传染性法氏囊病病毒AH1株vp2基因在昆虫细胞中的表达与应用  

Recombinant Vp2 protein of infectious bursal disease virus AH1 strain expressed in insect cells: a vaccine candidate

作　　者：欧阳伟[1,2] 王永山[1] 周宇[1,2] 张海彬[2] 唐雨德[3] 

机构地区：[1]江苏省农业科学院兽医研究所农业部动物疫病诊断与免疫重点开放实验室国家兽用生物制品工程技术研究中心,南京210014 [2]南京农业大学动物医学院,南京210095 [3]南京军区军事医学研究所,南京210002

出　　处：《生物工程学报》2010年第5期595-603,共9页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30571371);江苏省自然科学基金(Nos.BK2008352;BK2009041);江苏省农业科技创新基金(No.CX(08)106)资助~~

摘　　要：将近期引起传染性法氏囊病(IBD)免疫预防失败的传染性法氏囊病病毒(IBDV)vp2基因,定向克隆入杆状病毒表达系统的供体质粒pFastBacHTA中,构建重组供体质粒pFastBacHTA-VP2,转化Escherichia coli DH10Bac感受态,筛选重组杆状病毒表达质粒pBac-VP2。用pBac-VP2转染Sf9昆虫细胞,获得重组杆状病毒vBac-VP2。对重组杆状病毒vBac-VP2感染的Sf9细胞,用间接免疫荧光试验(IFA)检测,具有特异性荧光;用IBDV抗体夹心ELISA检测,呈阳性反应,抗原效价达到1.6×103;用Western blotting分析,在53kDa处出现一条特异蛋白条带;电镜观察,重组Vp2蛋白能够自组装成病毒样颗粒,在感染细胞中发现了"包涵体样"结构。用HisTrap HP亲和层析柱纯化的重组Vp2蛋白作为包被抗原,建立的IBDV抗体间接ELISA检测方法具有良好的特异性。用重组杆状病毒感染的Sf9昆虫细胞裂解物,免疫2周龄SPF鸡,一次免疫14d后,ELISA检测抗体效价为8×102,中和抗体效价为1106,攻毒实验的存活率为30%;二次免疫14d后,ELISA抗体效价为3.2×103,中和抗体效价为2536,存活率为100%。在实验观察7d内,重组Vp2蛋白免疫保护鸡未显任何临床症状和病理变化,法氏囊/体重比高于对照组(P<0.05)。本实验制备的病毒样颗粒重组Vp2蛋白在研制新型IBD基因工程疫苗和检测试剂方面显示出了应用前景。Protective immune response of the available IBD vaccine is insufficient to fully protect against the prevailing strain of the infectious bursal disease virus （IBDV）. Such a vaccination escape IBDV field isolate idenfied from Anhui province of China in December 2007, where IBD broke out at 2 weeks post vaccination. The IBDV vp2 gene was cloned into pFastBacHTA donor plasmid, followed by generation of the recombinant bacmid DNA pBac-VP2. The latter was used to transfect insect cell Sf9 with Lipofectamine to produce recombinant baculovirus vBac-VP2. The Sf9 cells infected with vBac-VP2 were stained positive against IBDV antibody using the indirect immunofluorescence assay （IFA）, which was also confirmed by the detection of IBDV Vp2 protein in the infected Sf9 cells by IBDV sandwich ELISA. Western blotting revealed that the calculated protein of approximately 53 kDa was in the expressed in the insect cells. Moreover, virus-like particles （VLPs） and ＂inclusion body-like＂structure in the infected Sf9 cells were observed under electron microscopy. We further developed an indirect ELISA for the detection of the IBDV antibodies, which was specific and sensitive. In addition, the lysates of vBac-VP2 infected cells was used to immunize 2-week-old SPF chickens, followed by challenging with the virulent IBDV, the survival rate was 30% at 14 days post primary immunization, however, the survival rate was 100% at 14 d after the booster vaccination. The ELISA antibody titers was up to 3.2×10^3 and neutralization antibody titer was 2536, significantly higher than those of one-shot vaccination, 8×10^2 and 1106, respectively. The immunized chickens did not show any clinical signs and histopathological changes of infection in 7-days trial time. The bursa/body-weight ratios were higher than those of the unimmunized control （P 〈0.05）. The virus-like-particle recombinant Vp2 protein expressed in insect cells promises to be a novel subunit vaccine and diagnostic reagent candidate for IBDV.

关 键 词：传染性法氏囊病病毒 VP2基因 病毒样颗粒 包涵体样结构 间接ELISA 免疫实验 

分 类 号：S852.65[农业科学—基础兽医学]