A type of agalactiae streptococcus Mu-20, its construction method and application

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a Streptococcus agalactiae Mu-20 and a construction method and application thereof. The present application constructs a fish-derived Streptococcus agalactiae strain with a capsule polysaccharide deficiency, no hyaluronidase activity, no hemolysis and no trehalose fermentation, and the strain number is Mu-20. The strain was preserved in the Guangdong Microbial Culture Collection Center on June 13, 2022, and the preservation number is GDMCC No: 62540. The strain does not carry a chloramphenicol resistance marker gene, has high biological safety, has no risk of virulence returning to strength, has high immunogenicity, can protect immunized fish against the invasion of Streptococcus agalactiae, and is easy to cultivate, simple to use and low in production cost. The strain involved in the present application can be used to prepare a live attenuated vaccine for fish streptococcosis, and has extremely high commercial application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a Streptococcus agalactiae Mu-20, its construction method, and its applications. Background Technology

[0002] Streptococcus agalactiae is the main pathogen causing streptococcal disease in fish.

[0003] Currently, the prevention and control of streptococcal disease in fish mainly relies on chemical and biological agents such as antibiotics, disinfectants, and probiotics. The extensive use of antibiotics during treatment not only leads to drug residues and food safety issues in aquatic products, but also increases the drug resistance of pathogens, significantly reducing the effectiveness of chemical treatments and further jeopardizing healthy aquaculture. Vaccines for streptococcal disease in fish are mainly inactivated vaccines, subunit vaccines, and live attenuated vaccines. Inactivated vaccines are limited in their use due to the need for injection, inconvenient administration, and unsatisfactory efficacy. Subunit vaccines have high production costs, weaker immune responses, and the need for injection, and therefore, no commercially available products have been developed and applied. Live attenuated vaccine strains have attracted widespread attention due to their low production costs, good immune efficacy, ease of use, and ability to induce maternal immunity. Currently, a search of academic papers and patents reveals live attenuated vaccine strains of *Streptococcus agalactiae* obtained through resistance pressure screening, live attenuated vaccine strains of *Streptococcus agalactiae* obtained through genetic engineering, and live attenuated vaccine strains obtained through natural selection. Therefore, developing effective live attenuated vaccines is of significant practical importance for the prevention of streptococcal disease in fish. Summary of the Invention

[0004] To address the aforementioned problems, one objective of this invention is to provide a fish-derived attenuated strain of Streptococcus agalactiae with a targeted gene deletion, namely Streptococcus agalactiae Mu-20. This strain exhibits low virulence, strong immunogenicity, and significantly reduced pathogenicity to fish, while remaining viable in fish for an extended period. It can stimulate the production of antibodies in fish, thus providing excellent protection.

[0005] The second objective of this invention is to provide a method for constructing the aforementioned Streptococcus agalactiae Mu-20.

[0006] A third objective of this invention is to provide the application of the aforementioned agalactia-resistant streptococcus Mu-20.

[0007] This invention is achieved through the following technical solution:

[0008] The Streptococcus agalactiae of this invention is a capsular-deleted attenuated strain of Streptococcus agalactiae from fish, which has 20 deleted genes. It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 13, 2022, with the accession number GDMCC No: 62540, and its English Latin name is Streptococcus agalactiae.

[0009] The method for constructing the agalactia-resistant streptococcus Mu-20 includes the following steps:

[0010] (1) The recombinant plasmid pSET4s-cps was electroporated into competent cells of fish-derived Streptococcus agalactiae strain WC1535, spread on BHI plates containing spectinomycin, and incubated upside down at 28±2℃ for 30-40h.

[0011] (2) Select the obtained single clones into BHI liquid medium and culture them at 28±2℃ to the logarithmic growth phase. Then dilute them 10,000 to 100,000 times, spread them on non-resistant BHI plates, and culture them upside down at 37℃ to obtain agalactia streptococci WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD.

[0012] Single clones were selected for colony PCR detection using cps-dF and cps-dR primers.

[0013] cps-dF: 5′-TATGATTAAACGAAACTTCCTC-3′;

[0014] cps-dR: 5′-AGCATTCAATAGCAGCACTCC-3′.

[0015] Positive knockout clones amplified to the expected PCR product size of 2213 bp. Sequencing analysis of the PCR amplification product of the deletion strain revealed a deletion of the cps gene cluster, indicating the successful construction of a cps-deleted mutant strain, named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD. Samples of strains WC1535 and WC1535△cps were fixed, negatively stained, and their capsules were observed using transmission electron microscopy. The results showed that the capsule of the mutant strain WC1535△cps was missing compared to the wild-type strain.

[0016] (3) The recombinant plasmid pSET4s-hylB was electroporated into fish-derived Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD competent cells, plated on BHI plates containing spectinomycin, and incubated upside down at 28±2℃ for 30-40h;

[0017] (4) Pick the single clones obtained from the above culture and put them into BHI liquid medium. Culture them at 28±2℃ to the logarithmic growth phase, then dilute them 10,000 to 100,000 times, spread them on BHI plates without resistance, and culture them upside down at 37℃; to obtain the agalactia-free streptococcus WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB.

[0018] Single clones were selected for colony PCR detection using hylB-dF and hylB-dR primers.

[0019] hylB-dF: 5′-TTCACCTGCCTCCAGTTTACCGC-3′;

[0020] hylB-dR: 5′-GTGAAGAACTCGGCTGGGAACC-3′.

