in a three-dimensional human skin model:Characteristic cellular alterations and interference with keratinocyte differentiation[J].PLoS One,2019,14(1):e0210504.doi:10.1371/journal.pone.0210504.
[2]	Long M J,Wang Y Y,Chen D X,Wang Y,Wang R X,Gong D Y,He H J,Rock D L,Hao W B,Luo S H.Identification of host cellular proteins LAGE3 and IGFBP6 that interact with Orf virus protein ORFV024[J].Gene,2018,661:60-67.doi:10.1016/j.gene.2018.03.089.
[3]	董佳易.羊口疮的免疫致病机制研究进展[J].山东畜牧兽医,2020,41(1):61-62.doi:10.3969/j.issn.1007-1733.2020.01.034.
	Dong J Y.Research progress on the immunopathogenic mechanism of sheep aphthous[J].Shandong Journal of Animal Science and Veterinary Medicine,2020,41(1):61-62.
[4]	Bergqvist C,Kurban M,Abbas O.Orf virus infection[J].Reviews in Medical Virology,2017,27(4):e1932.doi:10.1002/rmv.1932.
[5]	Şevik M. Orf virus circulation in cattle in turkey[J].Comparative Immunology,Microbiology and Infectious Diseases,2019,65:1-6.doi:10.1016/j.cimid.2019.03.013.
[6]	向华,黄忍,朱江江,岳华,汤承,张焕容.羊口疮病毒ORFV129锚蛋白在山羊睾丸细胞中的亚细胞定位[J].华北农学报,2020,35(4):222-229.doi:10.7668/hbnxb.20191063.
	Xiang H,Huang R,Zhu J J,Yue H,Tang C,Zhang H R.Subcellular localization of Orf virus 129 anchor protein in goat testis cells[J].Acta Agriculturae Boreali-Sinica,2020,35(4):222-229.
[7]	Bala J A,Balakrishnan K N,Abdullah A A,Mohamed R,Haron A W,Jesse F F A,Noordin M M,Mohd-Azmi M L.The re-emerging of Orf virus infection:A call for surveillance,vaccination and effective control measures[J].Microbial Pathogenesis,2018,120:55-63.doi:10.1016/j.micpath.2018.04.057.
[8]	黄忍,向华,杨发龙,张焕容.羊口疮病毒129蛋白对NF-kappaB信号通路介导的宿主细胞因子转录水平的影响[J].中国预防兽医学报,2021,43(6):584-590.doi:10.3969/j.issn.1008-0589.202007007.
	Huang R,Xiang H,Yang F L,Zhang H R.Effects of ORFV 129 protein on mRNA transcription level of four cytokines mediated by NF-κB signaling pathway in goat testis sertoli cells[J].Chinese Journal of Preventive Veterinary Medicine,2021,43(6):584-590.
[9]	Diel D G,Luo S,Delhon G,Peng Y,Flores E F,Rock D L.Orf virus ORFV121 encodes a novel inhibitor of NF-kappaB that contributes to virus virulence[J].Journal of Virology,2011,85(5):2037-2049.doi:10.1128/jvi.02236-10.
[10]	Lester S N,Li K.Toll-like receptors in antiviral innate immunity[J].Journal of Molecular Biology,2014,426(6):1246-1264.doi:10.1016/j.jmb.2013.11.024.
[11]	朱晓琴,李正莉,朱长庚,王效静.IL-1 β或IL-6对大脑皮质星形胶质细胞细胞周期的影响[J].神经解剖学杂志,2005,21(3):313-316.doi:10.3969/j.issn.1000-7547.2005.03.019.
	Zhu X Q,Li Z L,Zhu C G,Wang X J.Effects of IL-1 β or IL-6 on the cell cycle of astrocytes in cerebral cortex of neonatal rats[J].Chinese Journal of Neuroanatomy,2005,21(3):313-316.
[12]	罗杰,刘维佳.IL-6、GATA6在大鼠肺动脉平滑肌细胞中的表达及对细胞增殖的影响[J].天津医药,2020,48(11):1025-1030,1137.doi:10.11958/20200939.
