侵染猕猴桃的柑橘叶斑驳病毒外壳蛋白多克隆抗体的制备及检测应用

Polyclonal antibody preparation and application of coat protein of citrus leaf blotch virus infecting kiwi plant

柑橘叶斑驳病毒(Citrus leaf blotch virus,CLBV)是β线形病毒科(Betaflexiviridae)柑橘病毒属(Citrivirus)的代表种,前期研究表明CLBV在陕西栽培猕猴桃中广泛发生。为进一步简化CLBV的检测,并探究其致病机制,通过RT-PCR扩增得到CLBV猕猴桃分离物外壳蛋白(coat protein,cp)基因,将其插入原核表达载体pET-30a(+),导入E.coli BL21(DE3)感受态细胞,成功表达出约59 kDa的融合蛋白。用纯化后的融合蛋白对大白兔进行皮下多点免疫注射,获得的抗血清经纯化浓缩后,最终得到浓度为3.78 mg·mL-1的多克隆抗体。将抗体1∶1000稀释后可检测500 pg抗原,使用Dot-ELISA可直接检测田间猕猴桃样品。多克隆抗体的制备为CLBV的检测提供了便捷,也是后续研究CLBV致病机理的重要工具。

Citrus leaf blotch virus(CLBV)is the type species of the genus Citrivirus,family Betaflexiviridae.Previous studies have shown that CLBV occurs widely in kiwi plants cultivated in Shaanxi.To further simplify the detection of CLBV and explore its pathogenic mechanism,the coat protein(cp)gene of CLBV isolates from kiwi was obtained by RT-PCR amplification,and inserted into the prokaryotic expression vector pET-30 a(+),which was transferred into the E.coli BL21(DE3)cells.A 59 kDa fusion protein was successfully expressed.The purified fusion protein was used for subcutaneous multi-point immunization of rabbits,and the obtained antiserum was purified and concentrated to finally obtain a polyclonal antibody with a concentration of 3.78 mg·mL-1.After diluting antibody 1∶1000,500 pg of antigen can be detected,and Dot-ELISA can directly be used for on-field virus detection in kiwi samples.The preparation of polyclonal antibodies provides convenience for the detection of CLBV,and is also an important tool for the subsequent study of the pathogenic mechanism of CLBV.