Cloning, Identification And Expression Analysis Of Anti-Virus And Immune Related Genes In Flounder Paralichthys Olivaceus

Flounder Paralichthys olivaceus is an important aquaculture species in China. It has a wide distribution and makes great benefits to the domestic industry of aquaculture. While at present, the main indoors breeding manner brought lots of problems including bad quality of the water and environment, which directly leads to the breakout and transmission of all kinds of fish diseases. Although the diseases can be controlled in some extent by using chemical drugs, keeping using drugs will lead to the environment pollution and drug-fast pathogens appearance, which make the condition become worse. So it is important and necessary to work on the fish immunology. The present study aims to study the flounder immunity and importantly to clone non-specific immune genes from the fish. Firstly, the relationship between the SMRV and fish cells was investigated, and the results indicated that the main death manner of SMRV infected CLC cells is through apoptosis, the existence of apoptosis has little influences on the virus duplication, while the virus duplication is necessary for the occurrence of apoptosis. Secondly, using suppression subtractive hybridization (SSH), a subtractive cDNA library was constructed with the flounder embryo cells treated with the UV-inactivated fish rhabdovirus as tester and the control cells as driver. The mRNA was isolated from tester and driver cells respectively and double-strand cDNA molecules were synthesized by reverse transcription. After two times of subtractive hybridization and then two times of PCR amplification, the forward subtracted PCR products were ligated with T vector (pMD-18-T) and the subtractive cDNA library construction was finished. 4200 clones of the total 7400 clones were detected by nest PCR, and about 4000 clones contained the inserted fragments with the length between 250bps and 1000bps. Dot blot was performed on the nest PCR products of the 4000 clones hybridized with the Dig-High Prime labeled probes and 668 clones were obtained as positive. These results showed that the subtractive cDNA library was suitable for further screening of genes related to the anti-virus function. Thirdly , by construction of SMART-cDNA library together with application of RACE technique several immune related gene including cystein proteinase, decorin, NDPK, RACK, chemokine were cloned, and all of them were identified in flounder for the first time. By using RT-PCR, the transcription level of the cloned genes in different tissues of health flounder was investigated and the results show that they have a different distribution with each member. Finally, detailed characteristics of flounder decorin gene were explored by successful prokaryotic expression and eukaryotic expression of its intact ORF. Western blot performed on the tissues of brain, headkidney, posterior kidney, heart, thymus, gill, muscle, intestine, spleen, liver, testis, and ovary with the multiple antibody prepared by injecting rabbit with the prokaryotic expression product indicated that the highest expression level exists in gill while the lowest in ovary among the detected tissues. By transfected the FE cells with the recombinant plasmid the founder decorin gene were successful transient expressed, and this technique will enable us to explore the function of the clone genes in the next step. In this thesis, SMRV, a fish rhabdovirus with the strong virulence, was used as the induce factors to explore the virus pathogenic mechanism and the immune responses of flounder both in the organism and cell level. At the same time several immune-related genes were cloned. All of these will not only supply basic data for searching the effective control methods of flounder diseases but also promote the fish developmental and comparative immunology for the reason of it specific evolution position.