	Luo J,Liu W J.Expressions of IL-6 and GATA6 in rat pulmonary artery smooth muscle cells and their effects on cell proliferation[J].Tianjin Medical Journal,2020,48(11):1025-1030,1137.
[13]	张先锋,吕智,冯毅.干扰素-γ联合顺铂对人骨肉瘤细胞U-2OS增殖抑制作用的研究[J].中国现代药物应用,2010,4(15):86-87.doi:10.14164/j.cnki.cn11-5581/r.2010.15.195.
	Zhang X F,Lü Z,Feng Y.Interferon-γ Inhibitory effect of combined cisplatin on proliferation of human osteosarcoma cell line U-2OS[J].Chinese Journal of Modern Drug Application,2010,4(15):86-87.
[14]	陈慧芹.羊口疮病毒119蛋白诱导细胞凋亡分子机制的研究[D].广州:南方医科大学,2017.
	Chen H Q.The molecular mechanisms of cell apoptosis induced by Orf virus(ORFV)119 protein[D].Guangzhou:Southern Medical University,2017.
[15]	Wang R X,Wang Y,Liu F,Luo S H.Orf virus:A promising new therapeutic agent[J].Reviews in Medical Virology,2019,29(1):e2013.doi:10.1002/rmv.2013.
[16]	杨璐,黄忍,张焕容,陈明,向华.新生山羊睾丸支持细胞分离培养方法研究[J].黑龙江畜牧兽医,2021(3):1—4,10,155.doi:10.13881/j.cnki.hljxmsy.2020.05.0151.
	Yang L,Huang R,Zhang H R,Chen M,Xiang H.Isolation and culture of testis sertoli cells in newborn goats[J].Heilongjiang Animal Science and Veterinary Medicine,2021(3):1—4,10,155.
[17]	冯平.羊口疮病毒的分离鉴定与B2L和F1L基因在昆虫杆状病毒系统中的表达及免疫原性分析[D].呼和浩特:内蒙古农业大学,2018.
	Feng P.Isolation and identification of Orf virus and the expression of B2L and F1L gene in Baculovirus system and its antigenicity analysis[D].Hohhot:Inner Mongolia Agricultural University,2018.
[18]	Pang F,Wang X Y,Chen Z,Zhang Z X,Zhang M M,Wang C Q,Yang X H,An Q,Du L,Wang F Y.Integrated analysis of differentially expressed miRNAs and mRNAs in goat skin fibroblast cells in response to Orf virus infection reveals that cfa-let-7a regulates thrombospondin 1 expression[J].Viruses,2020,12(1):118.doi:10.3390/v12010118.
[19]	Schneider L E,Protschka M,Müller U,Muhsen M,Magin T M,Anderegg U,Saalbach A,Büttner M,Alber G,Siegemund S.Orf virus infection of human keratinocytes and dermal fibroblasts:Limited virus detection and interference with intercellular adhesion molecule-1 up-regulation[J].Experimental Dermatology,2019,28(2):142-151.doi:10.1111/exd.13861.
[20]	王江林,王永,池永东,朱江江,林亚秋.KLF11抑制山羊肌内前体脂肪细胞分化[J].畜牧兽医学报,2020,51(5):976-986.doi:10.11843/j.issn.0366-6964.2020.05.009.
	Wang J L,Wang Y,Chi Y D,Zhu J J,Lin Y Q.KLF11 inhibits the differentiation of goat intramuscular preadipocyte[J].Acta Veterinaria et Zootechnica Sinica,2020,51(5):976-986.
[21]	Khoury G A,Baliban R C,Floudas C A.Proteome-wide post-translational modification statistics:frequency analysis and curation of the swiss-prot database[J].Scientific Reports,2011 13(1):90.doi:10.1038/srep00090.
[22]	韩妲丽,邢新,王晓云.睾丸支持细胞与间质细胞的相互作用[J].医学综述,2008,14(17):2567-2570.doi:10.3969/j.issn.1006-2084.2008.17.003.
