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

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∶64 000，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。

关键词： 鹅星状病毒;衣壳纤突蛋白VP27;多克隆抗体

中图分类号： S858.332.65+9.6  文獻标识码： A  文章编号： 1000-4440（2021）02-0412-06

Abstract： 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∶64 000. 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.

Key words： goose astrovirus;capsid spike protein VP27;polyclonal antibody

星状病毒（Astrovirus）是一种单股正链无囊膜的RNA病毒，可以感染哺乳动物和禽类。在哺乳动物中，星状病毒通常感染人、猪[1]、狗[2]和牛[3]等，主要引起胃肠炎和腹泻，在极个别条件下会引起脑炎[4]。在禽类动物中，星状病毒感染主要发生在鸡、鸭、鹅和火鸡中，引起鸡肾炎[5]、鸭致死性肝炎[6]、火鸡肠炎[7]等临床常见疾病。近年来，在安徽、江苏、福建等省份流行一种由鹅星状病毒（Goose astrovirs）感染引起雏鹅痛风为主要特征的疾病[8]。鹅星状病毒主要感染21日龄左右的雏鹅，死亡率可达30%，给养鹅产业带来极大的经济损失。

目前鹅星状病毒的检测主要依赖于RT-PCR方法，没有商品化的试剂盒[9]。衣壳纤突蛋白VP27作为存在于鹅星状病毒表面的衣壳纤突蛋白，含有中和抗原表位，介导鹅星状病毒粒子与细胞表面受体结合[10]。……