羊口疮病毒ORFV129锚蛋白在山羊睾丸 细胞中的亚细胞定位

doi:10.7668/hbnxb.20191063

摘要/Abstract

摘要： 为明确羊口疮病毒ORFV129锚蛋白在细胞中的定位，参照GenBank中收录的ORFV SY 17全基因组（登录号：MG 712417.1）序列设计引物，扩增ORFV129基因完整编码框并测序鉴定，利用生物信息学软件分析该基因编码蛋白的氨基酸序列、蛋白跨膜区域，预测其亚细胞定位；并将ORFV129完整基因连接到真核表达载体pEGFP-N1，经双酶切鉴定及测序鉴定是否成功构建重组表达质粒pEGFP-ORFV129；利用LipoGene^TM2000介导将pEGFP-ORFV129转染新生山羊睾丸原代细胞，通过Western Blot鉴定ORFV129蛋白是否正确表达，同时，经DAPI核酸染料对细胞核进行染色后，激光共聚焦显微镜观察ORFV129蛋白的亚细胞定位。结果显示，成功扩增ORFV129完整基因，该基因全长大小为1 551 bp，测序鉴定其正确；生物信息学分析发现，其编码516个氨基酸，含有8种锚蛋白ANKs重复基序（ANKs），不存在跨膜区域，亚细胞定位预测其蛋白主要定位于细胞质；Western Blot结果显示，转染了pEGFP-ORFV129重组质粒的山羊睾丸原代细胞中检测到了ORFV129蛋白的表达；亚细胞定位结果表明，ORFV129蛋白主要定位于细胞质，与预测结果一致。研究结果为进一步探明ORFV129蛋白在诱导机体免疫应答中的作用奠定了基础。

关键词: 山羊, 羊口疮病毒, ORFV129锚蛋白, 生物信息学分析, 真核表达, 亚细胞定位

Abstract: This experiment was conducted to study the localization of ORFV129 ankyrin repeat proteins from Orf virus on transfected cells. According to the sequence of ORFV SY 17 complete gene (GenBank accession number:MG 712417.1), primers were designed to amplify the 129 gene of ORFV,and ORFV129 gene was cloned into pEGFP-N1 eukaryotic expression vector directionally. The recombinant expression plasmid was identified by restriction enzyme digestion analysis,sequencing and named as pEGFP-ORFV129. Preliminary bioinformatic analysis of ORFV129 gene was predicted and analyzed,including the amino acid sequence,protein transmembrane region and subcellular localization. The recombinant plasmid pEGFP-ORFV129 was transfected into the primary testis cells of newborn goat,and identification of ORFV129 protein expression by Western Blot. Subcellular localization of ORFV129 was examined with laser confocal microscopy after DAPI staining. The sequence of ORFV129 gene was amplified and cloned successfully,with a total length of 1 551 bp. Amino acid sequence analysis showed that the gene encoded a protein containing 516 amino acids,composing of 8 multiple copies of the ankyrin repeat motifs(ANKs). The transmembrane region analysis showed that ORFV129 had no transmembrane regions,and subcellular localization predicted that the protein was mainly located in the cytoplasm. The result of Western Blot showed that recombinant plasmid pEGFP-ORFV129 transfected into the primary testis cells of newborn goat was successfully expressed. Subcellular localization experiment results showed that the ORFV129 protein was located in the cytoplasm,which was identical with the predicted ones.The results might lay a foundation for further research the function of ORFV129.

Key words: Goat, Orf virus, ORFV129 ankyrin repeat proteins, Bioinformatic analysis, Eukaryotic expression, Subcellular localization

中图分类号:

S855.3
Q78

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 Hua, HUANG Ren, ZHU Jiangjiang, YUE Hua, TANG Cheng, ZHANG Huanrong. Subcellular Localization of Orf virus 129 Anchor Protein in Goat Testis Cells[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(4): 222-229. doi: 10.7668/hbnxb.20191063.

