Cloning, Expressing And Identifying On Canine Parvovirus (CPV) VP₂ Gene

The disease of Canine parvovirus is a kind of acute infectious disease, which is caused by Canine parvovirus(CPV). The characteristic clinical symptoms of CPV are vomiting, bloody enteritis and serious lucopenia, which also cause acute cardiac muscle inflammation. The course of the disease is short, the mortality is high. The disease is one of the most infectious diseases in our country, which not only endanger the dogs but also make a lot of loss.A pair of primers was designed according to the sequence of VP₂ gene from Genebank, the genome DNA of CPV was extracted from CPV/CDV vaccine, then it was used as templates for polymerase chain reaction (PCR) to amplify the VP₂ gene. The VP₂ gene then was cloned into the restriction of plasmid pET22b(+). The recombinant plasmid was identified by restriction enzymes analysis and nucleotide sequence analysis, the results showed that the VP₂ gene has been correctly cloned into plasmid pET22b(+), the prokaryotic expression vector of pET22b/VP₂ was successfully constructed.The corrcet recombinant pET22b/VP₂ was transformed into Escherichia coli BL21(DE3)and Escherichia coli Rosetta^TM(DE3) with IPTG inducing. The expressed protein was measured by SDS-PAGE, the results showed that there was not obvious expression strip in Escherichia coli BL21 (DE3) ;While in Escherichia coli Rosetta^TM(DE3) , it can get high expression, the expressed protein was morn than 30% of total bacterial protein and existed in the form of inclusion bodies. Target proteins was purified by Ni²⁺ affinity chromatography. After renaturation, the protein can be recognized by the positive serum of CPV.The trail has successfully cloned the full segment of VP., gene, and made it got high expression in the prokaryotic expression system. All these work will laid foundation for the research and development of genetic engineering vaccine and new diagnose reagent.