[0021] Positive knockout clones could amplify PCR products of the expected size of 1659 bp. Sequencing analysis of the PCR amplification products of the deletion strain revealed that hylB was deleted in the mutant strain, indicating that the hylB-deleted mutant strain had been successfully constructed and named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB.

[0022] (5) The recombinant plasmid pSET4s-cyl was electroporated into fish-derived Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB competent cells, plated on BHI plates containing spectinomycin, and incubated upside down at 28±2℃ for 30-40 h;

[0023] (6) Pick the single clones obtained from the above culture and put them into BHI liquid medium. Culture them at 28±2℃ to the logarithmic growth phase, then dilute them 10,000 to 100,000 times, spread them on BHI plates without resistance, and culture them upside down at 37℃; to obtain the agalactia-free streptococcus WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF.

[0024] Single clones were selected for colony PCR detection using primers cyl-dF and cyl-dR.

[0025] cyl-dF: 5′-ATGCTGATGGAGAAAGGCTGAGTA-3′;

[0026] cyl-dR: 5′-CACTTAACTCATCACAGCCACCAC-3′.

[0027] Positive knockout clones could amplify PCR products of the expected size of 1716 bp. Sequencing analysis of the PCR amplification products of the deletion strain revealed that cylE and cylF were deleted in the mutant strain, indicating that a mutant strain with cylE and cylF deletions had been successfully constructed, named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF.

[0028] (7) The recombinant plasmid pSET4s-treP was electroporated into fish-derived Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF competent cells, plated on BHI plates containing spectinomycin, and incubated upside down at 28±2℃ for 30-40 h;

[0029] (8) Pick the single clones obtained from the above culture and put them into BHI liquid medium. Culture them at 28±2℃ to the logarithmic growth phase, then dilute them 10,000 to 100,000 times, spread them on BHI plates without resistance, and culture them upside down at 37℃; to obtain the agalactia-free streptococcus WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF△treP.

[0030] Single clones were selected for colony PCR detection using treP-dF and treP-dR primers.

[0031] treP-dF: 5′-ATTGTGCGTTCATTACATCCAGAG-3′;

[0032] treP-dR: 5′-AGGAATCATCTTGTCCAAAACTA-3′.

[0033] Positive knockout clones amplified to the expected PCR product size of 1847 bp. Sequencing analysis of the PCR amplification product of the deletion strain revealed a deletion of treP in the mutant strain, indicating that a treP-deficient mutant strain had been successfully constructed. This strain was named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF△treP. For ease of writing and communication, this deletion strain was renumbered as Mu-20, indicating that the deletion strain (Mutant) contains the deletion of 20 genes.

[0034] The detailed method for preparing the fish-derived Streptococcus agalactiae competent cells includes the following steps:

[0035] (1) Fish-derived agalactia streptococcus WC1535 was revived in BHI and cultured overnight at 28±2℃;

[0036] (2) Add 1 mL of overnight cultured bacterial solution to 80–120 mL of fresh BHI liquid medium, and culture on a shaker (180 r / min) at 28±2℃ for 5–6 h to expand the culture. When OD 600 When the concentration is 0.6, collect the bacterial cells;

[0037] (3) Centrifuge the expanded bacterial culture at 5000 r / min for 8-12 min and discard the supernatant;

[0038] (4) The obtained bacterial cells were gently suspended in 10-20 mL of glycerol with a final concentration of 10%-15%, and the supernatant was removed by centrifugation; thus, the fish-derived Streptococcus agalactiae competent cells were obtained.

[0039] Furthermore, repeat step (4) twice; gently suspend the bacterial cells in 1 mL of 15% glycerol and dispense them into 1.5 mL centrifuge tubes (100 μL / tube) to obtain competent Streptococcus agalactiae cells.

[0040] Preferably, the recombinant plasmids pSET4s-cps, pSET4s-hylB, pSET4s-cyl, and pSET4s-treP are obtained by seamless cloning of the pSET4S plasmid with cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments, respectively. Specifically:

[0041] (1) Take the double-digested pSET4S plasmid and the cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments respectively, and then add them to the Seamless cloning kit ligation system. After mixing, treat at 50±2℃ for 25-35 min to obtain the ligation solution. The volume ratio between the double-digested pSET4S plasmid and the cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments and the Seamless cloning kit ligation system is 6:3:1; that is, the volume ratio between the Seamless cloning kit ligation system and the gene fragments [cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down)] and the double-digested pSET4S plasmid is 1:3:6.

[0042] (2) Take the ligation solution obtained in step (1) and add it to 10 times the volume of the ligation solution to E. coli DH5α competent cells. Incubate on ice for 25-35 min, then heat shock at 42℃ for 90 s, incubate on ice for 2-3 min, and then recover at 37℃ for 25-35 min. Spread the mixture on LB plates containing spectinomycin. PCR screening is performed to select positive clones, and the positive PCR products are sequenced to verify the recombinant plasmids, which are named pSET4s-cps, pSET4s-hylB, pSET4s-cyl, and pSET4s-treP.

[0043] Preferably, the construction of the double-digested pSET4S plasmid is as follows:

[0044] The pSET4S plasmid was extracted and double-digested with Hind III and EcoRI at 37°C for 3–5 h. The product was then recovered using a 0.8% agarose gel to obtain the double-digested pSET4S plasmid. The digestion system consisted of: 1 μg pSET4S, 1 U Hind III, 1 U EcoRI, 3 μL 10×Buffer, and ddH2O added to a final volume of 30 μL.

