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| 重组猪肺表面活性蛋白A在昆虫杆状病毒表达系统的表达及体外抗PRRSV活性初步鉴定 |
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李兰1,2, 郑其升1,2, 张元鹏1,2, 李鹏成1,2, 肖希龙3, 付言峰4, 侯继波1,2
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| (1.江苏省农业科学院 国家兽用生物制品工程技术研究中心, 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, 江苏 扬州 225009;3.中国农业大学 动物医学院, 北京 100193;4.江苏省农业科学院 畜牧研究所, 南京 210014) |
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| 摘要: |
| 为研究重组猪肺表面活性蛋白A（RpSP-A）的抗病毒活性，采用Bac-to-Bac系统对RpSP-A蛋白进行表达。首先PCR扩增目的基因并将其克隆至pFastBac1转移载体获得pFast-SP-A，通过转化DH10Bac感受态细胞获得重组Bacmid，然后转染sf9细胞获得重组杆状病毒rBV-SP-A，在sf9细胞上进行目的蛋白的表达，经镍柱纯化、脱盐柱处理后，采用蚀斑减少方法评价RpSP-A对PRRSV感染Marc-145细胞的抑制作用。结果显示RpSP-A在sf9细胞中得到正确表达，并且能够降低PRRSV在Marc-145细胞上的感染，补加Ca2+时30 μg/mL RpSP-A抑制率高达77%，15 μg/mL RpSP-A抑制率为43%，未补加Ca2+时RpSP-A没有表现出抑制作用。上述结果表明利用Bac-to-Bac系统可以稳定获得大量具有抗PRRSV作用的可溶性RpSP-A。 |
| 关键词: 猪 表面活性蛋白A 杆状病毒表达 猪繁殖与呼吸综合征病毒 抗病毒活性 |
| DOI：10.11841/j.issn.1007-4333.2017.04.11 |
| 投稿时间：2016-04-07 |
| 基金项目:农业部公益性行业（农业）科研专项经费项目（201303046） |
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| Expression of recombint pig lung surfactant protein A and its antiviral activity against PRRSV in vitro |
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LI Lan1,2, ZHENG Qisheng1,2, ZHANG Yuanpeng1,2, LI Pengcheng1,2, XIAO Xilong3, FU Yanfeng4, HOU Jibo1,2
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| (1.Jiangsu Academy of Agricultural Sciences, National Research Center of Engineering and Technology for Veterinary Biologicals, Nanjing 210014, China;2.Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China;3.College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;4.Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China) |
| Abstract: |
| Recombint pig surfactant protein A (RpSP-A),which belongings to collectin,was obtained using Bac-to-Bac baculovirus expression system to study its antiviral activity.Firstly,RpSP-A gene was amplified by PCR and cloned into pFastBac1 vector to express pFast-SP-A.pFast-SP-A was then transformed into DH10Bac competent cells,to produce recombinant Bacmid.The above Bacmid DNAs were transfected into Sf9 insect cells in next step.The protein RpSP-A was finally expressed in sf9 cells,purified using a Ni2+-Sepharose HisTrap HPTM affinity column and removed imidazole on a HiTrapTM desalting column.The antiviral properties of RpSP-A on PRRSV infection was also assessed in Marc-145 cells using plaque assay.The results showed that:Soluble SP-A was successfully obtained and it inhibited the infectivity of PRRSV in Marc-145 cells;30 μ g/mL of RpSP-A reduced plaques by 77% (P<0.000 1),whereas 15 μ g/mL RpSP-A reduced plaques by 43% (P<0.05);And no inhibition was observed of RpSP-A in the absence of Ca2+ as a negative control.It is concluded that large amount of soluble RpSP-A with anti-PRRSV action could be obtained by using the Bac-to-Bac baculovirus expression system,which provided a foundation for anti-PRRSV drug development. |
| Key words: pig surfactant protein A baculovirus expression porcine reproductive and respiratory syndrome virus antiviral activity |
