Genetic Diversity Analysis Of E2Gene Of CSFV Isolated In Shandong Province And Immunogencity Research Of Eukaryotic Expression Vetor Contained E2Gene Of CSFV

Classic swine fever (CSF) is a highly contagious disease caused by Classic swine fever virus(CSFV), which appears clinical symptom of fever, generalized haemorrhages ichoremia and high death rate. The epidemic of CSF has made a huge damage to pig industry and impacted the international trade of pigs or related products. China is one of epidemic zone of CSF. In China, the epidemic status of CSF is complicated and characterized by latent infection and sporadic outbreak. For prevention and control the epidemic of CSF, it is significant to make a systematical molecular epidemiological study, figure out the diversity of CSFV strains and develop novel maker vaccine against CSF. All the experiments and results are as follows:1. Isolation and identification of CSFV strains in Shandong ProvinceIn this study, we detected656samples which is collected from Shandong Province during2011-2012. The result of RT-PCR detection showedthat20.66%of these samples were CSF positive, which meant CSFV infection is common in pig farms in Shandong Province. The positive samples above refercd were inocubated to PK-15cell for isolating CSFV strains. Then isolates were identified by RT-PCR and real-time PCR.2. The diversity of E2geneE2sequences were amplified by RT-PCR and compaired with the reference strains recorded in GenBank to analyze the genetic diversity of CSFV.According to the result of comparison and analysis, nine field strains were isolated successfully. These nine isolates belonged to subgroup2.1and shared a quite highly homology.Conversely, the homology between isolated strains and HCLV is much lower,which shared79.5-81.6%identities in nucleotide sequence and81.2-85.9%identities in amino acid sequence respectively. The data indicated that the prevalent strains in Shandong Province had been considerable different from vaccine strain and they were still evolving towards the direction deviated from vaccine strain slowly.3. The construction and immunogenicity identification of recombinant eukaryotic expression vector contained E2gene of CSFVIn this study, a series of recombinant eukaryotic expression vectors were constructed by inserting E2fragments of different length in order to figure out whether it will change the immunogenicity of E2glycoprotein. The endocelluar expression of recombinant vectors was detected by RT-PCR and IFA test at first step. Then humoral immunity level and cell-mediated immunity level induced by recombinant vectors were measured by ELISA and lymphocytes transformation test in mice. At last,several results were acquired:all of the recombinant vectors were able to express E2glycoprotein in PK-15cell; the peptide between996amino acid site and1010amino acid site probablly played an important role in celluar immunity against CSFV while the peptide between870amino acid site and990amino acid site was independent of the immunogenicity of E2glycoprotein; recombinant vector named pVAX-999rE2could be regarded as a canadiate CSF DNA vaccine.