[0045] Preferably, the construction of the cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments is as follows:

[0046] The cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments were obtained by Guangzhou Aiji Biotechnology Co., Ltd. through gene synthesis.

[0047] The application of the aforementioned Streptococcus agalactiae Mu-20 involves using it for the prevention and control of streptococcal disease in fish, particularly as a live attenuated vaccine for streptococcal disease in fish.

[0048] The mutant strain Mu-20 described in this invention cannot synthesize a capsule normally due to the deletion of gene clusters related to capsule synthesis. Since the capsule is the most important virulence factor for *Streptococcus agalactiae*, this capsule-deficient strain significantly reduces its pathogenicity. The Mu-20 strain cannot synthesize hyaluronidase due to the deletion of hylB. Since hyaluronidase can degrade hyaluronic acid between tissue cells, *Streptococcus agalactiae* utilizes secreted hyaluronidase to facilitate diffusion between tissue cells, thereby increasing the strain's pathogenicity. Therefore, the Mu-20 strain's ability to diffuse between tissues due to the lack of hyaluronidase activity is significantly reduced, further weakening the pathogenicity of *Streptococcus agalactiae*. The Mu-20 strain lacks hemolytic activity due to the deletion of cylE and cylF. Because cylE and cylF are involved in the synthesis of rhamnolipids with hemolytic activity, the deletion of cylE and cylF prevents *Streptococcus agalactiae* from hemolysing on blood agar plates, further reducing the strain's pathogenicity. Furthermore, the Mu-20 strain is unable to utilize extracellular trehalose due to the absence of the trehalose transport-related gene treP, which reduces the strain's carbon source utilization capacity and affects its survival ability in the natural environment. This indicates that the release of the Mu-20 strain into the natural environment will not cause negative impacts on the microecological environment, and its biosafety is higher.

[0049] The main advantages of this invention are as follows: This invention deletes all 20 genes from strain WC1535, including the capsular polysaccharide gene cluster cps, the hyaluronidase gene hylB, the hemolysin synthesis-related genes cylE and cylF, and the trehalose utilization-related gene treP, to construct the mutant strain Mu-20. This mutant strain exhibits a significantly reduced virulence against tilapia and zebrafish. Because the Mu-20 strain lacks important virulence-related genes and carbon source utilization-related genes from *Streptococcus agalactiae*, it has high safety and no risk of virulence reversion. This strain has high immunogenicity, effectively protecting immune tilapia against *Streptococcus agalactiae* invasion. Furthermore, the large-scale fermentation process is simple and the production cost is low, laying a solid foundation for effectively controlling the occurrence and spread of streptococcal disease in fish in my country. Intraperitoneal injection of the Mu-20 mutant strain into tilapia allows it to survive in the fish for an average of more than 11 days, effectively stimulating tilapia to develop immunity over a prolonged period. Furthermore, when tilapia (10g, 50g, and 100g) were artificially infected with this deletion strain, no tilapia died and no clinical symptoms were observed. Attached Figure Description

[0050] Figure 1 Images of hyaluronic acid degradation from wild-type Streptococcus agalactiae strain WC1535 and deletion strain Mu-20. The wild-type strain has a clear zone, while the deletion strain does not.

[0051] Figure 2 Images showing the hemolytic activity of wild-type strain WC1535 and deletion strain Mu-20 on defatted sheep blood agar plates from Columbia. The wild-type strain shows a hemolytic zone; the deletion strain does not.

[0052] Figure 3 The figure shows the results of the trehalose utilization test of wild-type strain WC1535 and deletion strain Mu-20. The wild-type strain is positive and the deletion strain is negative.

[0053] Figure 4 Images showing the growth competition test between the Mu-20 deletion strain and pathogenic bacteria. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to specific embodiments to help those skilled in the art to understand the present invention, but without limiting the scope of protection of the present invention.

[0055] 1. Materials

[0056] Streptococcus agalactiae strain WC1535 was isolated from diseased tilapia by the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences. Its GenBank accession number is CP016501.2. Plasmids pSET4S and pSET5S were kindly provided by Dr. Daisuke Takamatsu of the National Institute of Animal Health, Japan. Reference: Takamatsu D., Osaki M., Sekizaki T. Thermosensitive suicide vectors for gene replacement in Streptococcus suis. Plasmid. 2001, 46, 140–148.

[0057] 2. Primer design and synthesis

[0058] The genome sequence of *Streptococcus agalactiae* strain WC1535 (Genbank accession number: CP016501) was used as a template. Primers for PCR amplification of the target fragments were designed using Primer 5.0 software. Using cps-dF and cps-dR primers, PCR amplification was used to detect the deletion of the cps gene cluster; using hylB-dF and hylB-dR primers, PCR amplification was used to detect the deletion of hylB; using cyl-dF and cyl-dR primers, PCR amplification was used to detect the deletion of cylE and cylF; and using treP-dF and treP-dF primers, PCR amplification was used to detect the deletion of treP.

[0059] The primer sequences are as follows:

[0060] cps-dF: 5′-TATGATTAAACGAAACTTCCTC-3′ (SEQ ID NO.1)

[0061] cps-dR: 5′-AGCATTCAATAGCAGCACTCC-3′ (SEQ ID NO.2)

[0062] hylB-dF: 5′-TTCACCTGCCTCCAGTTTACCGC-3′ (SEQ ID NO.3)

[0063] hylB-dR: 5′-GTGAAGAACTCGGCTGGGAACC-3′ (SEQ ID NO.4)

[0064] cyl-dF: 5′-ATGCTGATGGAGAAAGGCTGAGTA-3′ (SEQ ID NO.5)

[0065] cyl-dR: 5′-CACTTAACTCATCACAGCCACCAC-3′ (SEQ ID NO.6)

[0066] treP-dF: 5′-ATTGTGCGTTCATTACATCCAGAG-3′ (SEQ ID NO.7)

[0067] treP-dR: 5′-AGGAATCATCTTGTCCAAAACTA-3′ (SEQ ID NO. 8)

[0068] 3. Synthesis of cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments.

