| In recent years,with the sustained development of grouper aquaculture industry,the production of grouper in China has approached 100,000 tons.However,with the gradually expansion of scale farming,various diseases such as bacteria,viruses and parasites are frequent,resulting in a large number of deaths and causing serious economic losses to the grouper aquaculture industry.In particular.Vibrio harveyi is one of the most important pathogens causing grouper disease.V.harveyi CJG-1 was isolated and identified from the Black sea bass(Centropristis striata),and its growth characteristics and the best conditions for preparation of inactivated vaccine were explored.V.harveyi CJG-1 was used to infect Black sea bass,which effected on the serum indicators and the immune relevant genes expression in related tissues were studied,as well as to explore the effect of immune response of immune-related genes in different tissues,Black sea bass were injected with inactivated whole-cell vaccine of V.harveyi CJG-1,then immune effect of Black sea bass immunized with vaccine that were studied,the main contents are as follows:(1)Under conditions:28℃and 180 rpm speed oscillation cultured V.harveyi CJG-1,and gradully monitored bacterial concentrations,the result shows that logarithmic growth phase of V.harveyi CJG-1 is from 4h to 12h;using bacteria solution that has cultured for 12h,to prepare the inactivated bacterin vaccine.The concentration of formaldehyde was set at 0,0.05%,0.1%,0.2%,0.3%,0.4%and 0.5%,respectively placed in 4℃and 28℃.After the vaccine quality and safety testing,the optimal conditions for preparing the inactivated vaccine were formaldehyde concentration of 0.2%,at 28℃and inactivated for 24h.(2)The artificial infection test which Black sea bass were intraperitoneal injected with 1×10~8 cfu/ml V.harveyi CJG-1 was carried out.Some biochemical indicators like what alkaline phosphatase,aspartate aminotransferase,alanine aminotransferase and Lysozyme activity in Serum samples were detected,at 0h(before infection),6h,12h,18h,24h,36h,48h,72h and 96h after infection,the results showed that the four kinds of enzyme activity were increased to varying degrees and significantly higher than of the control group,indicating that all the indicators of anti-infection of Black sea bass are increased to resist the relevant damage factors caused by infection.(3)After artificial infection,venous blood sampling respectively at 0h(before infection),6h,12h,18h,24h,36h,48h,72h and 96h,and then,the spleen,head kidney,liver and gill were dissected rapidly,and those tissues samples were collected in centrifuge tubes containing RNA preservation solution for RNA extraction and reverse transcribed into cDNA.Primer Premier 5.0 software was used to design sequence-specific primers of TLR2,MyD88 and HSP70,their relative expression were determined by quantitative real-time polymerase chain reaction(qRT-PCR)withΒ-action as internal control.The results showed that the up-regulation ratios of the expression of TLR2 and MyD88 in spleen and liver were 9.3 times,21 times and 9.9 times,5.9 times respectively,and the expression of MyD88 in head kidney reached the peak to 2.3 times at 6h,the expression of MyD88 in gill reached 8.3 times at 36h;the expression of HSP70 in head kidney and gills rapid responsed to 11.2 and 10.7 times at 18h,and its expression in the liver and spleen were 9.7 and 2.8 times at 72h respectively,all above indicating that TLR2,MyD88 and HSP70 involved in the immune response of V.harveyi infection,in addition to that,besidses head kidney and spleen,liver and gills in the immune response process is also important tissues.(4)Black sea bass were intraperitoneal injected with 1×10~9 cfu/ml inactivated V.harveyi CJG-1 vaccine which were prepared at a formaldehyde concentration of 0.2%at 28℃for 24 h.the spleen,head kidney,liver and gill were dissected rapidly at 0h(before infection),6h,12h,18h,24h,36h,48h,72h and 96h,and those tissues samples were collected in centrifuge tubes containing RNA preservation solution for RNA extraction and reverse transcribed into cDNA.Two weeks later,booster immunization used the same dose and manner.At the fourt week,using 1×10~8cfu/ml of V.harveyi CJG-1 challenged all fish to detect the protective effect of the vaccine.The results showed that the expression of TLR2 and MyD88 in the head and liver significantly up-regulated,including the expression of MyD88 was 73.2 and 95 times and reached peak at 36h,TLR2in liver was 62 times at 24h and in head kidney was 144.4 times at 36h;The expression of HSP70 in spleen,head kidney,liver and gill reached peak within 18-48h,including in the head kidney was 6.2 times at 48h higher than the control group,HSP70 in liver peak appeared at 18h;attacking Toxicity test results indicated that immunized Black sea bass obtained strong immune protective and with a protection rate of 85.71%.Therefore,analysis of immune gene expression from the molecular point of view shows that TLR2,MyD88 and HSP70 were involved in immune response of Black sea bass,the good immune protection rate also indicates that the vaccine has good immunoprotective effect.The inactivated vaccine prepared in this experiment is effective to the immunization of Black sea bass. |
PDF Full Text Request