Streptococcus agalactiae WC1535 delta cps delta hylB as well as construction method and application thereof

Abstract

The invention relates to the technical field of gene engineering, and particularly discloses streptococcus agalactiae WC1535 delta cps delta hylB as well as a construction method and application thereof. The streptococcus agalactiae WC1535 [delta] cps [delta] hylB is preserved in Guangdong Microbial Culture Collection Center on February 28, 2025, the preservation number is GDMCC NO.65955, and the address of the preservation unit is the 5th building, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. Compared with a traditional single-fragment deletion method, the toxicity of the deletion strain is not prone to being enhanced, and compared with a traditional injection immune vaccine, the gene deletion strain is used for preparing the tilapia oral live vaccine, the immune operation is simpler, and more labor cost is saved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to Streptococcus agalactiae WC1535ΔcpsΔhylB, a construction method thereof and an application thereof. Background Art

[0002] China is the largest producer, consumer and exporter of tilapia in the world. Due to the development of intensive farming and the deterioration of farming conditions, the incidence of tilapia streptococcosis has been relatively high in recent years in China. The main pathogen of tilapia streptococcosis is Streptococcus agalactiae. Clinically, the treatment of this disease mainly relies on antibiotics at present. However, the problems of drug resistance caused by the unreasonable use of antibiotics and food safety are becoming increasingly severe. There is an urgent need for safe, efficient and practical prevention and control measures. Among them, vaccines are important products for reducing and replacing antibiotics in the prevention and control of tilapia Streptococcus agalactiae disease.

[0003] Currently, the forms of Streptococcus agalactiae vaccines under research include inactivated vaccines, attenuated live vaccines, genetic engineering subunit vaccines, DNA nucleic acid vaccines, live bacterial vector vaccines, etc. However, there is only one commercial injectable inactivated vaccine against Streptococcus agalactiae in tilapia on the market at present, and it has obtained usage licenses in Indonesia, Brazil and a few countries in Central America (Liu et al., 2016). Due to problems such as cumbersome injection immunization operation, high labor cost and low profit margin of tilapia farming, it is expected that the acceptance of farmers will be very low.

[0004] Oral immunization has the advantages of simple immunization operation, being not restricted by the farming mode and low labor cost. Currently, there are very few oral vaccines for fish on the global market. The main reason is that the immunoprotective effect of oral immunization is relatively low. Existing literature has proved that Streptococcus agalactiae may cause tilapia to be infected with streptococcosis by invading through the intestine (Achado et al., 2019). Oral immunization simulates the natural infection route of Streptococcus agalactiae in fish, and oral immunization of Streptococcus agalactiae is feasible.

[0005] Capsular polysaccharide is a layer of mucus outside the cell wall of Streptococcus agalactiae, which can protect Streptococcus agalactiae from antibacterial or bactericidal substances and phagocytosis by host phagocytes, help the bacteria adhere to the surface of host cells and induce infection, and protect the bacteria from the killing effect of the host immune system. Studies have shown that the deletion of the capsular gene cluster of Streptococcus agalactiae can lead to a basic loss of its pathogenicity to tilapia (Zhang et al., 2019), and the live vaccine prepared from it can produce good protection when injected into tilapia (Zhang et al., 2020). Hyaluronate lyase (HAase) is widely present in streptococci (Yildirim et al., 2002). It can degrade the β-1,4 glycosidic bonds of hyaluronic acid and certain chondroitin sulfates through enzymatic action, helping Streptococcus agalactiae break through the biophysical barrier of host tissues (Li et al., 2001), and it is an important virulence factor of Streptococcus agalactiae (Girish et al., 2007).

[0006] Most of the in - research vaccine types for Streptococcus agalactiae in tilapia are injection - immunization vaccines, which are cumbersome to operate and have high labor costs. Moreover, the in - research live vaccines for Streptococcus agalactiae in tilapia are generally attenuated by continuous passage in vitro under antibiotic pressure or without antibiotic pressure, and the mechanism of virulence attenuation is unclear, or they are derived from single - sequence fragment deletions, with a high risk of virulence reversion. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to propose a Streptococcus agalactiae WC1535ΔcpsΔhylB, which uses genetic engineering methods to purposefully delete the related gene cluster for capsular synthesis and the hyaluronidase - encoding gene. Using this strain to prepare an oral live vaccine for tilapia to solve the problems of cumbersome immunization of current Streptococcus agalactiae injection - type vaccines and the safety risks of live vaccine immunization.

[0008] In addition, the present invention also proposes a preparation method and an application of the above - mentioned Streptococcus agalactiae WC1535ΔcpsΔhylB.

