A recombinant strain, a preparation method thereof and application of the recombinant strain in preparing a vaccine for lactating streptococcus agalactiae of tilapia

Abstract

The application discloses a recombinant strain and a preparation method thereof and application of the recombinant strain in preparation of a vaccine for Streptococcus agalactiae of tilapia, and the recombinant strain is successfully constructed by the following steps: designing an ORF4 primer, amplifying an ORF4 gene fragment from a DNA template of Streptococcus agalactiae, editing the ORF4 gene fragment into Escherichia coli by means of a CRISPR / Cas9 gene editing technology, and connecting a green fluorescent protein (GFP). The recombinant strain is subjected to immunoprotection research on the Streptococcus agalactiae of tilapia, and it is shown that the recombinant strain has a good protection effect, and can provide a solid theoretical basis and technical support for application and development of the vaccine for Streptococcus agalactiae of tilapia in the field of aquaculture.

Description

Technical Field

[0001] This invention relates to the field of bioengineering strain technology, and in particular to a recombinant strain, its preparation method, and its application in the preparation of alactamase-free streptococcal vaccine for tilapia. Background Technology

[0002] Streptococcus agalactiae (GBS), a key pathogen in tilapia farming, is widely distributed in the natural environment and can colonize the human urogenital and digestive tracts. It is also one of the major pathogens in humans and other animals. This pathogen is mainly transmitted horizontally through broken skin or via the fecal-oral route, easily infecting healthy fish populations. GBS accounts for over 90% of clinically isolated strains, with a cumulative mortality rate of 30%–80%, causing significant economic losses to the aquaculture industry. Vaccination is a core technical means of controlling pathogenic diseases in aquaculture. Currently, various types of vaccines have been developed for GBS in tilapia, including inactivated vaccines, DNA vaccines, and live attenuated vaccines. In recent years, the emergence of CRISPR / Cas9 technology has brought revolutionary changes to the field of gene editing. With its advantages of high efficiency, simplicity, and high specificity, this technology has rapidly become a hot topic in life science research. CRISPR / Cas9 technology has also been widely used in bacterial gene editing, providing a powerful tool for basic research and applied development in bacteria.

[0003] Currently, there are no publicly reported applications of gene-editing technology to produce vaccines in the prevention and control of Streptococcus agalactiae in tilapia. Summary of the Invention

[0004] This invention discloses a recombinant bacterial strain, its preparation method, and its application in the preparation of Streptococcus agalactiae vaccine for tilapia. The recombinant bacterial strain DH5α-ORF4-GFP constructed in this invention has shown good protective effects in the immunoprotective study against Streptococcus agalactiae in tilapia.

[0005] The first aspect of the present invention provides a recombinant strain (Escherichia coli DH5α-ORF4-GFP), which was deposited at the China Center for Type Culture Collection on October 28, 2025, with accession number CCTCC NO: M20252368, and its sequence listing is shown in SEQ ID NO:1.

[0006] A second aspect of the present invention provides a method for preparing the recombinant strain described above, comprising the following steps:

[0007] (1) Using Streptococcus agalactiae HN016 strain as a template, promoter fragment QDZ and fragment ORF4 were obtained by amplification;

[0008] Using Escherichia coli DH5α strain as a template, fragments LHA and RHA were obtained by amplification;

[0009] Using pBAV1K-T5-sfGFP plasmid as a template, the GFP gene fragment was obtained by amplification;

[0010] Using pSC101-PBAD-sgRNA-Donor26D plasmid as a template, fragments PD1, PD2, PD3, PD4, and PD5 were obtained by amplification.

[0011] The promoter fragment QDZ is shown in SEQ ID NO:2, the fragment ORF4 is shown in SEQ ID NO:3, the fragment LHA is shown in SEQ ID NO:4, the fragment RHA is shown in SEQ ID NO:5, the fragment GFP is shown in SEQ ID NO:6, the fragment PD1 is shown in SEQ ID NO:7, the fragment PD2 is shown in SEQ ID NO:8, the fragment PD3 is shown in SEQ ID NO:9, the fragment PD4 is shown in SEQ ID NO:10, and the fragment PD5 is shown in SEQ ID NO:11;

[0012] (2) The promoter fragment QDZ, the fragment ORF4, the fragment LHA, the fragment RHA, the GFP, the fragment PD1, the fragment PD2, the fragment PD3, the fragment PD4 and the fragment PD5 are ligated and transformed into Escherichia coli to obtain plasmid pSC101-PBAD-sgRNA-ORF4-GFP, as shown in SEQ ID NO:12;

[0013] (3) p15A-PBAD-Cas9-PT5-Redγβα and plasmid pSC101-PBAD-sgRNA-ORF4-GFP were transformed into Escherichia coli, and the recombinant strain DH5α-ORF4-GFP was induced and screened.

