Preparation And Immune-protective Effect Of LrrG-Sip Fusion Protein Against Streptococcus Agalactiae Of Tilapia

Streptococcus agalactiae, known as group B streptococcus(Group B streptococcus, GBS), is gram-positive bacterium and distributed widely in the world. It could cause the aquatic animals, mammals and even human beings suffering from sepsis,pneumonia and meningitis. In recent years, tilapia aquaculture has been badly affected by streptococcusis, which has also caused severe economic losses in aquaculture of many species of fresh water, marine and estuarine fish world wide. Currently, control of tilapia Streptococcus disease mainly relies on antibiotics, but the irrational use of antibiotics increases the antibiotic resistance of the pathogens. Vaccine is one potentially effective mean of preventing tilapia Streptococcus disease becauseit is efficient, safe,and without residue.S.agalactiae surface immunogenic protein Lrr G(Leucine-rich repeat protein from GBS) and Sip(Surface Immunogenic Protein) exist in a variety of serotypes. They are highly conserved, and both have immune protection against S.agalactiae. Studies have shown that the immunogenicity of fusion protein expressed by two or more immunogenic genes is superior to a single immunogenic protein. This study attempted to integrate S.agalactiae immunogenicity gene Lrr G and Sip, produce Lrr G-Sip fusion protein, explore whether the fusion protein provide a better immune protection rate.The Lrr G and Sip gene was amplified from genome DNA of S. agalactiae isolated from tilapia by PCR with specific primers. In order to obtain the Lrr G-Sip fusion protein, the prokaryotic expression vector p Cold II-Lrr G-Sip was constructed and Sip and Lrr G gene were cloned into vector p Cold II one by one using double enzyme method of gene splicing technology, adding biological protection hydrophobic polypeptide linker(Gly4Ser) 3 linker sequence. After optimizingthe expression conditions, Lrr G-Sip fusion proteinwas successfully induced, and Nile tilapia was immunized by the purified fusion protein. The main contents and conclusions are as follows.1 Construction and Expression of the Prokaryotic Expression Vector for Lrr G-Sip Fusion Gene of Tilapia Streptococcus agalactiaeIn this study, the Lrr G, Sip gene is amplified from genome DNA of S.agalactiae isolated from tilapia by PCR with specific primers. For keeping the biological activityof the fusion protein being similaras the natural protein, biological protection hydrophobic polypeptide linker(Gly4Ser) 3 linker sequence was added in the upstream primer of Sip. As Lrr G and Sip gene are large, it is easy to cause mutation by overlap extension PCR technology. Therefore, Lrr G and Sip were inserted into p Cold II vector using double digestion method to construct prokaryotic expression vector p Cold II-Lrr G-Sip. Then it was transformed into competent cells BL21(DE3). SDS-PAGE showed that the fusion protein was in two kinds of forms, i.e. soluble protein and inclusion body. Induced conditions of fusion protein expression was optimized by the method of control variables including IPTG concentration, induction temperature and induction time.The results showed that 9hours, 15 ℃, 0.5 mmol·L-1IPTG was the optimal inducing condition with the most soluble and abundant fusion protein. The Lrr G-Sip fusion protein was purified and evaluated through Western blotting. The fusion protein was about 160 k Da. which is consistent with the prediction(162 k Da).This suggested the prokaryotic expression vector p Cold II-Lrr G-Sip was constructed successfully and could be used for next step of the immunogenicity study in tilapia.2 Immunogenicity of Lrr G-Sip Fusion Protein of Streptococcus agalactiae isolated from TilapiaLrr G and Sip are both surface immunogenic protein of S. agalactiae isolated from tilapia. In order to evaluate the immunogenicity of Lrr G-Sip recombinant fusion protein, Nile tilapia(O. niloticus) were immunized by intraperitoneal(IP) injection with200μL Lrr G-Sip recombinant fusion protein at dose of 0.5μg / g(R1 group), 1.0μg / g(R2 group) and 1.5μg / g(R3 group) respectively. Mean while, control groups of Nile tilapia were IP injected with 200μL Sip protein(1.0μg/g, S group), 200μL Lrr G protein(1.0μg/g, L group) and 200μL sterile phosphate buffered saline(PBS,P group)respectively. At 15 d, all of the immunized fish(groups of R, S, L, and P) were challenged with S. agalactiaeby IP injection at LD50 dose of 100μL(containing4.0×108cfu/m L). Results showed that the relative percent survival(RPS) of fish from group R1(0.5μg/g) was the highest(89.14%). The OD values of serum antibody of fish from group R1 were 0.63 and 0.64 at 14 d and 28 d post-immunization,respectively, significantly higher than that of other groups(S and L)(P<0.05).The activity of peroxidase and alkaline phosphatase of fish serum from this group were also significantly higher than that of other groups(P<0.05)in the above two time points.There was no significantly difference of the activity of lysozyme and total superoxidedismutase of fish serum among different protein immunization groups(P>0.05). The results suggested that Lrr G-Sip fusion protein had better immunogenicity than single protein Lrr G or Sip, which could also reduce the injection dose effectively.