[0009] To solve the above - mentioned technical problems, the present invention adopts the following technical solutions to achieve:

[0010] A Streptococcus agalactiae WC1535ΔcpsΔhylB, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on February 28, 2025, with the deposit number: GDMCC NO.65955, and the address of the deposit unit is the 5th floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. Classification and naming: Streptococcus agalactiae.

[0011] The present invention provides a method for constructing the above-mentioned Streptococcus agalactiae WC1535ΔcpsΔhylB, which includes the following steps:

[0012] (1) Construction of Streptococcus agalactiae WC1535Δcps deletion strain: Using the genomic DNA of Streptococcus agalactiae wild strain WC1535 as a template, the upstream and downstream homologous arm sequences of the capsular synthesis gene cluster were amplified with primer pairs cps-up-F / R and cps-down-F / R respectively; pSET4S was double-digested with restriction enzymes EcoRⅠ and HindⅢ to linearize it; The upstream homologous arm, downstream homologous arm of the amplified capsular synthesis gene cluster and the double-digested linearized pSET4S vector were ligated using a seamless cloning kit, and the ligated vector was transformed into DH5α Escherichia coli competent cells. The culture was spread on an LB plate containing spectinomycin (100 μg / mL). Colony PCR was performed using universal primers M13F / M13R to screen for positive clones. After extracting the plasmid from the positive colonies, it was sent for sequencing verification. The constructed positive plasmid was named pSET4S-cps. Take 10 μL of the recombinant plasmid pSET4S-cps and add it to 100 μL of WC1535 competent cells, mix gently, incubate on ice for 30 min, then transfer it into an electroporation cuvette for electroporation. Immediately add 900 μL of BHI medium, transfer the mixture to a sterile centrifuge tube, and incubate at 28°C and 200 r / min for 2 - 3 h. The culture was spread on a BHI plate containing spectinomycin (100 μg / mL) and cultured overnight at 28°C until single colonies grew. Pick a single colony and inoculate it into 1 mL of BHI medium containing spectinomycin (100 μg / mL), and culture it inverted at 28°C for 12 h. Inoculate the overnight culture 1:100 (V / V) into 1 mL of BHI medium containing spectinomycin (100 μg / mL), and culture it with shaking at 37°C and 180 rpm. Inoculate the overnight culture 1:100 (V / V) into 1 mL of BHI medium, and culture it with shaking at 28°C and 180 rpm. From the 3rd generation onwards, take 100 μL of the bacterial liquid from each generation, dilute it with sterile PBS in gradient, and spread it on a BHI plate without antibiotics and a BHI plate containing spectinomycin (100 μg / mL), and culture it overnight at 37°C. When the colonies growing on the BHI plate without antibiotics are significantly more than those on the BHI plate containing spectinomycin (100 μg / mL), pick the suspected mutant strains that can grow on the BHI plate without antibiotics and inoculate them into 1 mL of BHI medium, and culture them overnight at 28°C and 180 rpm. Then perform PCR identification with cps-dF / cps-dR. The amplified fragment size of the gene deletion strain is 2089 bp, and the amplified fragment of the wild strain is enlarged and cannot be amplified within a limited amplification time, without amplification products. Send the PCR products of the suspected deletion strains for sequencing to compare whether the target fragment is deleted. Retain the Streptococcus agalactiae WC1535Δcps deletion strain with gene deletion after comparison;

[0013]