[0014] Furthermore, step (1) specifically includes:

[0015] Using Streptococcus agalactiae HN016 as a template, the S. agalactiae HN016 promoter fragment QDZ was amplified using primer QDZ, and the fragment ORF4 was amplified using primer ORF4.

[0016] Using Escherichia coli DH5α strain as a template, fragment LHA was amplified using primer LHA, and fragment RHA was amplified using primer RHA.

[0017] Using pBAV1K-T5-sfGFP plasmid as a template, the GFP gene fragment was amplified by primer GFP.

[0018] Using pSC101-PBAD-sgRNA-Donor26D plasmid as a template, fragments PD1, PD2, PD3, PD4, and PD5 were amplified using primers yw1, yw2, yw3, yw4, and yw5, respectively.

[0019] The forward and reverse primers of primer QDZ are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively; the forward and reverse primers of primer ORF4 are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively; the forward and reverse primers of primer LHA are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively; the forward and reverse primers of primer RHA are shown in SEQ ID NO:19 and SEQ ID NO:20, respectively; the forward and reverse primers of primer GFP are shown in SEQ ID NO:21 and SEQ ID NO:22, respectively; the forward and reverse primers of primer yw1 are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively; the forward and reverse primers of primer yw2 are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively; and the forward and reverse primers of primer yw3 are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively. As shown in NO:27 and SEQ ID NO:28, the forward and reverse primers of primer yw4 are shown in SEQ ID NO:29 and SEQ ID NO:30, respectively, and the forward and reverse primers of primer yw5 are shown in SEQ ID NO:31 and SEQ ID NO:32, respectively.

[0020] Furthermore, in step (2), the ligation reaction is carried out using the pEASY®-Uni Seamless Cloning and Assembly Kit.

[0021] Further, in step (3), p15A-PBAD-Cas9-PT5-Redγβα and plasmid pSC101-PBAD-sgRNA-ORF4-GFP were transformed into Escherichia coli. The cells were then revived and cultured in LB medium at 30°C for 0.5-2 h, and then inoculated into LB agar plates containing 50-200 μg / mL ampicillin and 30-100 μg / mL kanamycin sulfate, and cultured statically at 30°C for 36-60 h.

[0022] Furthermore, in step (3), the induced screening specifically includes:

[0023] A single colony was picked from the LB agar plate and inoculated into LB medium. The culture was then shaken at 30°C for 2-4 hours to obtain culture A.

[0024] Add 80-150 mg / mL ampicillin solution, 80-150 mg / mL kanamycin solution, and 80-150 mg / mL IPTG solution to culture A. The volume ratio of LB agar plate medium, ampicillin solution, kanamycin sulfate solution, and IPTG solution is 1000:0.5-5:0.2-3:1-6. Continue culturing at 30℃ for 0.6-2 h. Add 1 mol / L L-arabinose and continue induction culture at 30℃ for 12-30 h to obtain culture B.

[0025] Culture B was transferred to LB medium and cultured at 37°C with shaking for 20-36 h to obtain culture C.

[0026] Dilute culture C in sterile water and spread it on LB agar plates containing 50 μg / mL kanamycin sulfate. Incubate at 40°C for 20-36 h. Pick a single colony that emits green fluorescence from the LB agar plate to obtain the recombinant strain DH5α-ORF4-GFP.

[0027] Furthermore, in step (2) and / or step (3), the Escherichia coli strain is Escherichia coli DH5α.

[0028] A third aspect of the present invention provides a tilapia agalactiae streptococcus vaccine, comprising the recombinant strain described above, or the recombinant strain prepared by the method described above.

[0029] The fourth aspect of this invention provides the application of the recombinant strains described above or the recombinant strains prepared by the methods described above in the prevention and control of streptococcal disease in tilapia.

[0030] The fourth aspect of this invention provides the application of the recombinant strains described above or the recombinant strains prepared by the methods described above in the preparation of a tilapia streptococcal vaccine.

[0031] Based on the research of this invention, C5a peptidase is a highly conserved serine protease on the surface of Streptococcus agalactiae. The C5a peptidase of Streptococcus agalactiae is abbreviated as ScpB (Streptococcal C5apeptidase from group B Streptococcus), secreted onto the bacterial surface by ScpB gene editing. It is currently the largest GBS surface protein discovered and is present in all serotypes of GBS. Analysis of Streptococcus agalactiae strain HN016 (accession number: CP011325.1) shows that the C5a protein is encoded by the ScpB gene, and its full-length ORF is 3703 bp. The C5a protein is expressed by ORF1 (1275 bp) and ORF4 (2403 bp), with ORF4 encoding the largest protein subunit, which is also the largest protein subunit of the C5a protein. Therefore, in this experiment, ORF4 primers were designed to amplify the ORF4 gene fragment from the DNA template of Streptococcus agalactiae. The ORF4 gene fragment was then edited into Escherichia coli using CRISPR / Cas9 gene editing technology and ligated with green fluorescent protein (GFP), thus successfully constructing the DH5α-ORF4-GFP strain.

