马铃薯卷叶病毒缺失突变CP基因的原核表达及抗血清的制备  

Prokaryotic Expression of Deletion Mutation CP Gene of Potato leafroll virus and Preparation of Antiserum

作　　者：隋炯明[1] 何心凤[1] 郭真[1] 李广存[2] 王晶珊[1] 郭宝太[1] 

机构地区：[1]青岛农业大学生命科学学院,山东省植物生物技术高校重点实验室,山东青岛266109 [2]山东省农业科学院蔬菜研究所,济南250100

出　　处：《园艺学报》2012年第10期1949-1957,共9页Acta Horticulturae Sinica

基　　金：山东省自然科学基金项目(Y2008D43)

摘　　要：利用RT-PCR扩增马铃薯卷叶病毒(PLRV)外壳蛋白(CP)基因(PLRV-CP),回收大小约630bp的特异性扩增片段并进行T-A克隆,测序表明该基因长度为627bp,与已报道的36个PLRV-CP基因的核苷酸序列的同源性大于96%。以pBAD/Thio-TOPO为起始载体,构建了PLRV-CP基因的原核表达载体pBAD-LRCP。以pBAD-LRCP为模板,用PCR法删除了该基因富含精氨酸稀有密码子的第52～177核苷酸,获得了PLRV缺失突变CP基因的原核表达载体pBAD-LRCP-126。用阿拉伯糖诱导工程菌TOP10(pBAD-LRCP-126),获得了34kD的诱导表达的融合蛋白(重组CP)。用镍离子亲和层析法从包涵体中纯化出了高纯度的重组CP,用纯化的重组CP作抗原免疫家兔获得了PLRV特异性的抗血清,间接ELISA检测显示效价为1︰12800。本研究结果为利用重组CP作抗原大量制备PLRV抗血清奠定了基础。Potato leafroll virus（PLRV）coat protein（CP）gene（PLRV-CP）was amplified by RT-PCR,and the specific product about 630 bp was cloned through T-A ligation. DNA sequencing showed that its length was 627 bp,and the homology of nucleotide sequences between the cloned PLRV-CP gene and other 36 previously published ones was above 96%. Using pBAD/Thio-TOPO as initial vector,prokaryotic expression vector of PLRV-CP gene was constructed and named pBAD-LRCP. DNA fragment（52–177 nucleotides）rich in rare codons of arginine was deleted from PLRV-CP gene by PCR using pBAD-LRCP as template,the yielding recombinant plasmid pBAD-LRCP-126 was indeed the prokaryotic expression vector of deletion mutation CP gene of PLRV. After induction of the engineered strain TOP10（pBAD-LRCP-126）with arabinose,the mutation gene was successfully expressed and 34 kD fusion protein（the recombinant CP）was obtained. High-purity recombinant CP was purified from inclusion bodies with nickel affinity chromatography column,and the purified recombinant CP was used as antigen to immune rabbits. Antiserum specific to PLRV was obtained,its titer was 1︰12 800 in indirect ELISA detection. This research has laid the foundation for preparation of antiserum against PLRV by using recombinant CP as antigen in a large scale.

关 键 词：马铃薯卷叶病毒 CP基因 缺失突变 原核表达 抗血清 

分 类 号：S432.41[农业科学—植物病理学] S532[农业科学—农业昆虫与害虫防治]