羊口疮病毒<i>115</i>基因的克隆及原核表达

林裕胜; 黄丽丽; 江锦秀; 张靖鹏; 刘维巍; 刘庆华; 胡奇林

doi:10.19303/j.issn.1008-0384.2022.007.005

羊口疮病毒115基因的克隆及原核表达

doi: 10.19303/j.issn.1008-0384.2022.007.005

1.
福建省农业科学院畜牧兽医研究所，福建　福州　350013
2.
新疆生产建设兵团第十一师第五中学，新疆　乌鲁木齐　830000
3.
福建农林大学动物科学学院，福建　福州　350002

基金项目: 福建省农业高质量发展超越“5511”协同创新工程项目（XTCXGC2021008）；福建省科技计划公益类专项（2021R1026007）；福建省自然科学基金项目（2021J01484）；福建省农业科学院科技创新团队项目（CXTD2021007-2）

详细信息

作者简介:
林裕胜（1988−），男，硕士，助理研究员，主要从事动物传染病研究（E-mail：369807285@qq.com）

通讯作者:
胡奇林（1963−），男，硕士，研究员，主要从事动物传染病学和免疫学研究（E-mail：hql562713@163.com）

中图分类号: S 852.65
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出版历程
- 收稿日期: 2022-02-18
- 修回日期: 2022-04-20
- 网络出版日期: 2022-08-07
- 刊出日期: 2022-07-28

Cloning and Prokaryotic Expression of ORFV 115 Gene

1.
Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
2.
No. 5 Middle School, 11^th Division, Xinjiang Production and Construction Corps, Urumqi, Xinjiang　830000, China
3.
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的获得羊口疮病毒（ORFV）115基因表达蛋白。方法利用Oligo 7软件设计并筛选出115基因特异性扩增引物，以ORFV FJ-ND株基因组为模板，通过PCR技术扩增获得其基因序列，再将115基因克隆至pGEX-6p-1上，重组质粒pGEX-6p-115，并对其进行测序鉴定，鉴定正确后转化RosettaganmiB（DE3）感受态细胞，通过优化IPTG浓度和诱导时间获得重组融合蛋白最佳表达条件，用SDS-PAGE对表达的目的蛋白进行分析。结果 115基因全长450 bp，编码149个氨基酸；115基因可以在RosettaganmiB中表达，重组蛋白分子大小约42 kDa，主要以包涵体形式表达。结论成功克隆了ORFV 115基因，构建了pGEX-6P-115原核表达质粒，优化了重组蛋白表达条件并进行纯化，为进一步探索ORFV 115蛋白在感染宿主过程中的机制和作用奠定基础。
- 羊口疮病毒 /
- 115基因 /
- 克隆 /
- 原核表达
Abstract: Objective ORFV 115 was cloned for a study on the gene. Method Oligo 7 software was used to design and select primers for specific amplification of the gene. Using the genome of ORFV FJ-ND as template, the sequence was obtained by PCR. The gene was then cloned into pGEX-6p-1 to obtain the recombinant plasmid pGEX-6P-115 for sequencing and identification prior to transformation to RosettaganmiB (DE3) receptor cells. Expression conditions of the recombinant fusion protein were optimized with respect to IPTG concentration and induction time. SDS-PAGE was used to analyze the expressed target protein. Result ORFV 115 was 450 bp encoded 149 amino acids and could be expressed in RosettaganmiB. It was approximately 42 kDa in molecular weight and mainly expressed as inclusion body. Conclusion ORFV 115 was successfully cloned, the pGEX-6P-115 prokaryotic expression plasmid constructed, the recombinant protein purified, and the expression conditions optimized to facilitate further studies on the mechanism and role it plays in the infection on host animals.
- Orf virus /
- ORFV 115 /
- cloning /
- prokaryotic expression

HTML全文

图 1 115基因PCR扩增结果

M为1 000 DNA Ladder，1为PCR扩增产物，2为阴性对照。

Figure 1. Amplification of ORFV 115 by PCR

M=1 000 DNA Ladder, 1= PCR product, 2= negative control.

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图 2 重组质粒PCR产物

M为1 000 DNA Ladder，1为PCR鉴定产物，2为阴性对照。

Figure 2. PCR identification of recombination plasmid

M=1 000 DNA Ladder, 1=PCR identification product, 2=negative control.

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图 3 重组质粒酶切鉴定结果

M为5 000 DNA Ladder，1为双酶切产物。

Figure 3. Identification of recombination plasmid by restriction endonuclease digestion

M=1 000 DNA Ladder, 1= double digestion product.

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图 4 重组蛋白表达IPTG浓度优化结果

M为蛋白分子质量标准，1为未诱导pGEX-6P-1，2为诱导pGEX-6P-1，3为未诱导pGEX-6P-115，4~7为IPTG终浓度为0.4、0.6、0.8、1.0 mmol·L⁻¹。

Figure 4. Optimization of recombinant protein expression by varying IPTG concentration

M=protein molecular weight Marker, 1: uninduced pGEX-6P-1, 2: induced pGEX-6P-1, 3: uninduced pGEX-6P-115, 4 –7: IPTG concentration of 0.4, 0.6, 0.8, 1.0 mmol·L⁻¹.

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图 5 重组蛋白表达时间的优化结果

M为蛋白分子质量标准，1为未诱导pGEX-6P-1，2为诱导pGEX-6P-1，3为未诱导pGEX-6P-115，4~8为pGEX-6P-115诱导2、3、4、5、6 h。

Figure 5. Optimization of recombinant protein expression by varying induction time

NM=protein molecular weight Marker, 1：uninduced pGEX-6P-1, 2: induced pGEX-6P-1, 3: uninduced pGEX-6P-115, 4–8:induced pGEX-6P-115 of 2, 3, 4, 5, 6 h.