[0032] The recombinant strain of this invention showed a relative protection rate of 73.12% after being tested for immunoprotection against Streptococcus agalactiae in tilapia, demonstrating a significant protective effect. Its application in vaccine preparation can provide a solid theoretical basis and technical support for the application and development of Streptococcus agalactiae vaccines in the field of aquaculture. Attached Figure Description

[0033] Figure 1 This involves the construction of a recombinant bacterial strain. (A) PCR identification of the gene-edited *E. coli* DH5α-ORF4-GFP strain was performed using HA-F / ORF4-R primers. 1: *E. coli* DH5α strain; 2-3: *E. coli* DH5α-ORF4-GFP strain. (B) The expression of ORF4 protein in the *E. coli* DH5α-ORF4-GFP strain was detected. 1: *E. coli* DH5α strain; 2: *E. coli* DH5α-ORF4-GFP strain; 3: recombinant ORF4 protein.

[0034] Figure 2 These are the growth curves of Escherichia coli DH5α strain and DH5α-ORF4-GFP strain.

[0035] Figure 3 These are the histochemical staining results of the liver and kidneys of tilapia. (A) Liver, (B) Kidney.

[0036] Figure 4 shows the effect of DH5α-ORF4-GFP strain on the gut microbiota composition of tilapia. (A) Chao index; (B) Shannon index; (C) Principal component analysis (PCA); (D) Microbial community composition at the phylum level for each sample; (E) Microbial community composition at the genus level for each sample; (F) Histogram of LDA value distribution. The CK group was the blank control group, and the MY group was the single-immersion immunization group, using a concentration of 1.5 × 10⁻⁶ GFP. 4 CFU / mL. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0038] Example 1 Preparation of engineered strains

[0039] 1.1 Main Test Materials

[0040] Streptococcus agalactiae HN016 strain (NCBI accession number: CP011325.1), Escherichiacoli DH5α strain (NCBI accession number: CP026085), recombinant ORF4 protein, and murine ORF4 protein polyclonal antibody were provided and preserved by the Clinical Laboratory of the College of Animal Science and Technology, Guangxi University. pBAV1K-T5-sfGFP plasmid was purchased from HonorGene (HG-VYH1347), and p15A-PBAD-Cas9-PT5-Redγβα and pSC101-PBAD-sgRNA-Donor26D plasmids were purchased from Shandong Qibang Biotechnology Co., Ltd. pEASY®-Uni Seamless Cloning and Assembly Kit was purchased from TransGen Biotech (Beijing, China); DNA polymerase and reagents used for PCR were purchased from Takara Bio (Beijing, China).

[0041] 1.2 Preparation method

[0042] (1) Using Streptococcus agalactiae HN016 strain as a template, the promoter fragment QDZ (408bp) of S. agalactiae HN016 was amplified by primer QDZ, and the fragment ORF4 (2432bp) was amplified by primer ORF4.

[0043] Using Escherichia coli DH5α strain as a template, the LHA fragment (512 bp) was amplified by primer LHA and the RHA fragment (539 bp) was amplified by primer RHA.

[0044] Using pBAV1K-T5-sfGFP plasmid as a template, the gene fragment GFP (757 bp) was amplified by primer GFP.

[0045] Using pSC101-PBAD-sgRNA-Donor26D plasmid as a template, fragments PD1 (2127bp), PD2 (367bp), PD3 (301bp), PD4 (1118bp), and PD5 (341bp) were amplified using primers yw1, yw2, yw3, yw4, and yw5, respectively.

[0046] The promoter fragment QDZ is shown in SEQ ID NO:2, fragment ORF4 is shown in SEQ ID NO:3, fragment LHA is shown in SEQ ID NO:4, fragment RHA is shown in SEQ ID NO:5, fragment GFP is shown in SEQ ID NO:6, fragment PD1 is shown in SEQ ID NO:7, fragment PD2 is shown in SEQ ID NO:8, fragment PD3 is shown in SEQ ID NO:9, fragment PD4 is shown in SEQ ID NO:10, and fragment PD5 is shown in SEQ ID NO:11.

[0047] The forward and reverse primers for primer QDZ are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively; the forward and reverse primers for primer ORF4 are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively; the forward and reverse primers for primer LHA are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively; the forward and reverse primers for primer RHA are shown in SEQ ID NO:19 and SEQ ID NO:20, respectively; the forward and reverse primers for primer GFP are shown in SEQ ID NO:21 and SEQ ID NO:22, respectively; the forward and reverse primers for primer yw1 are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively; the forward and reverse primers for primer yw2 are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively; and the forward and reverse primers for primer yw3 are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively. As shown in ID NO:28, the forward and reverse primers of primer yw4 are shown in SEQ ID NO:29 and SEQ ID NO:30, respectively, and the forward and reverse primers of primer yw5 are shown in SEQ ID NO:31 and SEQ ID NO:32, respectively.

