Fusion Expression And Preparation Of Antibody Of Ascorbate Peroxidase Gene From Chinese Wild Vitis Pseudoreticulata In E.coli

Powdery Mildew is one kinds of plants fungus disease, originated from North American, now it spreads all over the world, course serious commercial loss to the grape products. China is the major product area of grape, and the Chinese Wild Vitis Species has excellent quality to resist disease. With the development of bio-technology, we can transform some gene resist disease into plants which essential to disease by gene engineering, thus cultivate some new strains link resist disease to excellent quality, it also avoids the weakness of traditional way. But, for the examination of transgenic plants and security evaluation, it is necessary to analyze the peculiarity of expression and biological function of purpose gene in vivo before transformed.According to the sequences of Ascorbate Peroxidase (APX) gene cloned from Chinese Wild Vitis Species, Design and synthesize special primers, we obtained the APX gene from cDNA by polymerase chain reaction(PCR) , and further cloned it into the pGEM-T easy vector. The cloned APX gene was cut out again with two restriction enzymes and was insert into the prokaryotic expression vector pGEX-4T-1, then transferred into E.coli BL21.GST-APX fusion protein was obtained after being induced by IPTG, and it counts up to 35% of the total protein in E.coli. The fusion protein has two different kinds of forms-soluble protein and inclution body. We have obtained high purity fusion protein after the soluble protein was purified by the GST affinity resin. After injecting rabbit, the polycloned antibody was produced. The results are as follows1 Recombinant cloning vector of pGEM-T/APX was successfully constructed by ligating the APX gene into pGEM-T easy vector and transformed into DH5α...