[0069] Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to synthesize cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments based on the provided gene sequences.

[0070] 4. Construction of recombinant plasmids pSET4s-cps, pSET4s-hylB, pSET4s-cyl, and pSET4s-treP

[0071] 4.1 Double digestion of pSET4S plasmid

[0072] pSET4S plasmid was extracted and digested with Hind III and EcoRI. The digestion system is shown in Table 1. The digestion was carried out at 37℃ for 3 h, and the digestion products were recovered by gel extraction.

[0073] Table 1. Double enzyme digestion system of pSET4S plasmid

[0074]

[0075]

[0076] 4.2 Seamless cloning of pSET4S plasmid with cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments: 6 μL of double-digested pSET4S plasmid was mixed with 3 μL of each of the cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments, and then 1 μL of the seamless cloning kit was added. After mixing, the mixture was treated at 50°C for 30 min.

[0077] 4.3 Transformation and screening of pSET4s-cps, pSET4s-hylB, pSET4s-cyl and pSET4s-treP recombinant plasmids

[0078] 10 μL of ligation buffer was added to 100 μL of *E. coli* DH5α competent cells. After incubation on ice for 30 min, the cells were heat-shocked at 42°C for 90 s, incubated on ice for 2 min, and then recovered at 37°C for 30 min. The resulting plasmids were then plated on LB agar plates containing chloramphenicol. Positive clones were screened by PCR, and the positive PCR products were sequenced for verification. The obtained recombinant plasmids were named pSET4s-cps, pSET4s-hylB, pSET4s-cyl, and pSET4s-treP, respectively.

[0079] 5. Construction of the multi-gene deletion Streptococcus agalactiae Mu-20 strain

[0080] The specific steps for preparing a fish-derived Streptococcus agalactiae WC1535 competent cell are as follows:

[0081] (1) Fish-derived agalactia streptococcus WC1535 was revived in BHI (brain heart infusion medium) and incubated overnight at 30°C;

[0082] (2) Add 1 mL of the overnight culture to 100 mL of fresh BHI liquid medium and culture on a shaker at 30°C for 5–6 hours to increase OD. 600 When the concentration is 0.6, collect the bacterial cells;

[0083] (3) Centrifuge the expanded bacterial culture at 5000 r / min for 10 min and discard the supernatant;

[0084] (4) The bacterial cells were gently suspended in 20 mL of 15% glycerol, centrifuged, and the supernatant was discarded;

[0085] (5) Repeat step (4) twice;

[0086] (6) Gently suspend the bacterial cells in 1 mL of 15% glycerol and dispense them into 1.5 mL centrifuge tubes to obtain competent Streptococcus agalactiae cells.

[0087] The gene deletion method for Mu-20 strain is as follows:

[0088] The recombinant plasmid pSET4s-cps was electroporated into competent *Streptococcus agalactiae* strain WC1535 cells, then plated on BHI plates containing spectinomycin and incubated upside down at 28°C for 36 h. Single colonies were picked and cultured in BHI liquid medium at 28°C to the logarithmic growth phase, then diluted 10,000–10,000 times and plated on antibiotic-free BHI plates, incubated upside down at 37°C. Single colonies were picked for colony PCR detection using primers cps-dF: 5′-TATGATTAAACGAAACTTCCTC-3′ (SEQ ID NO. 1) and cps-dR: 5′-AGCATTCAATAGCAGCACTCC-3′ (SEQ ID NO. 2). Positive knockout strains amplified the expected PCR product of 2213 bp. The positive PCR product was sequenced and verified, and the results showed that the cps gene cluster deletion mutant strain was successfully constructed and named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD.

[0089] Similar to the preparation method for competent cells of the wild-type strain WC1535, competent cells of the *Streptococcus agalactiae* strain with a deleted cps gene cluster were prepared. The recombinant plasmid pSET4s-hylB was electroporated into WC1535 competent cells containing ΔcpsA, ΔcpsB, ΔcpsC, ΔcpsD, ΔcpsE, ΔcpsF, ΔcpsG, ΔcpsH, ΔcpsI, ΔcpsJ, ΔcpsK, ΔcpsL, ΔneuA, ΔneuB, ΔneuC, and ΔneuD. These cells were then plated on BHI plates containing spectinomycin and incubated upside down at 28°C for 36 hours. Single colonies were picked and cultured in BHI liquid medium at 28°C until the logarithmic growth phase. The culture was then diluted 10,000–10,000 times and plated on antibiotic-free BHI plates, incubated upside down at 37°C. The *Streptococcus agalactiae* WC1535 was obtained with the following Δcps: AΔcpsBΔcpsCΔcpsDΔcpsEΔcpsFΔcpsGΔcpsHΔcpsIΔcpsJΔcpsKΔcpsLΔneuAΔneuBΔneuCΔneuDΔhylB. Furthermore, this strain exhibited no clear zone on hyaluronic acid medium, indicating that it lacked hyaluronidase activity. Figure 1Single clones were selected for colony PCR detection using primers hylB-dF: 5′-TTCACCTGCCTCCAGTTTACCGC-3′ (SEQ ID NO.3) and hylB-dR: 5′-GTGAAGAACTCGGCTGGGAACC-3′ (SEQ ID NO.4). Positive knockout strains amplified PCR products of the expected size of 1659 bp. Sequencing of the positive PCR products confirmed the successful construction of the cps gene cluster and hylB deletion mutant strain, which was named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB.