[0048] (2) The promoter fragment QDZ, fragment ORF4, fragment LHA, RHA, GFP, fragment PD1, fragment PD2, fragment PD3, fragment PD4 and fragment PD5 were ligated and transformed into Escherichia coli to obtain plasmid pSC101-PBAD-sgRNA-ORF4-GFP, as shown in SEQ ID NO:12.

[0049] Specifically, the ligation reaction was performed using the pEASY®-Uni Seamless Cloning and Assembly Kit (incubated at 50°C for 15 min in a PCR instrument), and the resulting product was transformed into Escherichia coli DH5α strain. The corresponding plasmid was extracted using the Plasmid Mini Kit II (OMEGA, USA) and named pSC101-PBAD-sgRNA-ORF4-GFP.

[0050] (3) p15A-PBAD-Cas9-PT5-Redγβα and plasmid pSC101-PBAD-sgRNA-ORF4-GFP were transformed into Escherichia coli, and the recombinant strain DH5α-ORF4-GFP was induced and screened.

[0051] Specifically, the process of conversion to E. coli includes:

[0052] p15A-PBAD-Cas9-PT5-Redγβα and plasmid pSC101-PBAD-sgRNA-ORF4-GFP were transformed into Escherichia coli DH5α using a 42℃ heat shock method. The cells were then revived and cultured in 1 mL LB medium at 30℃ for 1 h, and then inoculated into LB agar plates containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate. The cells were then incubated statically at 30℃ for 48 h for resistance selection.

[0053] Specifically, the induced screening process includes the following steps:

[0054] Single colonies were picked from LB agar plates containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate and inoculated into 2 mL of LB medium. The culture was incubated at 30°C with shaking for 2 h to obtain culture A. 2 μL of 100 mg / mL ampicillin, 1 μL of 100 mg / mL kanamycin, and 5 μL of 100 mg / mL IPTG were added to culture A, and the culture was incubated at 30°C for another 1 h. 40 μL of 1 mol / L L-arabinose was added, and the culture was induced at 30°C for another 16 h to obtain culture B. 50 μL of culture B was transferred to LB medium and incubated at 37°C with shaking for 24 h to obtain culture C. 1 μL of culture C was diluted in 100 μL of sterile water and spread entirely onto LB agar plates containing 50 μg / mL kanamycin sulfate. The plates were incubated at 40°C for 24 h. From LB... A single colony emitting green fluorescence is picked from an agar plate to obtain the recombinant strain DH5α-ORF4-GFP.

[0055] The success of gene editing was verified by PCR using primers HA-F / ORF4-R, and PCR and sequencing were performed using primers HA-F / HA-R. The sequence listing is shown in SEQ ID NO:1.

[0056] The PCR reaction system in the above preparation method is shown in Table 1:

[0057] Table 1 PCR reaction system

[0058]

[0059] Pre-denaturation at 98℃ for 5 min, denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 72℃ for 45 s, 35 cycles, and final extension at 72℃ for 10 min.

[0060] The PCR products were detected by 1% agarose gel electrophoresis at 120 V for 30 min.

[0061] The primers are shown in Table 2:

[0062] Table 2 Primer sequence listing

[0063] 1.3 Determination of growth curve of recombinant strain

[0064] The target bacteria were cultured in LB liquid medium at 37℃ with shaking at 180 r / min until the logarithmic growth phase. They were then transferred to fresh LB medium at a 1% inoculum and cultured under the same conditions with shaking. Samples were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, and 24 h, with uninoculated LB medium serving as a blank control. The absorbance (OD) at 600 nm was measured using a spectrophotometer. 600 (Value). Three parallel samples were set at each time point, and the mean was taken.

[0065] 1.4 Protein extraction and Western blot analysis of recombinant strains

[0066] Cultured bacteria were collected by centrifugation at 6000×g for 10 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in 1×PBS. The cells were then sonicated. After sonication, the supernatant was collected by centrifugation at 10000×g for 10 minutes at 4°C. Protein concentration was determined using the Bradford method. The extracted total protein was added to 6x sample buffer and denatured at 95°C for 10 minutes. The denatured protein was separated on a 4% (w / v) polyacrylamide gel at 80 V for 2 hours, followed by semi-dry transfer to an NC membrane. The transferred NC membrane was blocked with 5% skim milk powder. Detection was performed using a mouse-derived ORF4 protein polyclonal antibody as the primary antibody and HRP-conjugated Goat anti-Mouse IgG (H+L) (ABclonal, China) as the secondary antibody, with color development using BeyoECL Star (Beyotime, China).

