Construction And Evaluation Of Channel Catfish Virus (CCV) ORF6 Gene Suicide DNA Vaccine

In this study, channel catfish (Ictalurus punctatus), suicide DNA vaccine and conventional DNA vaccine were constructed and their expression levels in the cells were compared. The immunity effects were evaluated and compared after injection of conventional and suicide DNA vaccines. The main contents are as follows:1. The construction of suicide DNA vaccine of Channel Catfish Virus (CCV) ORF6 gene The suitable primers were designed according to CCV ORF6 gene sequence. The gene was cloned into suicide DNA vaccine vector pSFV and conventional DNA vaccine vector pcDNA3.1 respectively. After transformation into E. coli DH5a and extraction plasmid, the suicide DNA vaccines ps-ORF6 and conventional DNA vaccine pcd-ORF6 were constructed. The methods of PCR, double enzymes digestion and DNA sequencing were used to confirm that CCV ORF6 gene was correctly inserted into the vector.2. The expression of CCV suicide DNA vaccine in the cells. The recombinant plasmids were transfected into 293T cells(human embryo kidnet cell). Indirect immunofluorescence assay showed that both were expressed, but the express level of the suicide DNA vaccine was lower than that of conventional DNA vaccines.3. The effects of CCV suicide DNA vaccine to immunity of channel catfish. The channel catfish from 70 g to 90 g was divided into five groups and immunized by intramuscular injection using the suicide DNA vaccines (group III) and conventional DNA vaccines (group IV) of channel catfish and the other three control groups were injected with other three reagents, the vector of pSFV for group I, the vector of pcDNA3.1 (+) for group II and PBS for group V. At the weeks of 0,2,4,6,8, the phagocytic activity of leucocytes and bactericidal activity of phagocytes were tested by using MTT dying method, lysozyme and protein were detected by using kit.In the test, the significant difference was found between the suicide DNA vaccines and conventional DNA vaccines (P＜0.05), which were both higher than the three control groups. The results showed that the suicide DNA vaccines may enhance the phagocytic activity of leucocytes of the channel catfish. The results also indicated that the phagocytic activity of leucocytes of the channel catfish was influenced by various factors, as the phagocytic activities of leucocytes in all the groups have been upgraded. In the test of determine the lysozyme activity, the suicide DNA vaccines and conventional DNA vaccines had significant difference with the other control groups (P<0.05), and the value in the group III was significantly higher than that in the group IV (P<0.05). The results showed that the suicide DNA vaccines can improve the content of serum lysozyme to some extent. All the groups increased the total protein of the channel catfish, while the results showed the suicide DNA vaccines had no advantage and the environments did influence the value of the total protein.4. The impacts of the suicide DNA vaccines on the growth performance of channel catfishAfter the suicide DNA vaccines injection, the growth performance were evaluated during the following next days. Respectively, According to statistical analysis, there was no significant difference among the five groups (P>0.05).