水稻黑条矮缩病毒外壳蛋白基因的克隆、原核表达及抗体制备

Cloning and prokaryotic expression of coat protein gene of Rice blackstreaked dwarf virus and preparation of its polyclonal antibody

为探讨水稻黑条矮缩病毒（Rice black-streaked dwarf virus，RBSDV）的种群变异，采用RT-PCR方法从感染RBSDV山东分离物RBSDV-JN1的水稻中克隆该病毒的S10片段，并进行序列分析，进而构建包含RBSDV-JN1的CP基因的原核表达载体，导入大肠杆菌Escherichia coli BL21（DE3）诱导表达；并以表达的融合蛋白为抗原，制备病毒的多克隆抗体。结果显示：S10片段全长为1 801 bp，包含1个1 677 bp编码框，编码558个氨基酸的外壳蛋白（CP）；与GenBank已注册的19个RBSDV的CP基因序列相比较发现，病毒各分离物间核苷酸的序列相似性为90.5%~99.8%，氨基酸的序列相似性为95.9%~100.0%；地理位置较近的分离物间序列相似性较高，RBSDV种群分布呈现区域性差异。原核表达载体pET-RBSDV-CP经IPTG诱导，获得了分子量约为63 kD带有His标签的目的蛋白。用该蛋白制备的多克隆抗体经ELISA、Western blot和Dot-blot ELISA检测显示，效价为1：81 000，且具有良好的特异性。

In order to investigate the population variability of Rice black-streaked dwarf virus （RBSDV）, the full length cDNA of RBSDV Shandong isolate （RBSDV-JN1） segment 10 （S10） was cloned from the virus-infected rice samples by RT-PCR, and the sequence was analyzed. Coat protein gene （CP） of RBSDV-JN1 was constructed into the prokaryotic expression vector PET-30a （＋）. The fusion protein, obtained from the induced-expression with the prokaryotic expression vector pET-RBSDV-CP, was used as an antigen to prepare polyclonal antibody for the virus. The results of sequence analysis showed that the complete nucleotide sequence of S10 was 1 801 bp, containing a 1 677 bp open reading frame which encoded a coat protein of 558 amino acids. The similarity of nucleotide and predicted protein were 90.5%-99.8% and 95.9%-100.0%, respectively, compared with the CP gene of other 19 RBSDV isolates registered in GenBank. The sequence similarity between the isolates is higher in the species close in geographic positions, and the population distribution of the RBSDV showed regional difference. A 63 kD target protein with His-tag was obtained from prokaryotic expression induced by IPTG. The antibody was detected by ELISA, Western blot and Dot-blot ELISA, and the results showed that the titer of the antiserum was 1：81 000 with high specificity to RBSDV.