Cloning, Sequence Analysis And Expression Of The Structure Protein Gene Of AEV

According to Three pair of premier were designed according to specific structural protein VP0, VP1 and VP3 gene sequence of Avian Encephalomyelitis virus(AEV) 1143 strain in represented articles. By RT-PCR the gene fragment from brains and guts of SPF chicken embryos, which were infected with AEV Van-Roekel strain., were cloned in vitro. After harvesting and purifying, VP0, VP1, VP3 gene fragment were connected into PMD18-Teasy Vector linearized, and then these were transferred into JM109 cells. Positive clones were.selected by LB plates with Ampicillin and proliferated, and plasmids were extracted from Positive clones. By digestion with enzyme and PCR identification, nucleic acid sequence of positive plasmids were analyzed with Sangers deoxynucleotidyl termination, and relevant amino acid sequences were also calculated. So the homology of relevant nucleic acid and amino acid sequence were compared and analyzed with that of AEV 1143 strain and others of small RNA virus family.Recombinant expression vectors PET30a-VP0 were constructed, by connecting VP0 gene sequences with prokaryotic Expression Vector PET30a, and transduced into BL21 by IPTG and heatshock. To collect inclusion bodies, bacteria with vector were collected and broken up, and then inclusion bodies were analyzed with SDS-PAGE and Western-blot electrophoresis. Results showed that recombinant plasmids could be expressed in Escherichia coli, which were in the form of inclusion bodies; Molecular weight of PET-VP0 expression protein was 52Ku by Western-blot electrophoresis, and relative protein content was 26.3% by TLCS. In addition, specific reaction of these expression products with AEV serum was positive.