[0067] Test results are as follows Figure 1 A and Figure 1 As shown in B. Figure 1 The experimental results show that the DH5α-ORF4-GFP strain successfully expressed the ORF4-GFP protein, which is the same as expected (116.3 kDa). Figure 2The results showed that the growth rate of the DH5α-ORF4-GFP strain was not significantly different from that of the E. coli DH5α strain, indicating that the insertion of the ORF4-GFP gene had no significant effect on Escherichia coli.

[0068] Example 2 Immunoprotection test of recombinant strains

[0069] 2.1 Test and detection methods

[0070] 2.1.1 Immunoprotective assay method

[0071] This experiment used "Hainan No. 1" tilapia (average body weight of about 20g), purchased from Guangxi Nanning Fish Fry Supply Co., Ltd., and verified by bacteriological analysis of brain and kidney tissue samples that there was no bacterial infection. They were temporarily raised in a disinfected fish pond (7.0 m long × 1.5 m wide × 1.2 m high) and fed with artificial compound feed every day, with oxygen provided 24 hours a day.

[0072] Previous immune challenge experiments showed no statistically significant difference in the immunoprotective rate of the DH5α-ORF4-GFP recombinant strain against streptococcal disease in tilapia when using three different immunization delivery methods: intraperitoneal injection, oral gavage, and immersion immunization (P > 0.05). Therefore, subsequent experiments uniformly adopted the immersion immunization method. (Animal Ethics Approval: Guangxi University, No. GXU-2025-282).

[0073] 2.1.1.1 Immunization dose

[0074] The experimental fish were acclimatized for 5 days in a recirculating aquaculture system (water temperature 28±1℃, dissolved oxygen ≥5 mg / L, pH 7.0~7.5, ammonia nitrogen ≤0.05 mg / L) (fasting for 1 day before immunization), with 6 groups (≥30 fish per group): low dose (1.5×10 2 CFU / mL), medium dose (1.5×10 3 CFU / mL), high dose (1.5×10) 4 CFU / mL) soaking group (soaking for 1 h), Escherichiacoli DH5α (1.5×10) 3 The groups were divided into three groups: 1 H (CFU / mL) immersion group (DH5α group), positive control group (no immunization, subsequent challenge) (PC group), and blank control group (no immunization, no challenge) (NC group). 35 days post-immunization, except for the blank control group, all groups received 1.5 × 10⁻⁶ CFU / mL immersion. 9CFU / mL GBS intraperitoneal injection challenge (0.2 mL / tail), record mortality for 14 days, calculate cumulative mortality rate and relative protection rate (RPS=[1-(cumulative mortality rate of immunized group / cumulative mortality rate of positive control group)]×100%).

[0075] 2.1.1.2 Number of immunizations

[0076] The rearing environment and immunization method were the same as in 2.1.1.1, with 6 groups (≥30 fish per group): three groups of DH5α-ORF4-GFP immunization groups, which received 1, 2 or 3 doses of immersion vaccine (1.5×10⁻⁶) administered at 14-day intervals. 4 CFU / mL; 1 hour), one group of Escherichia coli DH5α (DH5α) immunization groups received a single immersion vaccine (1.5 × 10⁻⁶ CFU / mL; 1 hour), 4 CFU / mL; 1 hour), positive control group, and blank control group. Seven days after the last immunization was completed, except for the blank control group, the patients were challenged with the virus as described above. Mortality was recorded after 14 days and RPS was calculated.

[0077] 2.1.1.3 Determination of the optimal number of immunizations, immunization dose, and protection rate

[0078] Let the experimental group (1.5×10) 4 CFU / mLDH5α-ORF4-GFP (single immersion immunization) and a blank control group (≥30 fish per group) were fed at a water temperature of 28±1℃ for 14 days. Six fish from each group were then sampled. The two groups were then challenged with the virus using the above method. Mortality was recorded and RPS was calculated after 14 days.

[0079] 2.1.2 Detection of routine blood tests and biochemical indicators

[0080] Blood was collected from tilapia in groups 2.1.1.3 using tail vein sampling. Blood physiological parameters were measured: the collected venous blood was mixed thoroughly in EDTA-K2 anticoagulant tubes to avoid hemolysis or air bubbles, and analyzed within 2 hours using a five-part differential automated blood cell analyzer. Biochemical parameters were measured: 200 μL of serum was aliquoted into sterile EP tubes and analyzed using a Celercare® M5 automated biochemical analyzer.