[0090] Similarly, competent cells of *Streptococcus agalactiae* WC1535ΔcpsAΔcpsBΔcpsCΔcpsDΔcpsEΔcpsFΔcpsGΔcpsHΔcpsIΔcpsJΔcpsKΔcpsLΔneuAΔneuBΔneuCΔneuDΔhylB were prepared. The recombinant plasmid pSET4s-cyl was electroporated into these competent cells, which were then plated on BHI plates containing spectinomycin and incubated upside down at 28°C for 36 h. Single colonies were picked and cultured in BHI liquid medium at 28°C until the logarithmic growth phase. The culture was then diluted 10,000–10,000 times, plated on antibiotic-free BHI plates, and incubated upside down at 37°C. The *Streptococcus agalactiae* WC1535 was obtained with the following Δcps: AΔcpsBΔcpsCΔcpsDΔcpsEΔcpsFΔcpsGΔcpsHΔcpsIΔcpsJΔcpsKΔcpsLΔneuAΔneuBΔneuCΔneuDΔhylBΔcylEΔcylF. Furthermore, this deleted strain did not exhibit a hemolytic zone on Columbia defatted sheep blood agar plates. Figure 2Single clones were selected for colony PCR detection using primers cyl-dF: 5′-ATGCTGATGGAGAAAGGCTGAGTA-3′ (SEQ ID NO.5) and cyl-dR: 5′-CACTTAACTCATCACAGCCACCAC-3′ (SEQ ID NO.6). Positive knockout strains amplified PCR products of the expected size of 1798 bp. Sequencing of the positive PCR products confirmed the successful construction of mutant strains lacking the cps gene cluster, hylB, cylE, and cylF, which were named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF.

[0091] Similarly, competent cells of *Streptococcus agalactiae* WC1535ΔcpsAΔcpsBΔcpsCΔcpsDΔcpsEΔcpsFΔcpsGΔcpsHΔcpsIΔcpsJΔcpsKΔcpsLΔneuAΔneuBΔneuCΔneuDΔhylBΔcylEΔcylF were prepared. The recombinant plasmid pSET4s-treP was electroporated into these competent cells, which were then plated on BHI plates containing spectinomycin and incubated upside down at 28°C for 36 h. Single colonies were picked and cultured in BHI liquid medium at 28°C until the logarithmic growth phase. The culture was then diluted 10,000–10,000 times, plated on antibiotic-free BHI plates, and incubated upside down at 37°C. The *Streptococcus agalactiae* WC1535 was obtained with the following parameters: ΔcpsAΔcpsBΔcpsCΔcpsDΔcpsEΔcpsFΔcpsGΔcpsHΔcpsIΔcpsJΔcpsKΔcpsLΔneuAΔneuBΔneuCΔneuDΔhylBΔcylEΔcylFΔtreP. Carbon source utilization experiments showed that this deletion strain could not utilize trehalose (…). Figure 3Single clones were selected for colony PCR detection using primers treP-dF: 5′-ATTGTGCGTTCATTACATCCAGAG-3′ (SEQ ID NO.7) and treP-dR: 5′-AGGAATCATCTTGTCCAAAACTA-3′ (SEQ ID NO.8). Positive knockout strains amplified PCR products of the expected size of 1847 bp. Sequencing of the positive PCR products confirmed the successful construction of mutant strains lacking the cps gene cluster, hylB, cylE, cylF, and treP. These mutants were named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF△treP. Because the naming number of this deletion strain is too long, it has been renumbered as Mu-20, which is the first two letters of Mutant plus the number of deleted genes, 20.

[0092] The reaction system for the above PCR detection is as follows: 25 μL of 2×PCR Master Mix, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of target bacterial culture, and finally, double-distilled water (H2O) to a final volume of 50 μL.

[0093] The PCR amplification procedure is as follows:

[0094] a. React at 95℃ for 4 min, repeat once.

[0095] b. React at 94℃ for 30 seconds, repeat 30 times.

[0096] c. React at 56℃ for 30 seconds, repeat 30 times.

[0097] d. React at 72℃ for 150 seconds, repeat 30 times.

[0098] e. React at 72℃ for 10 min, repeat once.

[0099] f. React at 4℃ for 10 min, repeat once.

[0100] Finally, the PCR amplification results were detected by 1% agarose gel electrophoresis, and positive PCR products could be sequenced for analysis.

[0101] 6. Competition test between the deletion strain Mu-20 and other strains.

