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  

Expression of recombint pig lung surfactant protein A and its antiviral activity against PRRSV invitro

作　　者：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] LI Lan ZHENG Qisheng ZHANG Yuanpeng LI Pengcheng XIAO Xilong FU Yanfeng HOU Jibo(Jiangsu Academy of Agricultural Sciences, National Research Center of Engineering and Technology for Veterinary Biologicals, Nanjing 210014, China College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China)

机构地区：[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

出　　处：《中国农业大学学报》2017年第4期101-108,共8页Journal of China Agricultural University

基　　金：农业部公益性行业(农业)科研专项经费项目(201303046)

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。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 pFastBacl 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 μg/mL of RpSP-A reduced plaques by 77% （P〈0.000 1） ,whereas 15 μg/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.

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 

分 类 号：S858.28[农业科学—临床兽医学]