马铃薯Y病毒脉坏死株系(PVY^N)外壳蛋白基因的克隆、表达及其间接ELISA方法的建立  

Clone, Expression of Coat Protein Gene of Potato Virus Y-vein necrosis (PVY^N) and its Establishment of Indirect ELISA Method

作　　者：高方方[1] 吴忠义[2] 黄丛林[2] 孙惠生 曾申明[1] 

机构地区：[1]中国农业大学动物科技学院,北京100094 [2]北京市农林科学院生物中心,北京100097 [3]北京中际国华马铃薯开发中心,北京102100

出　　处：《中国农学通报》2008年第3期82-86,共5页Chinese Agricultural Science Bulletin

摘　　要：【目的】制备马铃薯Y病毒脉坏死株系(PVYN)多克隆抗体,建立间接ELISA法用于检测PVYN病毒。【方法】依据PVYN的CP基因序列设计引物,利用RT-PCR方法获得CP基因并连接构建到原核表达载体,进行原核表达,以纯化的重组蛋白作为抗原免疫新西兰大白兔,获得PVYN的抗血清并纯化。【结果】测序结果与其他已知序列比较,PVYNCP基因的核苷酸同源性95%,推断氨基酸的同源性达98%。以纯化蛋白为抗原进行免疫,成功获得了PVYN外壳蛋白抗血清,纯化后的IgG采用间接ELISA法检测抗原,抗体效价达1:500,使用该抗体稀释度检测感染植株,结果呈阳性。【研究结论】通过分子生物学途径获得了PVYN的多克隆抗体,建立的间接ELISA方法可以用于PVYN的病毒检测。[Objective]The polyclonal antibody of potato virus Y-vein necrosis （PVYN） was produced, and its establishment of indirect ELISA method was used for detection of PVYN; [Method]The PVYN coat protein （CP） gene was obtained by RT-PCR with the specific primers. Amplicons were cloned into pGEX-T vector, then the proper insertion of the cDNA and fidelity of sequences were verified by DNA sequencing. The CP gene expression vector was constructed and expressed in BL21 （DE3）. The antiserum was produced by immunizing New Zealand white rabbits with the purified recombined protein and the IgG purified from obtained antiserum was used to detect PVYN by indirect ELISA;[Results]The results showed that it had high homology （98% and 95%） in nucleotide acid and amino acid compared to other genes reported, respectively. Proteins fused with 6-His tag were expressed in BL21（DE3） and purified by affinity column successfully. PVYN was detected by indirect ELISA, and its result was positive; [Conclusion]The polyclonal antibody of PVYTM was generated by molecular biology method,and its establishment of indirect ELISA method can be used to detect PVYN.

关 键 词：马铃薯Y病毒脉坏死株系 原核表达 多克隆抗体 ELISA 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治] TS207.3[农业科学—植物保护]