Development And Evaluation Of Immune Effects Of Chimeric Subunit Vaccine And DNA Vaccine Against Tilapia Source Streptococcus Agalactiae

Tilapia (Oreochromis spp.), which is native to Africa, is the tropics and subtropicswarm water fish. Because of it’s wide diets, rapid growth, stronger adaptability andtolerance the low oxygen, tilapia has become one of the most important farmed fish inthe world. Our country is the world’s largest aquaculture and exporter country of tilapia.After the1980s, the freshwater and seawater fish has became infected with Streptococcusagalactiae in the worldwide in succession, and the morbidity and mortality showed a risingtrend year by year. In recent years, the tilapia aquaculture area of south China occurred amore serious disease caused by Streptococcus agalactiae disease and resulted in a largescale death phenomenon, which lead to the farmers suffered huge economic losses.Therefore, it is urgent to take effective measures to control the spread of the Streptococcusagalactiae disease.Based on above problems, we used the surface protein SIP and GAPDH, were fromthe Streptococcus agalactiae of tilapia, as vaccine targets to develop the tilapiaStreptococcus agalactiae chimeric vaccine and evaluate the immunoprotection. By thetechnology of PCR, we cloned SIP and GAPDH gene, then using the splicing overlapextension technology (SOEing) to abtain the SIP-GAPDH fusion gene. Finally, wesuccessful constructed chimeric subunit vaccine and DNA vaccine and do immuneprotective experiment in vitro.The study contents and results are as follows:1.The GAPDH、SIP、SIP-GAPDH gene were cloned into the prokaryotic expressionvector pET-32a(+), transformed into Escherichia coli strain BL21(DE3), respectively, toobtain the candidate recombinant engineering bacterium (pET-32a-GAPDH、pET-32a-SIP、pET-32a-SIP-GAPDH). These three recombinant engineering bacteriumswere expression in the form of inclusion body. We used HisTrapTMHP column to purifiedthese three fusion proteins and got the concentrations as follows: SIP-GAPDH (670μg/mL), GAPDH (560μg/mL), SIP (520μg/mL). 2. We purified the GAPDH、SIP、SIP-GAPDH recombinant proteins, respectively,immunized tilapia by intraperitoneal injection and detectd the antibody titer by ELISAmethod. The results show that the antibodies can be detected in three groups of immunizedfish, and the antibody titers of immunized and nonimmunized groups were significantdifference, reaching the peak at28days antibody level in the immunized fish. Theantibody level of SIP-GAPDH group was the highest (1:8192), following by the antibodylevel of SIP group (1:4096) and GAPDH group (1:2048). Artificial infection experimentsshowed immune protective rate of subunit vaccine based on SIP-GAPDH, SIP, GAPDHprotein respectively is90%,86.7%and83.3%. Real-time PCR (qPCR) data suggested thatthe mRNA expression level of IgM, IL-1β and CD8gene of tilapia were raised afterinjection of subunit vaccine. After immunization1d, the expression mRNA level of IgMin head kidney reached the peak. After immunization2d, the expression mRNA level ofIgM in brain and spleen raipidly reached the peak, and gradually reduced to a stable level,but still higher than the control group. The mRNA expression level of IL-1β reached thepeak in brain and spleen after12h and in the head kidney after1d. In brain, spleen andhead kidney, the expression mRNA level of CD8reached the peak in2d, and thendeclined but still higher than a certain level.3. We successfully constructed of eukaryotic expression plasmid pcDNA-GAPDH,pcDNA-SIP, pcDNA-SIP-GAPDH, respectively, immunized tilapia (Oreochromisniloticus) by intramuscular injection. We were used the PCR and RT-PCR technique todetect the expression of above three kinds of recombinant eukaryotic plasmid in varioustissues (muscle, brain, head kidney, spleen, gill and liver). After immunized7,14,21,28,35,42d, we collected the serum and detected the serum titer with ELISA method. Afterimmunized42d, the fishes were challenged to calculate the immune protective rate. Theresults showed that corresponding plasmid could be detected in muscle, brain, head kidneyand spleen of the groups immunized7d and28d, which is surrounding the point ofinjection. The results of ELISA demonstrated that the antibodies to correspondingrecombinant proteins were existing in serum of tilapia, which indicates the DNA vaccinecan induce the fusion protein to produce the corresponding antibody (reached the peak in21d and the titer of pcDNA-GAPDH、pcDNA-SIP and pcDNA-SIP-GAPDH were:1:1024,1:2048and1:4096, respectively). After immuned the tilapia42d, we challenged the fishby artificial infection, and three groups of recombinant plasmid’s immune protective rateas follows: pcDNA-GAPDH is83.3%, pcDNA-SIP is86.7%, and pcDNA-SIP-GAPDH is 93.3%. Real-time PCR (qPCR) data suggested that the mRNA expression level of IgM,IL-1β and CD8gene of tilapia were raised after injection of DNA vaccine. Afterimmunization1d, the expression mRNA level of IgM in brain and head kidney reachedthe peak. After immunization2d, the expression mRNA level of IgM in spleen rapidlyreached the peak, and gradually reduced to a stable level, but still higher than the controlgroup. The mRNA expression level of IL-1β and CD8in the brain, head kidney and spleenreached the peak in12h and2d, respectively, then decreased a certain level but stillhigher than the control group.