新疆辣椒CMV基因组序列分析与CP基因多克隆抗体制备

doi:10.13560/j.cnki.biotech.bull.1985.2017-0206

摘要/Abstract

摘要： 克隆新疆辣椒LJ-10 CMV基因组片段，并进行序列分析，构建CMV CP基因的原核表达载体，并制备CP基因的多克隆抗体。利用RT-PCR技术，克隆新疆辣椒LJ-10 CMV的RNA1（3 357 nt）、RNA2（3 042 nt），RNA3（2 212 nt）全基因组片段，与GenBank上登陆的CMV亚组IA、亚组IB、亚组Ⅱ分离物进行序列分析和系统进化树分析。将LJ-10 CMV CP基因克隆到原核表达载体pET-22b中，在大肠杆菌BL21（DE3）中表达纯化，以纯化的重组蛋白为抗原免疫兔子，制备CMV特异性抗血清，并进行间接ELISA和Western blotting分析。结果显示，成功克隆了新疆辣椒LJ-10 CMV的RNA1、RNA2、RNA3全基因组片段，序列分析及系统进化树分析表明，该分离物属于CMV亚组IB。成功构建了LJ-10 CMV CP基因的原核表达载体pET-CMV-CP，在大肠杆菌BL21（DE3）中经IPTG诱导获得了与预期大小一致的分子量约为27 kD的重组蛋白，制备了CMV的特异性抗血清。间接ELISA和Western blotting分析表明，制备的抗血清效价为1：500-1 000。新疆辣椒LJ-10 CMV分离物属于CMV亚组IB，成功构建了LJ-10 CMV CP基因的原核表达载体，制备了相应的多克隆抗体。

关键词: 黄瓜花叶病毒, 序列分析, 外壳蛋白, 原核表达, 蛋白质印迹检测

Abstract: This work aims to clone gene fragment of Cucumber mosaic virus（CMV）from pepper LJ-10 in Xinjiang，to analyze the sequence，construct the prokaryotic expression vector of CMV CP gene，and to prepare CP’s polyclonal antibodies. The complete genome fragments of RNA1（3 357 nt），RNA2（3 042 nt），and RNA3（2 212 nt）were cloned by RT-PCR，and compared with different CMV isolates from CMV subgroup IA，subgroup IB and subgroup II in the GenBank by sequence analysis and phylogenetic tree analysis. The CP gene of CMV in LJ-10 was cloned into prokaryotic expression vector pET-22b and expressed in Escherichia coli BL21（DE3），and the expressed proteins were purified. Rabbit was immunized with the purified protein to prepare the antiserum with CMV-specificity，and detected by indirect ELISA test and Western blot. As results，the genomes of RNA1，RNA2，and RNA3 were successfully cloned，the sequence analysis and phylogenetic tree analysis showed that the isolates belonged to CMV subgroup IB. The prokaryotic expression vector pET-CMV-CP was successfully constructed and expressed as a 27 kD recombinant protein in E. coli BL21（DE3）by IPTG induction，and whose molecular weight was identical to the expected one. The indirect ELISA test and Western blotting showed that the antiserum’s titer was 1：500-1 000. In conclusion，the CMV isolate from LJ-10 belongs to CMV subgroup IB. The prokaryotic expression vector of CMV CP gene from LJ-10 is successfully constructed，and the corresponding polyclonal antibody is prepared.

Key words: Cucumber mosaic virus, sequence analysis, coat protein gene, prokaryotic expression, Western blot

周东, 刘贞, 刘丽, 许鹏程, 郑银英. 新疆辣椒CMV基因组序列分析与^CP基因多克隆抗体制备[J]. 生物技术通报, 2017, 33(8): 88-94.

ZHOU Dong, LIU Zhen, LIU Li, XU Peng-cheng, ZHENG Yin-ying. Genome Sequencing of CMV Isolated from Pepper in Xinjiang and Polyclonal Antibody Preparation of ^CP Gene[J]. Biotechnology Bulletin, 2017, 33(8): 88-94.

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