来源于桃和苹果的苹果褪绿叶斑病毒的部分分子生物学特性和cp基因的原核表达

 从桃和苹果上分离得到苹果褪绿叶斑病毒ACLSV-HBP和ACLSV-C2个分离物,采用RT-PCR法进行扩增,所获扩增片段经序列测定,其全长分别为1768nt(ACLSV-HBP)和1751nt(ACLSV-C)。这2个分离物扩增片段全长的同源性为83%,mp基因片段核苷酸和推导编码氨基酸序列同源性分别为82.6%和87.1%;cp基因均由582nt组成,其核苷酸和推导编码氨基酸序列同源性分别为87.8%和95.9%。将2个分离物的cp基因与已报道ACLSV分离物进行序列同源性比较,结果显示ACLSV-HBP与SX/2的cp基因核苷酸序列及推导编码氨基酸序列同源性最高,分别为94.0%和96.4%。将ACLSV-HBP分离物的cp基因克隆到原核表达载体pGEX-KG,在大肠杆菌BL21(DE3)中诱导表达,SDS-PAGE分析表明,融合蛋白大小约为46kDa。Western-blot分析表明,该基因在大肠杆菌内得到高效表达,融合蛋白具有抗原性。

Partial molecular characterization of Apple chlorotic leaf spot virus from peach and apple trees and prokaryotic expression for cp gene

Two isolates of Apple chlorotic leaf spot virus were obtained from apple(ACLSV-C)and peach(ACLSV-HBP)trees grown in China.Nucleic acid fragments of size in 1 768 nt and 1 751 nt from ACLSV-HBP and ACLSV-C,including 3'-end region of mp gene,582 nt CP-coding region and 5'-end region of 3'-NCR,were cloned and sequenced,respectively.Identity of the two nucleic acid fragments was 83%.The identities of partial nucleotide and deduced amino acid sequences from ACLSV-HBP and ACLSV-C were 82.6% and 87.1% for mp genes,87.8% and 95.9% for cp genes.The sequence data obtained in this study were compared with reported sequences of ACLSV isolates from different sources.The results showed that ACLSV-HBP had a similarity of 94.0% at nucleotide acid level and 96.4% at deduced amino acid level to SX/2.The recombinant plasmid pGEX-KG-CP containing cp gene from ACLSV-HBP isolate was transformed into E.coli BL21(DE3).A 46 kDa fusion protein was expressed under the induction of IPTG at 0.4 mmol/L.Western-blot analysis showed that the cp gene was expressed effectively,and the fusion protein could give positive reaction with polyclonal antibody raised against ACLSV particles.