	Han D L,Xing X,Wang X Y.Interaction between sustentacular cell of testis and leydig cells in testis[J].Medical Recapitulate,2008,14(17):2567-2570.
[23]	袁小丽,白汝岚,刘姗姗,张家骅.血睾屏障开放调节机制研究进展[J].动物医学进展,2006,27(12):31-34.doi:10.16437/j.cnki.1007-5038.2006.12.008.
	Yuan X L,Bai R L,Liu S S,Zhang J H.Progress on opening and closing of the blood-testis barrier(BTB)[J].Progress in Veterinary Medicine,2006,27(12):31-34.
[24]	焦涛.高脂饮食诱导的脂肪组织白色化对小鼠生精微环境的影响及其调控机制研究[D].北京:北京协和医学院,2019.
	Jiao T.Functional and mechanistical investigation of the whitening adipose tissues-induced by high-fat diet on the spermatogenic environment of mice[D].Beijing:Peking Union Medical College,2019.
[25]	Zhang Q B,Jia Q G,Wang H,Hu C X,Sun D,Jiang R D,Zhang Z L.High-mobility group protein box1 expression correlates with peritumoral macrophage infiltration and unfavorable prognosis in patients with hepatocellular carcinoma and cirrhosis[J].BMC Cancer,2016,16(1):880.doi:10.1186/s12885-016-2883-z.
[26]	王水明,彭瑞云,高亚兵,陈浩宇,马俊杰,周红梅,王德文.HPM辐照后大鼠睾丸组织细胞因子表达的定量研究[J].中国体视学与图像分析,2006,11(2):135-138.doi:10.13505/j.1007-1482.2006.02.018.
	Wang S M,Peng R Y,Gao Y B,Chen H Y,Ma J J,Zhou H M,Wang D W.Study on expression of testicular cytokines in rats irradiated by high power microwave[J].Chinese Journal of Stereology and Image Analysis,2006,11(2):135-138.
[27]	Herter J R,Fuchs S Y.Recognition of substrate and Skp1 by the Homologue of Slimb(HOS)ubiquitin ligase receptor D role of the F-box[J].Medical Science Monitor,2002,8(8):283-288.
[28]	Firpo M R,Mounce B C.Diverse functions of polyamines in virus infection[J].Biomolecules,2020,10(4):628.doi:10.3390/biom10040628.
[29]	Getts D R,Chastain E M L,Terry R L,Miller S D.Virus infection,antiviral immunity,and autoimmunity[J].Immunological Reviews,2013,255(1):197-209.doi:10.1111/imr.12091.
[30]	Gu H D,Jan Fada B.Specificity in ubiquitination triggered by virus infection[J].International Journal of Molecular Sciences,2020,21(11):4088.doi:10.3390/ijms21114088.
[31]	Lukhele S,Boukhaled G M,Brooks D G.Type I interferon signaling,regulation and gene stimulation in Chronic virus infection[J].Seminars in Immunology,2019,43:101277.doi:10.1016/j.smim.2019.05.001.
[32]	Wasilenko S T,Stewart T L,Meyers A F A,Barry M.Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis[J].PNAS,2003,100(24):14345-14350.doi:10.1073/pnas.2235583100.
[33]	Su J,Willert C,Comanita L,Peters A,Gilbert P A,Strathdee C,O'Connell P J,McFadden G D,Dekaban G A.Inclusion of the viral anti-apoptotic molecule M11L in DNA vaccine vectors enhances HIV Env-specific T cell-mediated immunity[J].Virology,2008,375(1):48-58.doi:10.1016/j.virol.2008.01.011.
[34]	Mehta N,Taylor J,Quilty D,Barry M.Ectromelia virus encodes an anti-apoptotic protein that regulates cell death[J].Virology,2015,475:74-87.doi:10.1016/j.virol.2014.10.023.
[35]	彭建伟.羊口疮病毒ORFV125抑制宿主细胞凋亡的研究[D].兰州:西北民族大学,2018.
	Peng J W.Research of the apoptosis for bcl-2 like protein ORFV125 inhibition of host cell apoptosis[D].Lanzhou:Northwest University for Nationalities,2018.

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