摘要
采用RT-PCR扩增获得鹅星状病毒(Goose astrovirus)的衣壳纤突蛋白基因vp27,并将其克隆至pCold-SUMO原核表达载体。重组表达载体pCold-vp27经过测序和双酶切验证正确后,转化到感受态细胞Rosetta。挑取阳性克隆子,利用异丙基硫代半乳糖苷诱导表达鹅星状病毒衣壳纤突重组蛋白,使用镍柱纯化衣壳纤突蛋白VP27,将定量的重组蛋白免疫BALB/c小鼠。利用ELISA、Western blot和IFA鉴定鼠抗衣壳纤突蛋白VP27多克隆抗体。结果显示,鼠抗衣壳纤突蛋白VP27多克隆抗体的效价大于1∶64000,Western blot试验结果证明了鼠抗衣壳纤突蛋白VP27多克隆抗体的特异性,IFA试验结果证明鼠抗衣壳纤突蛋白VP27多克隆抗体能够识别天然的鹅星状病毒衣壳纤突蛋白VP27。
The vp27 gene of capsid spike protein of goose astrovirus was amplified by RT-PCR and cloned into prokaryotic expressional vector pCold-SUMO.The recombinant expressional vector pCold-vp27 was transformed into competent cell Rosetta after verified to be correct by sequencing and double enzyme digestion.The positive clones were selected,and the capsid spike recombinant protein of goose astrovirus was induced to express by isopropyl-β-D-thiogalactoside(IPTG).The capsid spike protein VP27 was purified by nickel column and quantitative recombinant protein was used to immune BALB/c mice.The polyclonal antibody against capsid spike protein VP27 in mice was identified by ELISA,Western blot and IFA.The results showed that the titer of polyclonal antibody against capsid spike protein VP27 in mice was higher than 1∶64000.The specificity of polyclonal antibody against capsid spike protein VP27 in mice was proved by the testing results of Western blot.Results of IFA test proved that the polyclonal antibody against capsid spike protein VP27 in mice could recognize natural goose astrovirus capsid spike protein VP27.
作者
吕炫
张淼
胡增金
李佳明
张冲
朱英奇
魏娟文
吴琼
张瑞辰
夏伦志
王桂军
LYU Xuan;ZHANG Miao;HU Zeng-jin;LI Jia-ming;ZHANG Chong;ZHU Ying-qi;WEI Juan-wen;WU Qiong;ZHANG Rui-chen;XIA Lun-zhi;WANG Gui-jun(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;Anhui Provincial Key Laboratory of Veterinary Pathobiology and Disease Control,Hefei 230036,China;Institute of Animal Husbandry and Veterinary Medicine,Anhui Academy of Agricultural Sciences,Hefei 230036,China)
出处
《江苏农业学报》
CSCD
北大核心
2021年第2期412-417,共6页
Jiangsu Journal of Agricultural Sciences
基金
科技部国家重点研发计划项目(2018YFD0500100、2016YFD0500805)
安徽省家禽产业技术体系项目(2019-2020)。