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图 6 重组蛋白的可溶性分析

M为蛋白分子质量标准；1为诱导pGEX-6P-115，2为pGEX-6P-115菌体裂解沉淀，3为pGEX-6P-115菌体裂解上清。

Figure 6. Solubility of recombinant protein

M=protein molecular weight Marker, 1=induced pGEX-6P-115, 2=bacterial lysis precipitate of pGEX-6P-115, 3=lysate supernatant of pGEX-6P-115.

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图 7 重组蛋白的纯化

M为蛋白分子质量标准，1为诱导pGEX-6P-115，2为ORFV 115重组蛋白的纯化产物。

Figure 7. Purification of recombinant protein

M=protein molecular weight Marke, 1=induced pGEX-6P-115, 2=purification of ORFV 115 recombinant protein.

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参考文献(16)

[1]	张孟然, 丛潇, 顾月月, 等. 羊口疮病毒主要基因组编码蛋白及其功能 [J]. 动物医学进展, 2022, 43(4):96−101. doi: 10.3969/j.issn.1007-5038.2022.04.018 ZHANG M R, CONG X, GU Y Y, et al. Main genome coding proteins and functions of orf virus [J]. Progress in Veterinary Medicine, 2022, 43(4): 96−101.(in Chinese) doi: 10.3969/j.issn.1007-5038.2022.04.018
[2]	FLEMING S B, WISE L M, MERCER A A. Molecular genetic analysis of orf virus: A poxvirus that has adapted to skin [J]. Viruses, 2015, 7(3): 1505−1539. doi: 10.3390/v7031505
[3]	SPYROU V, VALIAKOS G. Orf virus infection in sheep or goats [J]. Veterinary Microbiology, 2015, 181(1/2): 178−182.
[4]	FRIEBE A, FRIEDERICHS S, SCHOLZ K, et al. Characterization of immunostimulatory components of orf virus (parapoxvirus ovis) [J]. The Journal of General Virology, 2011, 92(Pt7): 1571−1584.
[5]	SULLIVAN J T, MERCER A A, FLEMING S B, et al. Identification and characterization of an orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K [J]. Virology, 1994, 202(2): 968−973. doi: 10.1006/viro.1994.1420
[6]	成伟伟, 张克山, 刘永杰, 等. 羊传染性脓疱病毒基因组结构和主要基因功能研究进展 [J]. 动物医学进展, 2014, 35(7):82−85. doi: 10.3969/j.issn.1007-5038.2014.07.018 CHENG W W, ZHANG K S, LIU Y J, et al. Advance in genome structure and major gene function of orf virus [J]. Progress in Veterinary Medicine, 2014, 35(7): 82−85.(in Chinese) doi: 10.3969/j.issn.1007-5038.2014.07.018
[7]	PERALTA A, ROBLES C, MARTÍNEZ A, et al. Identification and molecular characterization of Orf virus in Argentina [J]. Virus Genes, 2015, 50(3): 381−388. doi: 10.1007/s11262-015-1189-6
[8]	TEDLA M, BERHAN N, MOLLA W, et al. Molecular identification and investigations of contagious ecthyma (Orf virus) in small ruminants, North west Ethiopia [J]. BMC Veterinary Research, 2018, 14(1): 13. doi: 10.1186/s12917-018-1339-x
[9]	CHEN D X, ZHENG Z W, XIAO B, et al. Orf virus 002 protein targets ovine protein S100A4 and inhibits NF-κB signaling [J]. Frontiers in Microbiology, 2016, 7: 1389.
[10]	DIEL D G, DELHON G, LUO S, et al. A novel inhibitor of the NF-κB signaling pathway encoded by the parapoxvirus orf virus [J]. Journal of Virology, 2010, 84(8): 3962−3973. doi: 10.1128/JVI.02291-09
[11]	NAGENDRAPRABHU P, KHATIWADA S, CHAULAGAIN S, et al. 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
[12]	DIEL D G, LUO S, DELHON G, et al. 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
[13]	DELHON G, TULMAN E R, AFONSO C L, et al. Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus [J]. Journal of Virology, 2004, 78(1): 168−177. doi: 10.1128/JVI.78.1.168-177.2004
[14]	CHI X L, ZENG X C, LI W, et al. Genome analysis of orf virus isolates from goats in the Fujian Province of Southern China [J]. Frontiers in Microbiology, 2015, 6: 1135.
[15]	CORADDUZZA E, SANNA D, ROCCHIGIANI A M, et al. Molecular insights into the genetic variability of ORF virus in a Mediterranean region (Sardinia, Italy) [J]. Life (Basel, Switzerland), 2021, 11(5): 416.
[16]	张高峰, 杨侃侃, 张谦, 等. 羊口疮病毒安徽株023基因的原核表达与生物信息学分析 [J]. 浙江农业学报, 2019, 31(1):30−38. doi: 10.3969/j.issn.1004-1524.2019.01.04 ZHANG G F, YANG K K, ZHANG Q, et al. Prokaryotic expression and bioinformatics analysis of orf virus Anhui strain 023 gene [J]. Acta Agriculturae Zhejiangensis, 2019, 31(1): 30−38.(in Chinese) doi: 10.3969/j.issn.1004-1524.2019.01.04