http://www.hbnxb.net/CN/Y2020/V35/I4/222

参考文献

[1] 陆承平. 兽医微生物学[M]. 北京:中国农业出版社,2007. Lu C P. Veterinary microbiology[M]. Beijing:China Agricultural Press,2007.
[2] Bergqvist C,Kurban M,Abbas O. Orf virus infection[J]. Reviews in Medical Virology,2017,27(4):e1932. doi:10.1002/rmv.1932.
[3] Mondal B,Bera A K,Hosamani M,Tembhurne P A,Bandyopadhyay S K. Detection of Orf virus from an outbreak in goats and its genetic relation with other Parapoxviruses[J]. Veterinary Research Communications,2006,30(5):531-539. doi:10.1007/s11259-006-3270-z.
[4] 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.
[5] Angella L C,Gustavo C,Nicole K,Isabel C,Esperanza P P,Antonio T. Erythema multiforme after Orf virus infection[J]. Pediatric Dermatology,2018,35(4):e237-e238. doi:10.1111/pde.13526.
[6] 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.
[7] 闫丰超,邵佳,窦永喜. 羊口疮病毒分子生物学的研究进展[J]. 中国兽医科学,2013,43(1):103-109. doi:10.16656/j.issn.1673-4696.2013.01.009. Yan F C,Shao J,Dou Y X. Progress in molecular biology of Orf virus[J]. Chinese Veterinary Science,2013,43(1):103-109.
[8] Sonnberg S,Seet B T,Pawson T,Fleming S B,Mercer A A. Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes[J]. Proceedings of the National Academy of Sciences of the United States of America,2008,105(31):10955-10960. doi:10.1073/pnas.0802042105.
[9] Shen Y W,Feng M,Wu X F. Bombyx mori nucleopolyhedrovirus ORF40 is essential for Budded virus production and Occlusion-derived virus envelopment[J]. Journal of General Virology,2018,99(6):837-850. doi:10.1099/jgv.0.001066.
[10] 白刚,贾怀杰,何小兵,王盈盈,何妍萍,吴润,景志忠. 羊口疮病毒 ORF129 基因重组质粒的构建及其在BHK-21细胞中的表达[J]. 甘肃农业大学学报,2013,48(5):8-13. doi:10.13432/j.cnki.jgsau.2013.05.002. B ai G,Jia H J,He X B,Wang Y Y,He Y P,Wu R,Jing Z Z. Construction and expression of the recombinant plasmid of Orf virus ORF129 gene in BHK-21 cells[J]. Journal of Gansu Agricultural University,2013,48(5):8-13.
[11] 杜国玉,刘永杰,吴锦艳,王光祥,尚佑军,张勇.羊口疮病毒 ORF129 基因的原核表达、纯化及鉴定[J]. 中国畜牧兽医,2018,45(7):1798-1803. doi:10.16431/j.cnki.1671-7236.2018.07.009. Du G Y,Liu Y J,Wu J Y,Wang G X,Shang Y J,Zhang Y. Prokaryotic expression,purification and identification of ORF129 gene of Orf virus[J]. China Animal Husbandry & Veterinary Medicine,2018,45(7):1798-1803.
[12] Lü Q Z,Guo K K,Xu H,Wang T,Zhang Y M. Identification of putative ORF5 protein of porcine Circovirus type 2 and functional analysis of GFP-fused ORF5 protein[J]. PLoS One,2015,10(7):e0134203. doi:10.1371/journal.pone.0127859.
[13] Lacek K,Bauer B,Bien 'kowska-Szewczyk K,Rziha H J. Orf virus (ORFV) ANK-1 protein mitochondrial localization is mediated by ankyrin repeat motifs[J]. Virus Genes,2014,49(1):68-79.doi:10.1007/s11262-014-1069-5.
[14] 王少鑫,靳宝锋,崔立红. 锚蛋白重复序列通过NF-κB信号通路参与肠炎相关肿瘤的调控[J]. 世界华人消化系统,2015,23(2):189-195. Wang S X,Jin B F,Cui L H. Gankyrin regulates colitis-associated cancer via NF-κB signaling pathway[J]. World Chinese Journal of Digestology,2015,23(2):189-195.
[15] Sonnberg S,Fleming S B,Mercer A A. Phylogenetic analysis of the large family of Poxvirus ankyrin-repeat proteins reveals orthologue groups within and across Chordopoxvirus genera[J]. Journal of General Virology,2011,92(11):2596-2607. doi:10.1099/vir.0.033654-0.
[16] Liu R K,Moss B. Vaccinia virus C9 ankyrin-repeat/F-box protein is a newly identified antagonist of the type Ⅰ interferon-induced antiviral state[J]. Journal of Virology,2018,92(9):e00053-e00018. doi:10.1128/JVI.00053-18.
[17] Odon V,Georgana I,Holley J,Morata J,Maluqyer M C. Novel class of viral ankyrin proteins targeting the host E3 ubiquitin ligase cullin-2[J]. Journal of Virology,2018, 92(23):e01374-e01318.doi:10.1128/JVI.01374-18.