[0081] 2.1.3 Histochemical staining

[0082] Liver and kidney samples from three fish in each group (2.1.1.3) were fixed in 4% paraformaldehyde and embedded in paraffin. 6-micron sections were stained with hematoxylin and eosin (HE). Images were acquired using an LG-S80 digital tissue scanner (Servicebi, China).

[0083] 2.1.4 Gut microbiota analysis

[0084] Intestinal contents were collected from six fish in each group (2.1.1.3), and total genomic DNA was extracted using the OMEGA EZNA® Soil DNA kit. The V3-V4 hypervariable region of bacterial 16S rRNA was amplified by PCR using primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR products were analyzed using a Microplate reader (BioTek, FLx800) with the Quant-iT PicoGreends DNA assay kit, followed by Illumina NovaSeq sequencing at Shanghai Meiji Biotechnology Co., Ltd. to determine absolute quantitative microbial abundance. Paired-end reads were analyzed using QIIME 2. Bioinformatics analysis was performed using OmicStudio (https: / / www.omicstudio.cn / tool), including alpha diversity, principal coordinate analysis (PCoA), and linear discriminant analysis (LDA).

[0085] 2.2 Test Results

[0086] 2.2.1 Immunoassay Results

[0087] This study systematically investigated the immunoprotective effect of recombinant strain DH5α-ORF4-GFP against Streptococcus agalactiae (GBS) HN016 infection in tilapia through immunodose screening, optimization of immunization frequency, and validation experiments. In the dose screening experiment, doses of 1.5 × 10², 1.5 × 10³, and 1.5 × 10⁻⁶ were set. 4 Three gradient dose groups of CFU / mL were administered for single-dip immunization, with unimmunized fish serving as positive controls and E. coli DH5α strain as a control. 35 days post-immunization, fish were intraperitoneally injected with 3×10⁻⁶ CFU / mL. 8 CFU / tail challenge. Results showed that DH5α-ORF4-GFP immunization exhibited significant protective effects in all groups (P<0.05) (Table 3), and the protective effect was dose-dependent. The relative protection rate (RPS) of the high-dose immersion group reached 73.12%, while the RPS of the DH5α blank control group was 0, confirming its lack of anti-GBS activity. In the experiment based on the optimal dose and number of immunizations, the RPS of 1, 2, and 3 immersion immunizations (14-day interval) were 60.00%, 53.30%, and 48.00%, respectively (Table 4). The protective effect decreased with increasing number of immunizations, but statistical analysis showed that the number of immunizations had no significant effect on survival (P>0.05). Further validation experiments confirmed that 1.5×104 The recombinant protein saturation (RPS) of a single immersion immunization with CFU / mL was 65.3% (Table 5), which was not significantly different from the results of dose and number of immunizations (P>0.05). In conclusion, the recombinant strain DH5α-ORF4-GFP has a clear protective effect against GBS infection in tilapia, and at 1.5×10⁻⁶ CFU / mL... 4 A single immersion immunization with CFU / mL is the optimal approach, and its stability and reliability provide important technical support for the immune control of GBS disease in tilapia.

[0088] Table 3. Protective effect of recombinant strain DH5α-ORF4-GFP against Streptococcus agalactiae in tilapia at different immersion doses

[0089]

[0090] Table 4. Protective effect of recombinant strain DH5α-ORF4-GFP against Streptococcus agalactiae in tilapia after different immunization cycles.

[0091]

[0092] Table 5. Protective effect of a single immersion of DH5α-ORF4-GFP against Streptococcus agalactiae in tilapia.

[0093]

[0094] 2.2.2 Results of routine blood tests and blood biochemical characteristics

[0095] This experiment compared and analyzed the physiological and biochemical characteristics of tilapia in the control group and the immunized group through blood routine and blood biochemistry tests. The results showed that the immunization treatment had a multi-dimensional regulatory effect on the blood system of tilapia. As shown in Table 6, in terms of blood routine, there were significant differences in the platelet system (P<0.05). The total platelet count (PLT) in the MY group increased significantly by 161.40% compared with the CK group, and the monocyte ratio (+56.01%) and basophil ratio (+88.74%) increased simultaneously, indicating that the immune function was effectively activated. In contrast, the erythrocyte system (hemoglobin HGB decreased by 10.14% and red blood cell count RBC decreased by 5.92%) only showed slight physiological fluctuations. Blood biochemical indicators showed functionally correlated changes. As shown in Table 5, MY histoglobulin (GLO) increased by 25.90% and alanine aminotransferase (ALT) increased by 31.62%, reflecting enhanced synthesis of immune proteins and increased liver metabolic activity. At the same time, cholesterol (CHOL) decreased by 16.54% and glucose (GLU) fluctuated slightly, indicating that energy metabolism was shifted towards immune demand. Although some data showed slight decreases in total protein (TP) and albumin (ALB), the synergistic changes in core immune and metabolic indicators confirmed that immune treatment could maintain the basic stability of physiological functions while activating the tilapia's immune defense system, providing both hematological and biochemical evidence for the effectiveness of tilapia immune regulation.