[0014] (2) Construction of Streptococcus agalactiae WC1535ΔcpsΔhylB deletion strain: Using the genomic DNA of Streptococcus agalactiae wild strain WC1535 as a template, the upstream and downstream homologous arm sequences of the hyaluronidase-encoding gene hylB were amplified with primer pairs hylB-up-F / R and hylB-down-F / R respectively; pSET4S was digested with restriction enzymes EcoRⅠ and HindⅢ to linearize it; the upstream homologous arm, downstream homologous arm of the hlyB gene and the digested and linearized pSET4S vector were ligated using a seamless cloning kit, and the ligated vector was transformed into DH5α Escherichia coli competent cells. The culture was spread on an LB plate containing spectinomycin (100 μg / mL). Colony PCR was performed using universal primers M13F / M13R to screen for positive clones. After extracting the plasmid from the positive colonies, it was sent for sequencing verification. The constructed positive plasmid was named pSET4S-hylB. Take 10 μL of the recombinant plasmid pSET4S-hylB and add it to 100 μL of WC1535Δcps competent cells, mix gently, incubate on ice for 30 min, then transfer it into an electroporation cuvette for electroporation. Immediately add 900 μL of BHI medium, transfer the mixture to a sterile centrifuge tube, and incubate at 28 °C and 200 r / min for 2 - 3 h. The culture was spread on a BHI plate containing spectinomycin (100 μg / mL) and cultured overnight at 28 °C until single colonies grew. Pick a single colony and inoculate it into 1 mL of BHI medium containing spectinomycin (100 μg / mL), and culture it inverted at 28 °C for 12 h. Inoculate the overnight culture 1:100 (V / V) into 1 mL of BHI medium containing spectinomycin (100 μg / mL) and culture it with shaking at 37 °C and 180 rpm. Inoculate the overnight culture 1:100 (V / V) into 1 mL of BHI medium and culture it with shaking at 28 °C and 180 rpm. From the third generation onwards, take 100 μL of the bacterial solution each generation, dilute it with sterile PBS in gradient, and spread it on BHI plates without antibiotics and BHI plates containing spectinomycin (100 μg / mL), and culture it overnight at 37 °C. When the number of colonies growing on the BHI plate without antibiotics is significantly more than that on the BHI plate containing spectinomycin (100 μg / mL), pick the suspected mutant strains that can grow on the BHI plate without antibiotics and inoculate them into 1 mL of BHI medium, and culture them overnight at 28 °C and 180 rpm. Perform PCR identification with hylB-dF / hylB-dR. The amplified fragment size of the gene deletion strain is 1933 bp, and the amplified fragment size of the wild strain is 3934 bp. Send the PCR products of the suspected deletion strains for sequencing to check whether the target fragment is deleted, and retain the Streptococcus agalactiae WC1535ΔcpsΔhylB deletion strain with gene deletion after comparison.

[0015]

[0016]

[0017] The present invention also provides an application of Streptococcus agalactiae WC1535ΔcpsΔhylB in the preparation of an oral live vaccine for tilapia.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The Streptococcus agalactiae gene deletion strain WC1535ΔcpsΔhylB of the present invention is constructed by the multi-fragment deletion method. Compared with the traditional single-fragment deletion method, the virulence of this deletion strain is not easily reverted to strong. Using this gene deletion strain to prepare an oral live vaccine for tilapia, compared with the traditional injection immunization vaccine, the immunization operation is simpler and the labor cost is more saved. Description of the Drawings

[0020] Figure 1 It is the PCR identification result of the deletion strain using the primers cps-dF / R; Note: M: DNA Marker DL5000; 1-3: WC1535ΔcpsΔhylB; 4-5: WC1535; 6-7: WC1535Δcps;

[0021] Figure 2 It is the PCR identification result of the deletion strain using the primers hylB-dF / R; Note: M: DNA Marker DL5000; 1-3: WC1535ΔcpsΔhylB; 4: WC1535Δcps; 5: WC1535;

[0022] Figure 3 It is the growth curves of the wild-type strain WT (WC1535), Δcps, and ΔcpsΔhylB of Streptococcus agalactiae.

[0023] Figure 4 It is the detection result of the hyaluronidase activity of the three strains by the plate method;

[0024] Figure 5 It is the relative activity of hyaluronidase of the wild-type strain WT (WC1535), Δcps, and ΔcpsΔhylB;

[0025] Figure 6 It is the survival curve graph of tilapia in different immunization dose groups;

[0026] Figure 7 It is the survival curve graph of tilapia in different immunization temperature groups. Detailed Embodiments

[0027] To enable those skilled in the art to more clearly and intuitively understand the present invention, the present invention will be further described below in conjunction with the drawings.

[0028] Construction of WC1535ΔcpsΔhylB gene deletion strain in Example 1

[0029] 1. Construction of Streptococcus agalactiae WC1535Δcps deletion strain

[0030] 1.1 Construction of knockout plasmid pSET4S-cps: Using the genomic DNA of Streptococcus agalactiae wild strain WC1535 as a template, the upstream and downstream homologous arm sequences of the capsular synthesis gene cluster were amplified using primer pairs cps-up-F / R and cps-down-F / R respectively; pSET4S was digested with EcoRⅠ and HindⅢ restriction endonucleases to linearize it; the upstream homologous arm, downstream homologous arm of the amplified capsular synthesis gene cluster and the double-digested linearized pSET4S vector were ligated using a seamless cloning kit, and the ligated vector was transformed into DH5α Escherichia coli competent cells. The culture was spread on an LB plate containing spectinomycin (100 μg / mL), and colony PCR was performed using universal primers M13F / M13R to screen for positive clones. After extracting the plasmid from the positive colonies, it was sent for sequencing verification. The constructed positive plasmid was named pSET4S-cps.