[0102] To evaluate the competitive ability of the deletion strain Mu-20 when co-cultured with other strains, the deletion strain Mu-20 was inoculated with wild-type Streptococcus agalactiae WC1535, Aeromonas hydrophila GKY1, Aeromonas vesiculosus Ci1701, Edwardsiella tarda GD1701, Escherichia coli ATCC 25922, and Shigella spp. LS1 at a 1:1 ratio. After activation, the deletion strains Mu-20, Streptococcus agalactiae WC1535, Aeromonas hydrophila GKY1, Aeromonas vesiculosus Ci1701, Edwardsiella tarda GD1701, Escherichia coli ATCC 25922, and Shigella spp. LS1 were inoculated into BHI liquid medium and cultured overnight at 30°C using a shaker. The bacterial concentrations were then measured. The deletion strain Mu-20 was inoculated into six 200 mL bottles of 0.2×BHI liquid medium, with a final bacterial concentration of 1×10⁻⁶. 6 CFU / mL. *Streptococcus agalactiae* WC1535, *Aeromonas hydrophila* GKY1, *Aeromonas vesiculosus* Ci1701, *Edwardsiella tarda* GD1701, *Escherichia coli* ATCC 25922, and *Shigella spp.* LS1 were inoculated into 200 mL of the above 0.2×BHI liquid medium, respectively, to achieve a final bacterial concentration of 1×10⁻⁶ CFU / mL for WC1535, GKY1, Ci1701, GD1701, ATCC25922, and LS1. 6 The concentration of CFU / mL was determined, meaning the ratio of the deleted strain Mu-20 to its competing strain was 1:1. After inoculation, the bacterial culture was placed in a 28°C incubator for static incubation.

[0103] After co-culturing the deletion strain Mu-20 with competing strains for 1, 3, 5, 7, 10, and 15 days, the bacterial cultures were diluted and plated onto BHI plates for colony counting. Since the colony morphology of the deletion strain Mu-20 differed significantly from that of *Aeromonas hydrophila* GKY1, *Aeromonas vesiculosus* Ci1701, *Edwardsiella tarda* GD1701, *Escherichia coli* ATCC 25922, and *O. shigella* LS1 strains, it was easily distinguishable by visual observation and therefore colony counting could be performed directly. Considering that the colony morphology of the deletion strain Mu-20 was similar to that of the wild-type *Streptococcus agalactiae* strain, making visual identification impossible, PCR detection was used in this experiment. The bacterial cultures of the deletion strain Mu-20 and the wild-type strain were plated, and then 48 colonies were randomly selected for PCR amplification. The PCR amplification primers were cps-dF and cps-dR. When the extension time was 3 min, the PCR results showed positive fragments for the deletion strain Mu-20, and the PCR results showed negative fragments for the wild-type strain.

[0104] Based on the colony count results above, a survival trend of the vaccine strain and the competing strain was plotted with time on the x-axis and the percentage of Mu-20 deletion strain / competing strain on the y-axis. Figure 4 ).Depend on Figure 4 It can be seen that when the deletion strain Mu-20 was co-cultured with competing strains, the percentage of deletion strain Mu-20 began to decrease on day 3 and continued to decrease thereafter. By day 15, the percentages of deletion strain Mu-20 were 0.16%, 0%, 0.09%, 0%, 0.11%, and 0.24% of strains LS1, ATCC25922, GYK1, Ci1701, GD1701, and WC1535, respectively.

[0105] When the deletion strain Mu-20 was co-cultured with *Aeromonas hydrophila*, *Aeromonas vesiculosus*, *Edwardsiella tarda*, *Escherichia coli*, and *O. shigella*, the abundance of Mu-20 gradually decreased in the later stages of culture. In some experimental groups, Mu-20 was even undetectable after 15 days of co-culture. These results indicate that the deletion strain Mu-20 is at a competitive disadvantage when co-cultured with these bacteria, further demonstrating its high biosafety in application.

[0106] 7. Pathogenicity experiment of the Mu-20 deletion strain in tilapia.

[0107] Tilapia (approximately 20g in weight) were randomly divided into 4 groups of 30 fish each. The deletion strain Mu-20 and the wild-type strain WC1535 were resuspended in sterile PBS and then diluted to 2.0 × 10⁻⁶. 9 CFU / mL, 0.1 mL of bacterial suspension was injected intraperitoneally into each tilapia. The fish were raised at a water temperature of 28±0.5℃, and the survival and disease incidence were observed. The time and number of deaths were recorded, and the observation continued for 2 weeks. The results showed that the optimal concentration of the wild-type strain was 2.0 × 10⁻⁶ CFU / mL. 9 The mortality rate of tilapia was 100% at CFU / mL; the intraperitoneal injection concentration of the deletion strain Mu-20 was 2.0 × 10⁻⁶. 9 No tilapia died at CFU / mL levels, and no clinical symptoms were observed. The experimental results indicate that even at infection doses as high as 2.0 × 10⁻⁶ CFU / mL, [the disease / infection] was effective. 9 At CFU / mL, the deletion strain Mu-20 was not pathogenic to tilapia.

[0108] 8. Survival experiment of the Mu-20 deletion strain in tilapia

[0109] The deletion strain Mu-20, cultured to the logarithmic growth phase, was diluted to 1×10⁻⁶ with sterile PBS. 9CFU / mL, tilapia (30 fish, 0.1 mL / fish) were injected intraperitoneally and reared at a water temperature of 28±0.5℃. Three tilapia were dissected every two days, and their spleens and kidneys were collected to isolate bacteria. This process was repeated for 20 days. The results showed that the Mu-20 deletion strain had the longest survival time in the spleen and kidneys of tilapia, exceeding 11 days. This indicates that the Mu-20 deletion strain can continuously stimulate antibody production in tilapia, and this deletion strain has significant application value as a live attenuated vaccine against Streptococcus agalactiae.