[0096] Table 6. Results of routine blood tests (repeated n=6)

[0097]

[0098] The CK group served as the blank control group; the MY group served as the experimental group, with an immunization schedule of 1.5 × 10⁻⁶. 4 A single immersion immunization with CFU / mL for 14 days.

[0099] Table 7 Serum test results (repeated n=6)

[0100]

[0101] The CK group served as the blank control group; the MY group served as the experimental group, with an immunization schedule of 1.5 × 10⁻⁶. 4 A single immersion immunization with CFU / mL for 14 days.

[0102] 2.2.3 Histochemical staining

[0103] like Figure 2As shown, the degree of tissue damage in the MY group was not significantly different from that in the CK group. The sinusoidal structure of the liver was clear, and no obvious abnormalities were observed in the glomeruli and tubules of the kidneys. At the same time, blood biochemistry results showed no significant abnormalities in liver and kidney function indicators (such as ALT and CRE), indicating that the vaccination did not cause significant damage to the liver / kidneys of tilapia, demonstrating good biocompatibility.

[0104] 2.2.4 Effects of vaccines on the gut microbiota of tilapia

[0105] Vaccines can alter the composition of the gut microbiota, thus affecting host health. This study investigated the impact of this vaccine on the gut microbiota using 16S rRNA gene sequencing technology. Figure 4A , 4B In the study, no significant differences were found in the Chao and Shannon indices between the MY and CK groups, indicating that the microbial communities of the MY and CK groups did not change significantly. However, Figure 4C PCoA further revealed significant differences in community structure between the CK and MY groups. To determine the composition of the gut microbiota in the experimental groups, the gut microbiota composition at the phylum and genus levels was analyzed. Figure 4D , 4E The study presented the microbial composition of the CK and MY groups at the phylum and genus levels. At the phylum level, the dominant species in the CK group were *Fusobacteriota* (45.8%) and *Bacillota* (27.5%). Compared to the CK group, the MY group showed a decrease in the abundance of *Fusobacteriota* (27.3%) and an increase in the abundance of *Pseudomonadota* (23.9%). At the genus level, compared to the CK group, the MY group mainly showed a decrease in the abundance of *Cetobacterium* (from 45.8% to 27.3%). Linear discriminant analysis (LEfSe) was used to identify differentially expressed genera to investigate the characteristic changes in the gut microbiota in the experimental samples. Figure 4F The results showed that o_Enterobacterales, f_Enterobacteriaceae, and g_Ligilactobacillus were significantly enriched in the CK group; while c_Alphaproteobacteria, p_Actinomycetota, and c_Actinobacteria were significantly enriched in the MY group.

[0106] This invention utilizes CRISPR-Cas9 gene editing technology to successfully construct E. coli DH5α-ORF4-GFP expressing S. agalactiae ScpB protein, and determined its optimal immunization regimen to be 1.5 × 10⁻⁶. 4A single immersion at a concentration of CFU / mL was performed. This regimen not only induced effective immune protection (RPS up to 73.12%), but also verified its feasibility and application potential as a vaccine against Streptococcus agalactiae in tilapia through systematic immunization experiments, physiological and biochemical tests, and gut microbiota analysis, providing a new technical approach for the immunoprevention and control of streptococcal disease in tilapia.

[0107] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant bacterial strain, characterized in that: The recombinant strain was deposited at the China Center for Type Culture Collection on October 28, 2025, with accession number CCTCC NO: M 20252368, and its sequence listing is shown in SEQ ID NO:

1.

2. The method for preparing the recombinant strain according to claim 1, characterized in that... Includes the following steps: (1) Using Streptococcus agalactiae HN016 strain as a template, promoter fragment QDZ and fragment ORF4 were obtained by amplification; Using Escherichia coli DH5α strain as a template, fragments LHA and RHA were obtained by amplification; Using pBAV1K-T5-sfGFP plasmid as a template, the GFP gene fragment was obtained by amplification; Using pSC101-PBAD-sgRNA-Donor26D plasmid as a template, fragments PD1, PD2, PD3, PD4, and PD5 were obtained by amplification. The promoter fragment QDZ is shown in SEQ ID NO:2, the fragment ORF4 is shown in SEQ ID NO:3, the fragment LHA is shown in SEQ ID NO:4, the fragment RHA is shown in SEQ ID NO:5, the fragment GFP is shown in SEQ ID NO:6, the fragment PD1 is shown in SEQ ID NO:7, the fragment PD2 is shown in SEQ ID NO:8, the fragment PD3 is shown in SEQ ID NO:9, the fragment PD4 is shown in SEQ ID NO:10, and the fragment PD5 is shown in SEQ ID NO:11; (2) The promoter fragment QDZ, the fragment ORF4, the fragment LHA, the fragment RHA, the GFP, the fragment PD1, the fragment PD2, the fragment PD3, the fragment PD4 and the fragment PD5 are ligated and transformed into Escherichia coli to obtain plasmid pSC101-PBAD-sgRNA-ORF4-GFP, as shown in SEQ ID NO:12; (3) p15A-PBAD-Cas9-PT5-Redγβα and plasmid pSC101-PBAD-sgRNA-ORF4-GFP were transformed into Escherichia coli, and the recombinant strain DH5α-ORF4-GFP was induced and screened.