[0031] 1.2 Electrotransformation and screening of Streptococcus agalactiae WC1535Δcps deletion strain: Take 10 μL of the recombinant plasmid pSET4S-cps, add it to 100 μL of WC1535 competent cells, mix gently, incubate on ice for 30 min, then transfer to an electroporation cuvette for electrotransformation. Immediately add 900 μL of BHI medium, transfer the mixture to a sterile centrifuge tube, and incubate at 28°C with shaking at 200 r / min for 2 - 3 h. Spread the culture on a BHI plate containing spectinomycin (100 μg / mL) and incubate overnight at 28°C until single colonies appear. Pick a single colony and inoculate it into 1 mL of BHI medium containing spectinomycin (100 μg / mL), and incubate at 28°C in an inverted position for 12 h. Inoculate the overnight culture at a ratio of 1:100 (V / V) into 1 mL of BHI medium containing spectinomycin (100 μg / mL), and incubate at 37°C with shaking at 180 rpm. Inoculate the overnight culture at a ratio of 1:100 (V / V) into 1 mL of BHI medium, and incubate at 28°C with shaking at 180 rpm. From the third generation onwards, take 100 μL of the bacterial solution each generation, dilute it serially with sterile PBS, and spread it on BHI plates without antibiotics and BHI plates containing spectinomycin (100 μg / mL). Incubate overnight at 37°C. When the number of colonies growing on the BHI plate without antibiotics is significantly more than that on the BHI plate containing spectinomycin (100 μg / mL), pick the suspected mutant strains that can grow on the BHI plate without antibiotics into 1 mL of BHI medium, and incubate at 28°C with shaking at 180 rpm overnight. Then perform PCR identification using cps-dF / cps-dR (Reaction system: 25 μL of 2×Rapid Taq Master Mix, 2 μL of cps-dF, 2 μL of cps-dR, 1 μL of template, 20 μL of ddH2O. Reaction conditions: Pre-denaturation at 98°C for 10 min; denaturation at 95°C for 30 s, annealing at 59°C for 15 s, extension at 72°C for 1.5 min, 35 cycles of amplification; final extension at 72°C for 5 min). The amplified fragment size of the gene deletion strain is 2089 bp. The amplified fragment of the wild strain is too large and cannot be amplified within the limited amplification time, so there is no amplification product. Send the PCR product of the suspected deletion strain for sequencing to check whether the target fragment is deleted. Retain the screened Streptococcus agalactiae WC1535Δcps deletion strain with gene deletion.

[0032]

[0033] 2. Construction of Streptococcus agalactiae WC1535ΔcpsΔhylB deletion strain

[0034] 2.2 Construction of the knockout plasmid pSET4S-hylB: Using the whole genome DNA of Streptococcus agalactiae wild strain WC1535 as a template, the upstream and downstream homologous arm sequences of the hyaluronidase-encoding gene hylB were amplified with primer pairs hylB-up-F / R and hylB-down-F / R respectively; pSET4S was digested with restriction enzymes EcoRⅠ and HindⅢ to linearize it; the upstream homologous arm, downstream homologous arm of the hlyB gene and the double-digested and linearized pSET4S vector were ligated using a seamless cloning kit, and the ligated vector was transformed into DH5α Escherichia coli competent cells. The culture was spread on an LB plate containing spectinomycin (100 μg / mL), and colony PCR was performed using universal primers M13F / M13R to screen for positive clones. After extracting the plasmid from the positive colonies, it was sent for sequencing verification. The constructed positive plasmid was named pSET4S-hylB.