[0110] 9. Immunoprotective experiment of the Mu-20 deletion strain against tilapia

[0111] Tilapia (approximately 16.8 ± 4.2 g) were randomly divided into two groups of 100 fish each. The deletion strain Mu-20 was cultured in liquid BHI until it reached a stable growth phase, then resuspended in sterile PBS and diluted to 1.0 × 10⁻⁶. 9 CFU / mL. Tilapia in the immunized group were injected intraperitoneally with 0.1 mL of Mu-20 bacterial suspension, while tilapia in the control group were injected intraperitoneally with 0.1 mL of sterile PBS buffer. They were housed at a water temperature of 25.0±1.0℃ for 28 consecutive days. Then, a challenge experiment was conducted. Ninety tilapia from each group were artificially infected. Both the immunized and control groups were injected intraperitoneally with the wild-type strain WC1535 at a bacterial concentration of 3.5 × 10⁻⁶ CFU / mL. 8 CFU / mL. The mortality rate of tilapia in the immunized group and the control group was calculated separately, and then the relative immunoprotection rate (RPS) was calculated. The results showed that the RPS of tilapia immunized with the Mu-20 deletion strain was 92.4%, indicating that the Mu-20 deletion strain had a good immunoprotective effect on tilapia.

[0112] In this invention, the cps-(up-down) gene fragment sequence synthesized is as follows:

[0113]

[0114] The sequence of the hylB-(up-down) gene fragment synthesized by the gene:

[0115]

[0116]

[0117] The treP-(up-down) gene fragment sequence of gene synthesis:

[0118]

[0119] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the embodiments described herein, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.

Claims

1. A Streptococcus agalactiae Mu-20, characterized by, The attenuated fish-derived Streptococcus agalactiae strain with a capsule deletion has 20 deletion genes, and was preserved in the Guangdong Microbial Culture Collection Center on June 13, 2022, with a preservation number of GDMCC No: 62540 and an English Latin name of Streptococcus agalactiae .

2. The method for constructing Streptococcus agalactiae Mu-20 according to claim 1, wherein, comprising the following steps: (1) the recombinant plasmid pSET4s-cps obtained by seamless cloning of pSET4S plasmid and cps-(up-down) gene fragment with sequence as shown in SEQ ID NO. 9 is electrotransformed into fish-derived Streptococcus agalactiae WC1535 strain competent cells, coated on BHI plates containing spectinomycin, and placed at 28±2°C for inverted culture for 30-40 h; (2) The monoclonal obtained in the culture is picked again and inoculated into BHI liquid medium, and cultured at 28±2°C to the logarithmic growth phase, then diluted by 10000-100000 times, and inoculated on BHI plates without resistance and cultured at 37°C upside down; to obtain Streptococcus agalactiae WC1535Δ cpsA Δ cpsB Δ cpsC Δ cpsD Δ cpsE Δ cpsF Δ cpsG Δ cpsH Δ cpsI Δ cpsJ Δ cpsK Δ cpsL Δ neuA Δ neuB Δ neuC Δ neuD ; (3) The recombinant plasmid pSET4s-hylB obtained by seamless cloning of pSET4S plasmid and hylB-(up-down) gene fragment with sequence as shown in SEQ ID NO. 10 is electrotransformed into fish-derived Streptococcus agalactiae WC1535△ cpsA △ cpsB △ cpsC △ cpsD △ cpsE △ cpsF △ cpsG △ cpsH △ cpsI △ cpsJ △ cpsK △ cpsL △ neuA △ neuB △ neuC △ neuD The competent cells are spread on BHI plates containing spectinomycin and placed at 28±2°C for inverted culture for 30-40 h. (4) picking the monoclonal obtained in step (3) to BHI liquid medium, culturing at 28±2°C to logarithmic growth phase, then diluting 10000-100000 times, coating on non-resistant BHI plate, and culturing at 37°C upside down; obtaining the non-Streptococcus lactis WC1535△ cpsA △ cpsB △ cpsC △ cpsD △ cpsE △ cpsF △ cpsG △ cpsH △ cpsI △ cpsJ △ cpsK △ cpsL △ neuA △ neuB △ neuC △ neuD △ hylB ; (5) The recombinant plasmid pSET4s-cyl obtained by seamless cloning of the pSET4S plasmid and the cyl-(up-down) gene fragment with the sequence shown as SEQ ID NO. 11 is electrotransformed into fish-derived Streptococcus agalactiae WC1535△ cpsA △ cpsB △ cpsC △ cpsD △ cpsE △ cpsF △ cpsG △ cpsH △ cpsI △ cpsJ △ cpsK △ cpsL △ neuA △ neuB △ neuC △ neuD △ hylB The competent cells are coated on BHI plates containing spectinomycin and placed at 28±2°C for inverted culture for 30-40 h; (6) picking the monoclonal obtained in step (5) to BHI liquid medium, culturing at 28±2°C to logarithmic growth phase, then diluting 10000-100000 times, coating on non-resistant BHI plate, and culturing at 37°C upside down; obtaining the non-lactococcus WC1535△ cpsA △ cpsB △ cpsC △ cpsD △ cpsE △ cpsF △ cpsG △ cpsH △ cpsI △ cpsJ △ cpsK △ cpsL △ neuA △ neuB △ neuC △ neuD △ hylB △ cylE △ cylF ; (7) The recombinant plasmid pSET4s-treP obtained by seamless cloning of pSET4S plasmid and treP-(up-down) gene fragment with sequence as shown in SEQ ID NO. 12 is electrotransformed into fish-derived Streptococcus agalactiae WC1535△ cpsA △ cpsB △ cpsC △ cpsD △ cpsE △ cpsF △ cpsG △ cpsH △ cpsI △ cpsJ △ cpsK △ cpsL △ neuA △ neuB △ neuC △ neuD △ hylB △ cylE △ cylF The competent cells are coated on BHI plates containing spectinomycin and placed at 28±2°C for inverted culture for 30-40 h; (8) picking the monoclonal obtained in step (7) to BHI liquid medium, culturing at 28±2℃ to logarithmic growth phase, then diluting 10000-100000 times, coating on non-resistant BHI plate, and culturing at 37℃ upside down; obtaining the non-Streptococcus lactis WC1535△ cpsA △ cpsB △ cpsC △ cpsD △ cpsE △ cpsF △ cpsG △ cpsH △ cpsI △ cpsJ △ cpsK △ cpsL △ neuA △ neuB △ neuC △ neuD △ hylB △ cylE △ cylF △ treP , obtaining Mu-20 strain.

