经密码子优化的猪Ficolin α在昆虫细胞的表达及其体外抗病毒作用初步分析

doi:10.11843/j.issn.0366-6964.2017.12.015

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (12): 2358-2364.doi: 10.11843/j.issn.0366-6964.2017.12.015

经密码子优化的猪Ficolin α在昆虫细胞的表达及其体外抗病毒作用初步分析

李兰¹, 乔绪稳¹, 张元鹏¹, 陈瑾¹, 郑其升^1*, 侯继波^1,2*

1. 江苏省农业科学院动物免疫工程研究所, 南京 210014;
2. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2017-06-20 出版日期:2017-12-23 发布日期:2017-12-23
通讯作者: 侯继波,研究员,E-mail:houjibo@jaas.ac.cn;郑其升,研究员,E-mail:njcvc1302@163.com
作者简介:李兰(1983-),女,山东高密人,助理研究员,博士,主要从事兽用疫苗研究,E-mail:lanli99@foxmail.com
基金资助:
农业部公益性行业（农业）科研专项经费项目（201303046）

Expression of Codon-optimized Porcine Ficolin α in Insect Cell and Its Antiviral Activity in vitro

LI Lan¹, QIAO Xu-wen¹, ZHANG Yuan-peng¹, CHEN Jin¹, ZHENG Qi-sheng^1*, HOU Ji-bo^1,2*

1. Institute of Animal Immunology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2017-06-20 Online:2017-12-23 Published:2017-12-23

摘要/Abstract

摘要：

利用Bac-to-Bac昆虫杆状病毒表达系统表达具有天然构象的猪Ficolin α。首先构建重组穿梭质粒rBacmid-Ficolin α，转染sf9细胞获得重组杆状病毒rBV-Ficolin α；通过Image J软件对优化表达的Western blot图片进行灰度分析确定较佳表达条件；纯化目的蛋白质后，进一步评价其体外抗病毒活性。Western blot结果显示以7.5 MOI接种量感染的sf9细胞在96 h时获得了较高的表达，同时获得的重组猪Ficolin α具有抗猪繁殖与呼吸综合征病毒（PRRSV）活性。猪Ficolin α在sf9昆虫细胞中获得成功表达，且具有良好的抗PRRSV活性。本研究可以为猪Ficolin α的抗病毒活性及作用机制研究提供物质基础。

Abstract:

To get native porcine Ficolin α, Bac-to-Bac baculovirus expression system was used. Firstly, rBacmid-Ficolin α was constructed, and then transfected into sf9 insect cells to obtain recombinant baculovirus rBV-Ficolin α. The optimized expression conditions were detected by Western blot and analyzed by gray level using Image J software. Followed by purification, antiviral activity of target protein was assessed in vitro. Western blot results showed that the expression level of Ficolin α in transfected sf 9 insect cells was higher at the infection (MOI) of 7.5 when they were harvested at 96 h, and porcine Ficolin α had the antivirus activity against porcine reproductive and respiratory syndrome virus (PRRSV). In conclusion, porcine Ficolin α was successfully expressed in sf9 insect cells and showed strongly anti-PRRSV activity. This study provided a material basis for the study of antiviral activity and its mechanism of porcine Ficolin α.

中图分类号:

S852.42

李兰, 乔绪稳, 张元鹏, 陈瑾, 郑其升, 侯继波. 经密码子优化的猪Ficolin α在昆虫细胞的表达及其体外抗病毒作用初步分析[J]. 畜牧兽医学报, 2017, 48(12): 2358-2364.

LI Lan, QIAO Xu-wen, ZHANG Yuan-peng, CHEN Jin, ZHENG Qi-sheng, HOU Ji-bo. Expression of Codon-optimized Porcine Ficolin α in Insect Cell and Its Antiviral Activity in vitro[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(12): 2358-2364.

