利用菊花B病毒外壳蛋白特异片段进行多克隆抗体的制备与分析

Preparation and Analysis of Polyclonal Antibody Using Specific Fragments of Chrysanthemum Virus B Coat Protein

菊花B病毒(CVB)是乙型线状病毒科(Betaflexiviridae)麝香石竹潜隐病毒属(Carlavirus)成员,在浙江杭白菊基地普遍存在。为了能够特异、快速、简便检测CVB,利用SWISS-MODEL分析CVB外壳蛋白(CP)的三维结构,选择暴露在CVB CP三维空间结构外部的片段,片段之间使用连接肽串联以提高柔性,重复4个片段,根据大肠杆菌密码子的偏爱性将其相应的DNA序列进行优化,将合成的特异DNA序列连接表达载体pET-28a(+),转化至Escherichia coli BL21(DE3)菌株,经IPTG诱导和Ni-NTA重力柱层析获得了纯化后大小约为14 kDa的融合蛋白;将其作为抗原免疫家兔制备抗血清,纯化获得相应抗体;对获得的抗血清和抗体进行酶联免疫吸附测定(ELISA)和蛋白免疫印迹(WB)检测。间接ELISA检测结果表明,512000倍稀释的抗血清可检测到1μg抗原。纯化后最终得到浓度为15 mg·mL的抗体,WB检测结果,1∶1000稀释后的抗体可检测500 pg抗原,且能够特异性结合CVB CP蛋白。本研究制备的多克隆抗体为检测CVB提供了便利,也为后续CVB检测试纸条的开发提供了技术支撑。

Chrysanthemum virus B(CVB)is a member of the genus Carlavirus,which belongs to the family Betaflexiviridae.The CVB widely infected the Chrysanthemum in the Hangbaiju plantation areas of Zhejiang Province,China.In the present study,the tertiary structure of coat protein(CP)by SWISS-MODEL were analyzed,then a specific partial sequence exposed outside of the tertiary structure of CVB CP were selected and four repeats of thus fragment were connected with linker peptides to improve flexibility,and set the corresponding DNA sequence according to the codon preference of Escherichia coli.The specific DNA sequence was ligated to the expression vector pET-28 a(+)and transformed into E.coli BL21(DE3)strain,fusion protein was induced by IPTG and the purified fusion protein with a size of about 14 kDa was obtained by Ni-NTA gravity column chromatography.The antiserum was obtained after immunizing New Zealand white rabbits with this antigen;and the corresponding antibody was obtained after purification of the antiserum from the rabbit;the obtained antiserum and antibodies were detected by ELISA and western blot.Interestingly,we observed that indirect ELISA assay with 512000 folds dilution of antiserum could detect 1μg of the fusion protein.Furthermore,purified antibody of 15 mg·mLwere obtained.In addition,the western blot assay indicated that the purified antibody of 1∶1000 dilutions could detect 500 pg of the purified fusion protein extracted from infected leaves.Our findings showed that the obtained polyclonal antibody can facilitate the detection of CVB and thus enable the development of rapid identification techniques.