摘要
为制备禽传染性支气管炎病毒(IBV) S2蛋白的单克隆抗体(MAb),本研究通过原核表达系统表达并纯化重组S2蛋白,将其免疫BALB/c小鼠并通过间接ELISA方法筛选到4株能够稳定分泌S2蛋白MAb的杂交瘤细胞株,分别命名为1A7、4F12、4A7、4D1。亚类鉴定结果显示4株MAb的重链均为Ig G1,轻链均为κ。采用间接ELISA法检测上清及腹水效价,结果显示MAb细胞上清效价均不低于1∶12 800,腹水效价均不低于1∶51 200。采用western blot检测制备的S2蛋白MAb与IBV感染鸡胚尿囊液后天然表达的S2蛋白的反应原性;采用间接免疫荧光试验(IFA)检测IBV感染非洲绿猴肾细胞(Vero)后天然表达的S2蛋白的反应原性。Western blot结果显示,感染IBV的鸡胚尿囊液中出现了90 ku左右的特异性条带;IFA结果显示,感染IBV的Vero细胞中出现绿色荧光。以上结果表明,制备的S2蛋白MAb能够与天然表达的S2蛋白反应,反应原性较强。利用间接ELISA检测MAb与S2蛋白的亲和力,结果显示4株MAb对S2蛋白均有良好的亲和力。利用一系列表达部分重叠的重组S2片段并经western blot鉴定4株MAb识别的抗原表位,结果显示,1A7、4F12、4A7 MAb识别的抗原表位是^(69)VQINCLQYVCGSSLECRKLFQQ^(90),4D1MAb识别的抗原表位是^(175)YKKCTAGPLGTLKDLI^(190)。采用IFA检测MAb的中和活性,结果显示,4株MAb均不具有中和活性。通过western blot检测MAb的反应谱,结果显示,MAb均能够与所选不同基因型(GI-1、GI-7、GI-13、GI-19、GI-22、GI-23) IBV发生特异性反应,表明制备的MAb反应谱较广。本研究为进一步探究IBV S2蛋白的功能及病毒致病机制奠定了基础。
To prepare monoclonal antibody(MAb)against avian infectious bronchitis virus(IBV)S2 protein,the recombinant S2 protein was expressed and purified by prokaryotic expression system.BALB/c mice were immunized with the recombinant S2 protein and four hybridoma cells lines that secreted S2 protein MAb,which were named 1A7,4F12,4A7 and 4D1,were screened by indirect ELISA.Subclass identification results showed that heavy chains of all four MAbs were IgG1 and the light chains were κ chain. Supernatant and ascites were tested by indirect ELISA. The titers of the supernatant of MAb cells were more than 1∶12800, and the titers of ascites were more than 1∶51200. Western blot was used to detect the reactivity of S2 MAbs with S2 protein naturally expressed in chicken embryo infected with IBV. Indirect immunofluorescence assay (IFA) was used to detect the reactivity of S2 protein MAbs with S2 protein naturally expressed in Vero cells infected with IBV. These results showed the prepared S2 protein MAb was able to react with the naturally expressed S2 protein and was highly reactive. All four MAbs had good affinity to S2 protein by indirect ELISA. Using a series of restructuring S2 fragment expression overlap and the western blot antigen epitope identification of four strains of MAb recognition, the results showed that 1A7, 4F12, 4A7 MAbs recognized ^(69)VQINCLQYVCGSSLECRKLFQQ^(90), 4D1 MAb recognized ^(175)YKKCTAGPLGTLKDLI^(190). IFA was used to detect the neutraliz- ation activity of MAbs, the results showed that none of the MAbs had neutralizing activity. western blot analysis showed that MAbs could specifically react with the selected IBV genotypes (GI-1, GI-7, GI-13, GI-19, GI-22, GI-23). These results laid the foundation for further exploring the S2 protein function and the mechanism of virus pathogenic.
作者
李忠斐
周建卫
张焕东
彭莉
颜焰
周继勇
廖敏
LI Zhong-fei;ZHOU Jian-wei;ZHANG Huan-dong;PENG Li;YAN Yan;ZHOU Ji-yong;LIAO Min(Key Laboratory of Animal Virology,Ministry of Agriculture and Rural Affairs,Hangzhou 310058,China;Center for Veterinary Sciences,Zhejiang University,Hangzhou 310058,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2023年第9期942-950,共9页
Chinese Journal of Preventive Veterinary Medicine
基金
浙江省基础公益计划项目(LGN22C18003)。
