【摘要】 利用RT-PCR获得苹果褪绿叶斑病毒(ACLSV)库尔勒香梨分离物外壳蛋白(CP)基因。经序列分析表明,库尔勒香梨(PyrussinkiangensisYū)分离物CP基因由582个核苷酸组成,编码一个由193个氨基酸组成的蛋白质(GenBankaccessionNo.AY669389)。将其克隆到表达载体pET-28a(+)中,转化大肠杆菌(Escherichiacoli)BL21,筛选得到阳性克隆pET-ACP。用IPTG诱导表达,SDS-PAGE电泳分析表明,预期的22kD外壳蛋白在大肠杆菌中得到大量表达。回收原核表达的目的蛋白,免疫家兔,制备了ACLSV外壳蛋白的特异性抗血清。Westernblot分析表明,所制备的抗血清可用于检测样品中的ACLSV。更多还原

【Abstract】 Coat protein(CP) gene of Apple chlorotic leaf spot virus (ACLSV) isolated from Kuerle pear (Pyrus sinkiangensis Yū)was obtained by RT-PCR. Sequence analysis showed that the CP gene was 582 nt long, encoded a predicted protein of 193 amino acids(GenBank accession No.AY669389). It was constructed into expression vector pET-28a(+) and the recombinant plasmid pET-ACP was obtained. Escherichia coli BL21 transformed with the recombinant plasmid pET-ACP was induced by IPTG to specifically express the 22 kD protein at high level. The expressed protein was purified and the antiserum against the protein was raised in the rabbit. Western blotting analysis revealed that the antiserum could be used for detection of ACLSV.更多还原