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参考文献

[1] ANDRES J L, RÖNNSTRAND L, CHEIFETZ S, et al. Purification of the transforming growth factor-β(TGF-β) binding proteoglycan betaglycan[J]. J Biol Chem, 1991, 266(34):23282-23287.
[2] ICHIJO H, HELLMAN U, WERNSTEDT C, et al. Molecular cloning and characterization of ficolin, a multimeric protein with fibrinogen-and collagen-like domains[J]. J Biol Chem, 1993, 268(19):14505-14513.
[3] KILPATRICK D C, CHALMERS J D. Human L-ficolin (ficolin-2) and its clinical significance[J]. J Biomed Biotechnol, 2012, 2012:138797.
[4] BROOKS A S, HAMMERMUELLER J, DELAY J P, et al. Expression and secretion of ficolin β by porcine neutrophils[J]. Biochim Biophys Acta Gen Subj, 2003, 1624(1-3):36-45.
[5] MATSUSHITA M, ENDO Y, TAIRA S, et al. A novel human serum lectin with collagen-and fibrinogen-like domains that functions as an opsonin[J]. J Biol Chem, 1996, 271(5):2448-2454.
[6] MICHALSKI M, ST SWIERZKO A, LUKASIEWICZ J, et al. Ficolin-3 activity towards the opportunistic pathogen, Hafnia alvei[J]. Immunobiology, 2015, 220(1):117-123.
[7] LOZANO V, AGUADO L, HOORELBEKE B, et al. Targeting HIV entry through interaction with envelope glycoprotein 120(gp120):synthesis and antiviral evaluation of 1,3,5-triazines with aromatic amino acids[J]. J Med Chem, 2011, 54(15):5335-5348.
[8] BERTAUX C, DAELEMANS D, MEERTENS L, et al. Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions[J]. Virology, 2007, 366(1):40-50.
[9] HILLAIRE M L B, VAN EIJK M, VOGELZANG-VAN TRIERUM S E, et al. Assessment of the antiviral properties of recombinant surfactant protein D against influenza B virus in vitro[J]. Virus Res, 2015, 195:43-46.
[10] KEIRSTEAD N D, LEE C, YOO D, et al. Porcine plasma ficolin binds and reduces infectivity of porcine reproductive and respiratory syndrome virus (PRRSV) in vitro[J]. Antiv Res, 2008, 77(1):28-38.
[11] HAMED M R, BROWN R J P, ZOTHNER C, et al. Recombinant human L-Ficolin directly neutralizes hepatitis C virus entry[J]. J Innate Immun, 2014, 6(5):676-684.
[12] TAKAHASHI K, MOYO P, CHIGWESHE L, et al. Efficacy of recombinant chimeric lectins, consisting of mannose binding lectin and L-ficolin, against influenza A viral infection in mouse model study[J]. Virus Res, 2013, 178(2):495-501.
[13] VERMA A, WHITE M, VATHIPADIEKAL V, et al. Human H-ficolin inhibits replication of seasonal and pandemic influenza A viruses[J]. J Immunol, 2012, 189(5):2478-2487.
[14] MICHELOW I C, DONG M D, MUNGALL B A, et al. A novel L-ficolin/Mannose-binding lectin chimeric molecule with enhanced activity against ebola virus[J]. J Biol Chem, 2010, 285(32):24729-24739.
[15] 张家忠, 章晓联. 纤维胶凝蛋白(ficolin)在非特异性免疫中的作用[J]. 襄樊职业技术学院学报, 2006, 5(5):8-9.
ZHANG J Z, ZHANG X L. The role of ficolin in innate immunity[J]. Journal of Xiangfan Vocational and Technical College, 2006, 5(5):8-9. (in Chinese)
[16] 李兰, 郑其升, 付言峰, 等. 几种胶原样凝集素的生物学特性及其抗病原微生物的作用[J]. 中国兽医杂志, 2016, 52(11):76-78.
LI L, ZHENG Q S, FU Y F, et al. Biological characteristics and antimicrobial activity of several collagen lectins[J]. Chinese Journal of Veterinary Medicine, 2016, 52(11):76-78. (in Chinese)
[17] ENDO Y, IWAKI D, ISHIDA Y, et al. Mouse Ficolin B has an ability to form complexes with mannose-binding lectin-associated serine proteases and activate complement through the lectin pathway[J]. J Biomed Biotechnol, 2012, 2012:105891.

经密码子优化的猪Ficolin α在昆虫细胞的表达及其体外抗病毒作用初步分析

Expression of Codon-optimized Porcine Ficolin α in Insect Cell and Its Antiviral Activity in vitro

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