3. The method for constructing Streptococcus agalactiae Mu-20 according to claim 2, wherein After step (2), it further comprises the step of picking the single colony obtained in step (1) for colony PCR detection, and the primers are: cps-dF: 5'-TATGATTAAACGAAACTTCCTC-3' (SEQ ID NO. 1); cps-dR: 5'-AGCATTCAATAGCAGCACTCC-3' (SEQ ID NO. 2).

4. The method for constructing Streptococcus agalactiae Mu-20 according to claim 2, wherein After step (4), the single colony obtained in step (3) is picked for colony PCR detection, and the primers are: hylB-dF: 5'-TTCACCTGCCTCCAGTTTACCGC-3' (SEQ ID NO. 3); hylB-dR: 5'-GTGAAGAACTCGGCTGGGAACC-3' (SEQ ID NO. 4).

5. The method for constructing Streptococcus agalactiae Mu-20 according to claim 2, wherein After step (6), the single colony obtained in step (5) is picked for colony PCR detection, and the primers are: cyl-dF: 5'-ATGCTGATGGAGAAAGGCTGAGTA-3' (SEQ ID NO. 5); cyl-dR: 5'-CACTTAACTCATCACAGCCACCAC-3' (SEQ ID NO. 6).

6. The method for constructing Streptococcus agalactiae Mu-20 according to claim 2, wherein After step (8), the single clone obtained in step (7) is picked for colony PCR detection, and the primers are as follows: treP-dF: 5'-ATTGTGCGTTCATTACATCCAGAG-3' (SEQ ID NO. 7); treP-dR: 5'-AGGAATCATCTTGTCCAAAACTA-3' (SEQ ID NO. 8).

7. The method for constructing Streptococcus agalactiae Mu-20 according to claim 2, wherein The preparation of the fish-derived S. agalactiae WC1535 strain competent cells comprises the following steps: (1) The fish-derived S. agalactiae WC1535 strain is recovered in BHI and cultured at 28±2°C overnight; (2) Take 1 mL of the overnight culture and add to 80-120 mL of fresh BHI liquid medium, and incubate at 28±2°C for 5-6 h. When the OD 600 =0.6, collect the bacterial cells. (3) The bacterial solution obtained by the expansion culture is centrifuged at 5000 r / min for 8-12 min, and the supernatant is discarded; (4) The obtained bacterial cells are suspended with 10-20 mL of glycerol with a final concentration of 10%-15%, and the supernatant is removed by centrifugation; thus, the fish-derived S. agalactiae competent cells are obtained.

8. The method for constructing Streptococcus agalactiae Mu-20 according to claim 2, wherein comprises the following steps: (1) The pSET4S plasmid after double enzyme digestion is mixed with the cps-(up-down) gene fragment with the sequence of SEQ ID NO. 9, the hylB-(up-down) gene fragment with the sequence of SEQ ID NO. 10, the cyl-(up-down) gene fragment with the sequence of SEQ ID NO. 11, and the treP-(up-down) gene fragment with the sequence of SEQ ID NO. 12, then the Seamless cloning kit connecting system is added, and after mixing, it is treated at 50±2°C for 25-35 min to obtain a connecting liquid; the volume ratio between the pSET4S plasmid after double enzyme digestion and the cps, hylB, cyl and treP gene fragments and the Seamless cloning kit connecting system is 6:3:1; (2) The connecting liquid obtained in step (1) is added to 10 times the volume of E. coli DH5α competent cells, incubated on ice for 25-35 min, then heat shocked at 42°C for 90 s, incubated on ice for 2-3 min, then recovered at 37°C for 25-35 min, and then plated on LB plates containing spectinomycin; positive clones are selected by PCR, and the positive PCR products are sequenced to verify that the obtained recombinant plasmids are named pSET4s-cps, pSET4s-hylB, pSET4s-cyl and pSET4s-treP.

9. The method for constructing Streptococcus agalactiae Mu-20 according to claim 8, wherein, The construction of the pSET4S plasmid after double enzyme digestion comprises the following steps: The pSET4S plasmid is extracted, double enzyme cut by Hind III and EcoR I, enzyme cut at 37°C for 3-5 h, and finally the product is recovered by 0.8% agarose gel, to obtain the double enzyme cut pSET4S plasmid; wherein the enzyme cutting system is: pSET4S 1 μg, Hind III 1 U, EcoR I 1 U, 10×Buffer 3 μL, supplemented with ddH2O to 30 μL.

10. The use of the Streptococcus agalactiae Mu-20 of claim 1 in the preparation of a live attenuated vaccine for fish streptococcosis.

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