3. The method for preparing the recombinant strain according to claim 2, characterized in that: Step (1) specifically includes: Using Streptococcus agalactiae HN016 as a template, the S. agalactiae HN016 promoter fragment QDZ was amplified using primer QDZ, and the fragment ORF4 was amplified using primer ORF4. Using Escherichia coli DH5α strain as a template, fragment LHA was amplified using primer LHA, and fragment RHA was amplified using primer RHA. Using pBAV1K-T5-sfGFP plasmid as a template, the GFP gene fragment was amplified by primer GFP. Using pSC101-PBAD-sgRNA-Donor26D plasmid as a template, fragments PD1, PD2, PD3, PD4, and PD5 were amplified using primers yw1, yw2, yw3, yw4, and yw5, respectively. The forward and reverse primers of primer QDZ are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively; the forward and reverse primers of primer ORF4 are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively; the forward and reverse primers of primer LHA are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively; the forward and reverse primers of primer RHA are shown in SEQ ID NO:19 and SEQ ID NO:20, respectively; the forward and reverse primers of primer GFP are shown in SEQ ID NO:21 and SEQ ID NO:22, respectively; the forward and reverse primers of primer yw1 are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively; the forward and reverse primers of primer yw2 are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively; and the forward and reverse primers of primer yw3 are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively. As shown in NO:27 and SEQ ID NO:28, the forward and reverse primers of primer yw4 are shown in SEQ ID NO:29 and SEQ ID NO:30, respectively, and the forward and reverse primers of primer yw5 are shown in SEQ ID NO:31 and SEQ ID NO:32, respectively.

4. The method for preparing the recombinant strain according to claim 2, characterized in that: In step (2), the cloning reaction is carried out using the pEASY®-Uni Seamless Cloning and Assembly Kit.

5. The method for preparing the recombinant strain according to claim 2, characterized in that: In step (3), p15A-PBAD-Cas9-PT5-Redγβα and plasmid pSC101-PBAD-sgRNA-ORF4-GFP were transformed into Escherichia coli. The cells were then revived and cultured in LB medium at 30°C for 0.5-2 h. Subsequently, they were inoculated into LB agar plates containing 50-200 μg / mL ampicillin and 30-100 μg / mL kanamycin sulfate and cultured statically at 30°C for 36-60 h.

6. The method for preparing the recombinant strain according to claim 5, characterized in that: In step (3), the induced screening specifically includes: A single colony was picked from the LB agar plate and inoculated into LB medium. The culture was then shaken at 30°C for 2-4 hours to obtain culture A. Add 80-150 mg / mL ampicillin solution, 80-150 mg / mL kanamycin sulfate solution, and 80-150 mg / mL IPTG solution to culture A. The volume ratio of LB agar medium, ampicillin solution, kanamycin sulfate solution, and IPTG solution is 1000:0.5-5:0.2-3:1-6. Continue culturing at 30℃ for 0.6-2 h. Add 1 mol / L L-arabinose and continue induction culture at 30℃ for 12-30 h to obtain culture B. Culture B was transferred to LB medium and cultured at 37 °C with shaking for 20-36 h to obtain culture C. Dilute culture C in sterile water and spread it on LB agar plates containing 50 μg / mL kanamycin sulfate. Incubate at 40°C for 20-36 h. Pick a single colony that emits green fluorescence from the LB agar plate to obtain the recombinant strain DH5α-ORF4-GFP.

7. The method for preparing the recombinant strain according to claim 2, characterized in that: In step (2) and / or step (3), the Escherichia coli strain is Escherichia coli DH5α.

8. A tilapia agalactiae streptococcal vaccine, characterized in that: Includes the recombinant strain according to claim 1, or the recombinant strain prepared by the method of preparing the recombinant strain according to any one of claims 2-7.

9. The application of the recombinant strain according to claim 1 or the recombinant strain prepared by the preparation method of any one of claims 2-7 in the prevention and control of streptococcal disease in tilapia.

10. The use of the recombinant strain according to claim 1 or the recombinant strain prepared by the method of any one of claims 2-7 in the preparation of a tilapia streptococcal vaccine.

Images