| The classical swine fever (CSF) is one kind of serious diseases to threaten hog industry. Currently, the main way to prevent CSF is vaccinating by traditional attenuated vaccine. However, there is much shortage in traditional attenuated vaccine. So, to make more specific and effective development of new CSF vaccine has become a top priority. And CSFV DNA vaccine is one of the possible ways. CSFV belongs to genera Pestivirus of Flaviviridae family. The RNA of CSFV only contains one ORF to encode 11 proteins. E0, E1, E2 are envelope proteins of CSFV. E2 can induce the special antibodies, and it is the main antigen of CSFV. E0 can induce the special antibodies, too, and the gene order of E0 is more conservative. E1 plays an important role in processing of E0 and E2, although it can not induce special antibodies. In order to explore new method to control CSF using DNA vaccine, in this study, the recombinant plasmid was constructed containing all of the cDNA of CSFV envelope protein (E0/E1/E2) genes. Immunological experiments on mice and pigs with the recombinant plasmid were conducted. This study consists of three parts:1. The reverse transcription and polymerase chain reaction(RT-PCR)was used to amplify CSFV E0-E1-E2 genes of Shimen strain. The RT-PCR products were ligated into the eukaryotic expression vector pcDNA4.0 to get recombinant plasmid pcDNA4.0-E.2. After transforming to E.coli JM109, extraction and purification, pcDNA4.0-E was injected in hind legs muscle of mice, 100μg per mouse (50μg per leg). The second injection was done interval 15 days. In the day 10th, 20th, 30th after the second injection, mice were killed respectively. MTT was used for detecting the lymphocytes proliferation in spleen and peripheral blood of those mice. And indirect ELISA was also conducted for detecting the specific antibodies in peripheral blood. After injecting with pcDNA4.0-E, the lymphocytes proliferation in spleen and peripheral blood of mice was significantly higher than that of the control group (p<0.05). The antibodies of group pcDNA4.0-E increased from the day 10th after vaccinating. And furthermore, the antibody level of group pcDNA4.0-E was significantly higher than that of the control group (p<0.01). The above results confirmed that the envelope protein expressed by the recombinant plasmid can induce body to produce specific antibody and enhance cellular immunity.3. After transforming to E.coli JM109, extraction and purification, pcDNA4.0-E was injected in legs muscle of pigs, 4 mg per mouse (1 mg per leg). The second injection was done interval 15 days. The indirect ELISA was conducted for detecting the specific antibodies after the first vaccination. The antibody level of the immune group was significantly higher than that of the control group (p<0.05) from the day 20th after the first vaccination. It was indicated that the pcDNA4.0-E can induce specific antibody. In the day 40th after the first vaccinating, the CSFV of the Shimen strain was injected in all the pigs. The results showed that the time of appearance of hyperthermia was postponed. And compared with control, the symptoms and anatomic changes of immune pigs were slight. The results of histological detection showed that the changes of immune pigs were also slighter than control pigs. The pigs of control group died finally. However, there were 2 pigs of immune group survived. The virus-challenge tests showed that the pigs vaccinated by pcDNA4.0-E could partially make pigs to avoid challenge of CSFV.
|
PDF Full Text Request