传染性法氏囊病病毒新型变异株VP2蛋白原核表达及免疫效果评价  

Prokaryotic expression and immune effect evaluation of VP2 protein of a novel variant infectious bursal disease virus strain

作　　者：马景霞 王常伟 吴洪才 何吉鑫 牛晓赛 段志强[3] 朱杰 MA Jing-xia;WANG Chang-wei;WU Hong-cai;HE Ji-xin;NIU Xiao-sai;DUAN Zhi-qiang;ZHU Jie(Binzhou Argo-tech Extension Center,Binzhou,Shandong 256600,China;Shandong Binzhou Wohua Biological Engineering Co.,Ltd.,Binzhou,Shandong 256600,China;College of Animal Sciences,Guizhou University,Guiyang,Guizhou 550025,China)

机构地区：[1]滨州市农业技术推广中心,山东滨州256600 [2]山东滨州沃华生物工程有限公司,山东滨州256600 [3]贵州大学动物科学学院,贵州贵阳550025

出　　处：《南方农业学报》2023年第6期1876-1883,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31960698);滨州沃华生物基金项目([WH]201901QB01-02)。

摘　　要：【目的】构建传染性法氏囊病病毒新型变异株(vaIBDV)VP2蛋白原核表达质粒,并明确VP2蛋白的免疫原性,为vaIBDV亚单位疫苗的开发提供技术支持。【方法】从IBDV新型变异株(BZ)中扩增VP2基因片段,亚克隆至pET-28a(+)载体上构建重组原核表达质粒,并转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达后通过SDS-PAGE和Western blotting进行鉴定,并以vaIBDV阳性血清进行琼扩效价测定;经Ni-NTA亲和层析纯化及适当浓缩后,制备乳化疫苗并免疫21日龄SPF鸡,通过安全性检验、抗体效价测定及攻毒保护评价进一步验证融合蛋白VP2的免疫原性。【结果】在25℃下以IPTG进行诱导表达,获得的融合蛋白VP2主要呈可溶性表达,大小约40 kD,且表达上清液中的融合蛋白VP2能与vaIBDV阳性血清发生特异性反应,其琼扩效价可达1∶32;经Ni-NTA亲和层析纯化及适当浓缩后,融合蛋白VP2的琼扩效价高达1∶256。以融合蛋白VP2为抗原制备的亚单位疫苗稳定性好,安全性高,无免疫引起的不良反应,剖检也未发现接种部位有明显损伤、局部炎症或疫苗吸收不良等现象。免疫21 d后鸡血清琼扩效价平均值达1∶76.8,而血清抗体效价均在1∶20000以上,表明以融合蛋白VP2制备的疫苗可刺激机体产生强烈的体液免疫反应,抗体阳性率达100%;使用IBDV BZ株攻毒后7 d剖检观察鸡法氏囊病变情况,结果显示免疫组鸡只均未发病,其法氏囊也无明显萎缩现象,即免疫保护率达100%。【结论】通过原核表达系统能成功实现vaIBDV VP2蛋白的可溶性表达,且获得的融合蛋白VP2免疫原性良好,能对vaIBDV提供100%的免疫保护效果,为开发vaIBDV亚单位疫苗提供了技术支持。【Objective】This paper constructed the prokaryotic expression plasmid of VP2 protein of a novel variant of infectious bursal disease virus(vaIBDV),and clarified the immunogenicity of VP2 protein,so as to provide technical support for the development of vaIBDV subunit vaccine.【Method】The VP2 gene fragment was amplified from vaIBDV isolate(BZ strain)and cloned into vector pET-28a(+)to recombine prokaryotic expression plasmid,then transformed into the Escherichia coli BL21(DE3)competent cell,and induced by IPTG.The expressed products were identified by SDSPAGE and Western blotting,and the titer was determined by vaIBDV-positive serum through agar-gel precipitation(AGP).After purification by Ni-NTA affinity chromatography and proper concentration,the emulsified vaccine was prepared and used to immunize 21-day-old SPF chickens.The immunogenicity of the fusion protein VP2 was further verified by safety test,antibody titer determination and immune challenge experiment.【Result】The fusion protein VP2 was mainly expressed in soluble form at 25℃induced by IPTG,and the size was about 40 kD.The fusion protein VP2 in the expression supernatant could specifically react with vaIBDV positive serum,and its agar diffusion titer could reach 1∶32.After purification by Ni-NTA affinity chromatography and appropriate concentration,the agar diffusion titer of the fusion protein VP2 was as high as 1∶256.The subunit vaccine prepared with the fusion protein VP2 as the antigen had good stability,high safety,and no adverse reactions caused by immunization.No obvious damage,local inflammation,and poor vaccine absorption were found at the inoculation site.After 21 d of immunization,the average agar diffusion titer of chicken serum was 1∶76.8,and the serum antibody titer was above 1∶20000,indicating that the vaccine prepared with the fusion protein VP2 could stimulate the body to produce a strong humoral immune response,and the antibody positive rate was 100%.The pathological changes of bursa of Fabricius in chickens were ob

关 键 词：传染性法氏囊病病毒(IBDV) 新型变异株 VP2蛋白 亚单位疫苗 免疫效果 

分 类 号：S852.659.4[农业科学—基础兽医学]