Cloning And Construction Of Prokaryotic Expression Vector Of Coat Protein Gene Of Apple Stem Pitting Virus In Korla Pear And Its Expression In Escherichia Coli

A lot of trees in commercial fruit yards are infected commonly by viruses in China. Along with the continual enlargement of planting area towards fruit trees and the extension of their planting time, harmness from the viruses is more and more obvious and more attentions are paid by scientists and researchers, producers and businessmen of fruit trees. Foveavirus is a novel genus of plant viruses, typified by apple stem pitting virus (ASPV). Apple stem pitting virus (ASPV) is widely spread in commercial apple cultivars which are symptomlessly infected. Pear ring pattern mosoic virus(PRPMV), Pear vein yellow virus (PVYV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are distributed to different Taxonomic, but it is reportted by Jelkmann that these viruses are caused by ASPV. ASPV has many isolates because it spreads all over the world. Cloning and sequencing of coat protein of Apple stem pitting virus in Xinjiang Korla pear, and constructing the prokaryotic expression vector of coat protein, getting its expression in Escherichia coli, this essay studies the ORF5 last.1. Use young stems and leaves of Korla pear with ASPV as material, adopt the method used by EU095327 published in GenBank to synthesized primers, and nucleotide segments of coat protein are amplified. Target segments of coat protein are cloned in the vector, by enzyme digestion and sequencing, nucleotide segments are the ones that are needed. Sequncing of coat protein of apple stem pitting virus in Xinjiang Korla pear with MHczAp,ASPV isolate24 and ASPV isolate38, the homology is 81%-83%, and the homology of amino acid is 70%-71%.2. Through enzyme digestion of vector connected by restriction fragment, correct products are obtianed, then connect it with expression vector. The recombinant plasmid pET-3aCP is then transformed into BL21(Escherichia coli), and induced to express ASPV fusion protein by IPTG at 1～3h respectively, the expression product could react with specific antibody, and the relative molecular weight of the expression product is identical to expected value. In addition, the expression of this genus is limitted by time, the effective time is 1h-3h, and the products decrease after 4h. The molecular weight of ASPV CP is about 43.7kDa, the drift of the coat protein expression cassette, resulting in different expression of amino acids, but the weight of production is identical, and consistent with the recombinant protein molecular.This paper includes the study of coat protein of Apple Stem Pitting Virus (ASPV) in Korla pear, through cloned, established the vector and Expression in Escherichia coli, the isolates of coat protein of apple stem pitting virus in Xinjiang Korla pear are obtained and compared with other isolates.