桑花叶卷叶病相关病毒外壳蛋白基因的原核表达与免疫检测  

Prokaryotic Expression and Immunological Detection of Coat Protein-encoding Gene of Mulberry Mosaic Leaf Roll-associated Virus( MMLRa V)

作　　者：卢全有[1,2] 吴祖建[1] 夏志松[2] 谢联辉[1] 

机构地区：[1]福建省植物病毒学重点实验室福建农林大学,福州350002 [2]农业部蚕桑遗传改良重点实验室江苏科技大学,江苏镇江212018

出　　处：《蚕业科学》2015年第2期218-225,共8页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划"973"项目(No.2014CB1384-02)

摘　　要：桑花叶卷叶病相关病毒(mulberry mosaic leaf roll-associated virus,MMLRa V)是从发生桑花叶卷叶病的桑树植株中分离的一种线虫传多面体病毒属(Nepovirus)病毒,推测其外壳蛋白的分子质量约59 k D。以感染MMLRa V的桑树叶片的c DNA为模板,采用RT-PCR获得编码该病毒外壳蛋白C端38 k D的核苷酸序列片段,暂命名为CP38。将CP38连接到原核表达载体构建重组表达载体p GEX-4T-CP38,转化大肠杆菌BL21(DE3)并经1 mmol/L IPTG诱导,使融合蛋白GST-CP38高效表达。通过SDS-PAGE胶回收原核表达的融合蛋白,以其为抗原免疫新西兰大白兔制备MMLRa V的多克隆抗血清,采用间接ELISA法测定该抗血清的效价为1∶2 048,Western blot检测其能够与MMLRa V发生特异性反应。用制备的抗血清对感病桑树叶片粗汁液煮沸后离心获得的上清液进行间接ELISA检测,MMLRa V的检出率达到84%,可以应用于大田病样的检测。Mulberry mosaic leaf roll-associated virus（ MMLRa V）,which was isolated from mulberry tree infected with mulberry mosaic leaf roll disease,is a novel member of the genus Nepovirus. It was predicted that the molecular weight of MMLRa V coat protein was approximately 59 k D. In this paper,the partial nucleotide fragment（ tentatively named as CP38） encoding a 38 k D protein at C-terminal of MMLRa V coat protein was amplified from mulberry leaves infected with MMLRa V by RT-PCR. The amplified CP38 fragment was inserted into the prokaryotic expression vector for construction of recombinant plasmid p GEX-4T-CP38 and then transformed into Escherichia coli BL21（ DE3）. The fusion GST-CP38 was highly expressed in E. coli BL21 after induction with isopropyl-3-β-D-1-thiogalactoside（ IPTG） at the final concentration of1 mmol / L. The fusion protein was recovered by SDS-PAGE and was used as antigen to generate polyclonal antiserum in rabbit. The titer of the obtained antibody was 1∶ 2 048 measured by indirect ELISA. Western blotting analysis indicatedthat the polyclonal antiserum displayed good specific reactions against MMLRa V. The obtained antiserum was used to detect centrifugal supernatant of the boiled-treatment crude extract of MMLRa V-infected mulberry leaf by indirect-ELISA. The results showed that the detection rate of MMLRa V reached 84%,suggesting that the obtained polyclonal antiserum could be applied for MMLRa V detection of mulberry field samples.

关 键 词：桑花叶卷叶病相关病毒 外壳蛋白基因 原核表达 抗血清 酶联免疫吸附试验间接法 

分 类 号：S888.71[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]