Cloning and expression in e. coli of apple stem pitting virus cp gene in pyrus bretschneideri rehd.cv.yali | 鸭梨苹果茎痘病毒外壳蛋白基因克隆及原核表达

Chino. 摘　　要：为了进一步明确苹果茎痘病毒（Apple stem pitting virus,ASPV）的分子变异及株系分化,制备相应特异性抗血清。本研究以库尔勒地区种植的感染ASPV的鸭梨（Y）枝条韧皮部为试材,采用RT-PCR技术扩增ASPVCP基因,克隆、测序,获得了鸭梨分离物外壳蛋白（ASVPCP-Y）基因,将该CP基因连接到表达载体PET-28a上,转化到大肠杆菌BL21（DE3）,1mmol/LIPTG诱导表达,SDS-PAGE电泳分析。研究结果表明,获得的鸭梨ASPVCP基因全长1194bp,推测编码397个氨基酸,与已报道的ASPV分离物的氨基酸序列同源性为70%左右。进化分析结果显示,ASPVCP基因的分离物可聚为三个类群：第一类群的寄主为苹果,第二类群的寄主为梨（除了NC_003462,苹果）,ASPVCP-Y归入第三类群。CP基因在大肠杆菌中诱导表达,其融合蛋白分子量约为42kD。克隆的鸭梨ASPVCP基因及构建的原核表达载体为制备ASPV重组CP基因多克隆抗体及ASPV的分子生物学的进一步研究奠定了基础。

Inglés. In order to clarify evolutionary relationship and genetic diversity of apple stem pitting virus, and preparation the corresponding special antiserum. In this study, total RNA was extracted from phloem tissue of Ya （Y） pear which infected by ASPV and used as template for cDNA synthesis. The coat protein （CP） gene of ASPV was cloned and sequenced. The CP gene of Ya pear was cloned into expression vector PET-28a, and transformed into E. coli BL21 （DE3）, and SDS-PAGE electrophoresis analysis. The results showed that the complete CP gene consists of 1 194 nucleotides and encodes a polypeptide of 397 amino acid （aa）. Comparison of the amino acid sequences of Ya pear CP gene with other ASPV isolates showed approximately 70% similarity. Phylogenetic tree showed that all isolates of the coat protein （CP） genes of ASPV isolates at aa sequence revealed three groups. All ASPV isolates from apple were clustered to group I, whereas pear were clustered to groups Ⅱ （except NC_003462） and the Ya pear were clustered to group Ⅲ. Results of SDS-PAGE showed that specific expression of a 42 kD fusion protein was achieved by the inducing of 1 mmol/L IPTG. The complete CP gene and prokaryotic expression vector of Ya pear provided additional baseline data for preparation recombinant polyclonal antibody of ASPV and further study of ASPV molecular biology.