Identification And Protective Immunity Of Protective Antigens Against Streptococcus Agalactiae ZQ0910Based On Genome Sequence Analysis

Streptococcus is a genus of spherical Gram-positive bacteria and considered apathogen, which is able to infect a wide range of hosts, including human beings,beasts, birds and aquatic animals. In recent years, higher frequencies of streptococcaldiseases of cultured fish, especially tilapia diseases caused by Streptococcusagalactiae, incurred catastrophic losses to tilapia culture in South China. Andtherefore, prevention and control of S.agalactiae diseases has become a researchpriority. Vaccine is one of effective ways to control and eradicate infectious diseasesas well as fish diseases. However, little is known about the virulent-related factors andpathogenesis mechanisms of S.agalactiae from tilapias, and so far no effectivesubunit vaccines for S. agalactiae diseases have been developed and applied in fishculture.In this study, genotyping analysis was conducted for104S. agalactiae strainsisolated from tilapias cultured in different regions in2010and2011by Multilocussequence typing (MLST) technique, and the genetic evolution and variation of thesestrains were determined through comparison with other Streptococcus strains.According to the results of genotyping analysis, Six S. agalactiae strains wereselected to study the pathogenicity to tilapia in order to screen strains with higherimmunogenicity for vaccine preparation. Due to the higher pathogenicity, ZQ0910strain was selected for the follow-up studies, and its whole genome was sequenced byShotgun method. By bioinformatics analysis, five protective antigen genes wereselected from2022open reading frame (ORF), and then these protective antigenswere purified using genetic engineering techniques. Based on the immune experiment,SIP possesses the highest immune protective rate among these protective antigens,and thus sip gene was select to construct a eukaryotic expression plasmid pcDNA-Sip, and the immune protective effects of pcDNA-Sip were estimated through an in vivoexperiment.The results of this study are as follows:1) In2010and2011,104S.agalactiae strains were isolated from diseased tilapiasamples in Guangdong, Guangxi and Hainan provinces, and these strains wereidentified by methods of physiology&biochemistry and molecular biology.2) Genotyping analysis of104S.agalactae strains by MLST technique indicatedthat104strains contained three different types, including ST-7and two novelgenetypes (NST-1and NST-2). Among them, type ST-7contains8strains, and typeNST-1and NST-2contain92and4strains respectively. Allele image pattern showed aclose genetic relationship between novel genetypes and type ST-7, implied these threegenetypes belong to the same clone complex.3) Pathogenicity analysis was carried out within6selected strains to screen strainswith higher immunogenicity for vaccine preparation, and results indicated that onestrain named ZQ0910possesses a stronger virulence, which may be suitable for thevaccine preparation.4) The whole genome of ZQ0910strain was sequenced by Shotgun method and thesequence data were deposited in DDBJ/EMBL/GenBank database under the accessionnumber AKAP00000000.5) Twenty-one candidate proteins related to pathogenicity and immunogenicitywere found from the whole genome sequences of ZQ0910strain by BLAST analysis,and5proteins, including SIP, Hemolysin, FBP, VaA and HtrA, were selected for thefollow-up studies.6) The genes encoding target antigens (SIP, Hemolysin, FBP, VaA and HtrA) wereamplified by PCR respectively, and then ligated into the prokaryotic expression vectorpET28a (+) and pET32a (+). Recombinant plasmids were transformed intoEscherichia coli Rosetta, and the induced recombinant fusion proteins harboringhistidine tag were abundantly purified by Ni-NTA affinity chromatography.7) The purified target antigens were used to immunize Oreochromis niloticus, andantibody titer was determined by ELISA, together with results of the relative percentage survival (RPS) revealed that specific antibody titers against differentantigens were detected in all serum of immunized tilapias, and reached the highestlevel in the fourth week after immunization. Furthermore, the antibody levels in theimmunized groups were significantly higher than that in the negative group (P<0.01).After challenged by S. agalactiae, the RPS values of tilapias in SIP, HtrA, FBP,Hemolysin and VaA groups were80%,75%,74%,70%and70%, respectively.8) Sip gene cloned from S. agalactiae was selected to insert into pcDNA3.1(+)vector and the recombinant plasmid pcDNA-sip was injected into O.niloticus. ThepcDNA-Sip expression in tissues (including muscle, spleen, head kidney, gill andliver) was analyzed by PCR and RT-PCR at7and28d after immunization, andantibody titer was detected by ELISA at7,14,21,28d after immunization. Inaddition, the immune pretective rate of pcDNA-Sip was calculated at28d afterimmunization based on the mortality. The results indicated that pcDNA-Sipexpression could be detected in all studied tissues and antibody titer raised to the peak(1:5120) at21d after immunization. Additionally, the RPS value was90%accordingto the mortality by S. agalactiae challenge. The recombined plasmid pcDNA-Sipcould transfect O. niloticus tissues and keep expressing for a period of time，especially acquire higher RPS values.