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利用RT-PCR技术扩增得到了葡萄卷叶伴随病毒-3(Grapevine leafroll associated virus-3,GLRaV-3)中国分离物的外壳蛋白(coat protein,cp)基因。序列分析结果表明,cp基因的长度为942bp,与已报道的其它GLRaV-3分离物的cp核苷酸相似性为91%～99%,编码的氨基酸相似性为95%～100%。将此基因克隆到原核表达载体pET-28a(+)上,转化大肠杆菌BL21(DE3)plysS后用终浓度为1mmol/L的IPTG进行诱导表达,SDS-PAGE及Western blotting分析表明,cp在大肠杆菌中可表达出分子量约为35kDa的蛋白。纯化表达产物后免疫家兔制备抗血清。A蛋白酶联免疫吸附测定(PAS-ELISA)及斑点免疫结合测定(DBIA)结果显示,制备的特异抗血清可用于检测田间感病葡萄样品中的GLRaV-3。
The coat protein (cp) gene of Chinese isolates of Grapevine leafroll associated virus-3 (GLRaV-3) was amplified by RT-PCR. Sequence analysis showed that the length of cp gene was 942bp. The similarity of cp nucleotide with the other reported GLRaV-3 isolates was 91% -99%, and the amino acid similarity was 95% -100%. The gene was cloned into the prokaryotic expression vector pET-28a (+) and transformed into E. coli BL21 (DE3) plysS. The recombinant plasmid was induced with IPTG at a final concentration of 1 mmol / L. SDS- PAGE and Western blotting analysis showed that cp was expressed in the large intestine Bacillus can express a molecular weight of about 35kDa protein. After purification of the expressed product, rabbits were immunized to prepare antiserum. A proteinase-linked immunosorbent assay (PAS-ELISA) and dot immunobinding assay (DBIA) results showed that the prepared specific antiserum could be used to detect GLRaV-3 in field-sensitive grape samples.