Preparation of live recombinant Lactococcus lactis vaccine expressing Sip protein of Streptococcus agalactiae isolated from tilapia (Oreochromis niloticus) and immunogenicity analysis

Streptococcosis is a serious disease that threatens the development of Oreochromis niloticus industry in China. In order to make a vaccine of Streptococcus agalactiae with high immune efficiency and simple operation, we constructed a recombinant shuttle-plasmid pNZ8124-Sip which could express Sip protein of S. agalactiae. The recombinant plasmid was electro-transferred into Lactococcus lactis NZ9000 after being identified by enzyme digestion and sequencing analysis. The SDS-PAGE was used to obtain the optimumly induced concentration of nisin and induction time. The Sip protein was purified by Ni-chelating affinity chromatography and tested by Western blot. O. niloticus was vaccinated orally by gavage with different concentration of the recombinant L. lactis NZ9000-pNZ8124-Sip. The ELISA was used to test the change of serum antibody. The relative percent survival (RPS) was obtained by artificial abdominal injection with S. agalactiae.The SDS-PAGE showed that the molecular weight of expressed protein was 48 ku, which is equal to the expected protein size. The recombinant protein mainly existed as soluble protein and inclusion bodies. The concentration of purified protein could reach 7.65 mg/mL. The optimal condition was induction with 100 ng/mL nisin for 6 h. Western blot results showed that the recombinant Sip protein could be specifically combined with the mouse anti-His tag antibody. Oral immunization showed that the serum antibody and the resistance to S. agalactiae were significantly improved in the middle concentration group (2.24×10¹⁰ CFU/mL) and the low concentration group (2.24×10⁹ CFU/mL). The RPS of the middle concentration was 41.0%, which was the highest. This study can lay a foundation for research on oral vaccine of O. niloticus against S. agalactiae and has a broad prospect of application.