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

发布时间：2018-09-11 08:48

【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Ca~(2+)时30μg/mL RpSP-A抑制率高达77%,15μg/mL RpSP-A抑制率为43%,未补加Ca~(2+)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。
[Abstract]:To study the antiviral activity of recombinant porcine pulmonary surfactant protein A (RpSP-A), RpSP-A protein was expressed by Bac-to-Bac system. Firstly, the target gene was amplified by PCR and cloned into the pFastBac1 transfer vector to obtain pFast-SP-A,. The recombinant Bacmid, was obtained by transforming the DH10Bac receptive cells. Then the recombinant baculovirus rBV-SP-A, was transfected into sf9 cells to express the target protein on sf9 cells. The target protein was purified by nickel column. The inhibition effect of RpSP-A on PRRSV infected Marc-145 cells was evaluated by plaque reduction method after desalination column. The results showed that RpSP-A was correctly expressed in sf9 cells and could reduce the infection of PRRSV on Marc-145 cells. The inhibitory rate of 30 渭 g/mL RpSP-A on Marc-145 cells supplemented with Ca~ (2) was as high as 7715 渭 g/mL RpSP-A, but RpSP-A did not show inhibitory effect without Ca~ (2). These results indicate that a large number of soluble RpSP-A. with anti-PRRSV effect can be obtained stably by using Bac-to-Bac system.
【作者单位】：江苏省农业科学院国家兽用生物制品工程技术研究中心;江苏省动物重要疫病与人兽共患病防控协同创新中心;中国农业大学动物医学院;江苏省农业科学院畜牧研究所;
【基金】：农业部公益性行业(农业)科研专项经费项目(201303046)
【分类号】：S852.65

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