[18] Chang S J,Hsiao J C,Sonnberg S,Chiang C T,Yang M H,Tzou D L,Mercer A A,Chang W. Poxvirus host range protein CP77 contains an F-box-like domain that is necessary to suppress NF-κB activation by tumor necrosis factor alpha but is independent of its host range function[J]. Journal of Virology,2009,83(9):4140-4152. doi:10.1128/JVI.01835-08.
[19] Mohamed M R,Rahman M M,Lanchbury J S,Shattuck D,Neff C,Dufford M,van Buuren N,Fagan K,Barry M,Smith S,Damon I,Mcfadden G. Proteomic screening of Variola virus reveals a unique NF-κB inhibitor that is highly conserved among pathogenic orthopoxviruses[J]. Proceedings of the National Academy of Sciences of the United States of America,2009,106(22):9045-9050. doi:10.1073/pnas.0900452106.
[20] Mohamed M R,Rahman M M,Rice A,Moyer R W,Werden S J,Mcfadden G. Cowpox virus expresses a novel ankyrin repeat NF-κB inhibitor that controls inflammatory cell influx into virus-infected tissues and is critical for virus pathogenesis[J]. Journal of Virology,2009,83(18):9223-9236. doi:10.1128/JVI.00861-09.
[21] Shisler J L,Jin X L. The Vaccinia virus K1L gene product inhibits host NF-κB activation by preventing IκBα degradation[J]. Journal of Virology,2004,78(7):3553-3560. doi:10.1128/JVI.78.7.3553-3560.2004.
[22] Aravalli R N,Hu S X,Lokensgard J R. Inhibition of toll-like receptor signaling in primary murine microglia[J]. Journal of Neuroimmune Pharmacology,2008,3(1):5-11. doi:10.1007/s11481-007-9097-8.
[23] Pilato M D,Mejías-Pérez E,Sorzano C O S,Esteban M. Distinct roles of Vaccinia virus NF-κB inhibitor proteins A52,B15 and K7 in the immune response[J]. Journal of Virology,2017,91(13):e00575-e00617. doi:10.1128/JVI.00575-17.
[24] Nagendraprabhu P,Khatiwada S,Chaulagain S,Delhon G,Rock D L. A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling[J]. PLoS Pathogens,2017,13(12):e1006779. doi:10.1371/journal.ppat.1006779.
[25] Lamb S A,Rahman M M,McFadden G. Recombinant Myxoma virus lacking all Poxvirus ankyrin-repeat proteins stimulates multiple cellular anti-viral pathways and exhibits a severe decrease in virulence[J]. Virology,2014,464-465(1):134-145. doi:10.1016/j.virol.2014.06.021.
[26] Meng X Z,Xiang Y. Vaccinia virus K1L protein supports viral replication in human and rabbit cells through a cell-type-specific set of its ankyrin repeat residues that are distinct from its binding site for ACAP2[J]. Virology,2006,353(1):220-233. doi:10.1016/j.virol.2006.05.032.
[27] 冯园,贾怀杰,何小兵,成温玉,陈国华,王春燕,何玉林,景志忠. 口疮病毒B4R锚蛋白抑制细胞NF-κB信号通路的研究[J]. 中国兽医科学,2016,46(11):1412-1417. doi:10.16656/j.issn.1673-4696.2016.11.012. Feng Y,Jia H J,He X B,Cheng W Y,Chen G H,Wang C Y,He Y L,Jing Z Z. Poxvirus ankyrin protein B4R from Orf virus inhibits cell NF-κB signaling pathway[J]. Chinese Veterinary Science,2016,46(11):1412-1417.
[28] 安雪珂,贾怀杰,冯园,陈国华,何小兵,景志忠,王晓霞. 羊口疮病毒ORF128锚蛋白对HEK 293T 细胞NF-κB信号通路的调节作用及机制[J]. 细胞与分子免疫学杂志,2019,35(3):243-249. doi:10.13423/j.cnki.cjcmi.008759. An X K,Jia H J,Feng Y,Chen G H,He X B,Jing Z Z,Wang X X. Regulating effect of ORF128 on NF-κB signaling pathway of HEK293T cells and its mechanism[J]. Chinese Journal of Cellular and Molecular Immunology,2019,35(3):243-249.
[29] 葛士坤,张凯照,刘健新,王晴楠,于梦,陶攀,宁章勇. 羊口疮病毒 GDZC 株锚蛋白基因的克隆及序列分析[J]. 中国畜牧兽医,2016,43(12):3085-3093. doi:10.16431/j.cnki.1671-7236.2016.12.001. Ge S K,Zhang K Z,Liu J X,Wang Q N,Yu M,Tao P,Ning Z Y. Cloning and sequence analysis of ankyrin genes from Orf virus GDZC strain[J]. China Animal Husbandry & Veterinary Medicine,2016,43(12):3085-3093.
[30] Lacek K,Bauer B,Bieńkowska-Szewczyk K,Rziha H J. Orf virus (ORFV) ANK-1 protein mitochondrial localization is mediated by ankyrin repeat motifs[J]. Virus Genes,2014,49(1):68-79. doi:10.1007/s11262-014-1069-5.

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