Construction And Analysis Of Tilapia Streptococcus Agalactiae Vaccine

Tilapia has been introduced and raised by more than eighty countries in the world widespread because of its series of excellent features. China is one of the main countries of tilapia aquaculture, its environment and climate condition are suitable for growth of tilapia. However, with the rapid development of Chinese tilapia industry, Streptococcus disease has become the biggest threat to tilapia breeding. Recently, continuous outbreak of tilapia Streptococcus disease caused substantial economic losses to aquaculture industry in several southeast China provinces. Though current drug therapy can alleviate the immediate condition, it cannot cure the disease, since that using antibiotic causes drug-resistant strains and also leads to concern about food safety. Vaccine can stimulate immune antibodies of fish and enhance immunity of fish, compared with current drug therapy, using of vaccines to prevent and control tilapia Streptococcus disease is a relatively safe and effective way, it has become the main direction in the research of tilapia Streptococcus disease control.Surface immunogenicity protein SIP is a kind of immune related protein that exists in group B streptococcus, it is one of the important bacteria adhesion and engraftment factors. C5a is one of the complement activation products, it’s also an important mediator of inflammation and chemotactic factor, it can enhance the immune response obviously.In this study, molecular analysis of 16SrRNA gene were used for further identification for four strains Streptococcus agalactiae isolated from Guang Dong and Hai Nan provinces.BLAST showed that 16 SrRNA gene of all strains possessed highly homologous with their counterparts registered in GenBank (>99%). Sip gene was amplified from the genomic DNA of the four strains of Streptococcus agalactiae and the sequencing analysis show the Sip gene of BZ-1 strain Streptococcus agalactiae is the most conservative. Mature peptide gene of Sip was amplified from the Streptococcus agalactiae BZ-1.Prokaryotic expression vector pET23b-Sip was constructed and transformed into E. coli BL21 (DE3) strain, the recombinant protein was induced to express and SDS-PAGE analysis showed recombinant protein was solubly expressed. The purification of the recombinant protein was up to 95% after purified with nickel chelate affinity chromatography and gel filtration.The inactivated vaccine of BZ-1 strain Streptococcus agalactiae was constructed.Total RNA was extracted from the head kidney and spleen tissues of tilapia, the cDNA were synthysized through a series of reactions. The C5a gene was amplified from the cDNA through nestPCR and the SipC5a gene was amplified through the Overlap PCR. The DNA vaccine in the form of pVAX1-Sip and pVAXl-SipC5a were constructed, which express under the cytomegalovirus immediate-early promoter.Using Zebrafish as an animal model, the immunoprotective effect of DNA vaccine, inactivated vaccine and Surface immunogenicity protein SIP were examined. The fatality rate analysis showed that the mortality in vaccinated group was significantly lower than that of control group. Fish vaccinated with pVAXl-Sip and pVAXl-SipC5a exhibited a relative percent survival (RPS) of 65% and 70%, respectively, fish vaccinated with inactivated vaccines and recombinant protein SIP exhibited a relative percent survival (RPS) of 95% and 80%, respectively. These results indicate that these vaccines have good immunoprotective effect and complement C5a enhanced the immunoprotective effect of SIP protein. This study aims at providing effective vaccines and promoting the healthy development of tilapia aquaculture in China.