[0035] 1.2 Electrotransformation and screening of WC1535ΔcpsΔhylB deletion strain: Take 10 μL of the recombinant plasmid pSET4S-hylB and add it to 100 μL of WC1535Δcps competent cells, mix gently, incubate on ice for 30 min, then transfer to an electroporation cuvette for electrotransformation. Immediately add 900 μL of BHI medium, transfer the mixture to a sterile centrifuge tube, and incubate at 28 °C and 200 r / min for 2 - 3 h. Spread the culture on a BHI plate containing spectinomycin (100 μg / mL) and incubate overnight at 28 °C until single colonies appear. Pick a single colony and inoculate it into 1 mL of BHI medium containing spectinomycin (100 μg / mL), and incubate it in an inverted position at 28 °C for 12 h. Inoculate the overnight culture at a ratio of 1:100 (V / V) into 1 mL of BHI medium containing spectinomycin (100 μg / mL) and incubate at 37 °C with shaking at 180 rpm. Inoculate the overnight culture at a ratio of 1:100 (V / V) into 1 mL of BHI medium and incubate at 28 °C with shaking at 180 rpm. From the 3rd generation onwards, take 100 μL of the bacterial solution each generation, dilute it serially with sterile PBS, and spread it on BHI plates without antibiotics and BHI plates containing spectinomycin (100 μg / mL). Incubate overnight at 37 °C. When the number of colonies growing on the BHI plate without antibiotics is significantly more than that on the BHI plate containing spectinomycin (100 μg / mL), pick the suspected mutant strains that can grow on the BHI plate without antibiotics into 1 mL of BHI medium and incubate overnight at 28 °C with shaking at 180 rpm, and identify them by PCR using hylB-dF / hylB-dR (Reaction system: 25 μL of 2×Rapid Taq Master Mix, 2 μL of hylB-dF, 2 μL of hylB-dR, 1 μL of template, 20 μL of ddH2O. Reaction conditions: Pre-denaturation at 98 °C for 10 min; Denaturation at 95 °C for 30 s, Annealing at 60 °C for 15 s, Extension at 72 °C for 1.5 min, 35 cycles of amplification; Final extension at 72 °C for 5 min). The amplified fragment size of the gene deletion strain is 1933 bp, and that of the wild strain is 3934 bp. Send the PCR products of the suspected deletion strains for sequencing to check whether the target fragment is deleted. Retain the screened Streptococcus agalactiae WC1535ΔcpsΔhylB deletion strain.

[0036]

[0037] 3. Genetic stability analysis of the gene deletion strain WC1535ΔcpsΔhylB

[0038] Perform 10 consecutive passages and identify the genetic stability of this strain by PCR.

[0039] 4. Results

[0040] The deletion strains were verified by PCR using the primers cps-dF / cps-dR. A 2089bp band was amplified from WC1535Δcps and WC1535ΔcpsΔhylB, while no amplified band was obtained from the wild strain WC1535( Figure 1 ). The deletion strains were verified by PCR using the primers hylB-dF / hylB-dR. A 3933bp band was amplified from the wild strains WC1535 and WC1535Δcps, while a 1933bp band was amplified from WC1535ΔcpsΔhylB( Figure 2 ). ΔcpsΔhylB was subcultured continuously, and PCR identification was performed for each generation. After culturing for 10 generations, the PCR results showed no reverse mutation, indicating that the deletion strain had good genetic stability.

[0041] Example 2 Biological characteristic analysis of WC1535ΔcpsΔhylB

[0042] 1. Growth curve

[0043] The Streptococcus agalactiae wild strain WC1535 (abbreviated as WT), WC1535Δcps (abbreviated as Δcps), and WC1535ΔcpsΔhylB (abbreviated as ΔcpsΔhylB) were streaked on the BHI solid medium plate with an inoculation loop in a laminar flow hood and activated at 28°C for 24 h. Single colonies were picked and inoculated into a test tube containing 10 mL of BHI liquid medium, and cultured with shaking at 28°C and 200 r / min for 10 h. The above bacterial solutions were inoculated into BHI liquid medium at a ratio of 1:100. Then, 2 mL of each was taken and placed in a 12-well plate, and cultured with shaking at 28°C and 200 r / min. Three replicates were set for each strain. The absorbance value OD600 of the bacterial solution was measured with a spectrophotometer every 1 h for 13 consecutive hours to plot the growth curve.

[0044] 2. Detection of hyaluronidase activity

[0045] a. Plate method

[0046] Prepare BHI solid medium containing 0.04% hyaluronic acid (HA) and 5% bovine serum albumin (BSA) (HA was added before autoclaving, and BSA was filtered and sterilized with a 0.45μm filter membrane and added before pouring the plate). 10 μL of each of the three bacteria was dropped in different areas of the same plate and cultured overnight at 37°C. Then, the plate was soaked in 2 mol / L acetic acid for 15 minutes, and the degraded hyaluronic acid showed a transparent ring around the bacteria. This experiment was repeated three times.

[0047] b. Turbidity method

[0048] The acidified HA solution can form a stable colloidal solution with serum. When HA decomposes, the mixture with serum will become clear. After culturing each strain at 28 °C, 200 r / min, with shaking for 14 h, the supernatant was obtained by centrifugation as the test sample. The heat-inactivated bacterial liquid supernatant was used as the negative control group. BHI medium was used as the blank control group. The specific process was as follows: The HA working solution was added to a 96-well plate, 25 μL per well, and then 25 μL of the sample solution was added to each well. After thorough mixing, it was placed in a 37 °C water bath. After 30 min, 200 μL of the serum working solution was added to each well, mixed well, and left at room temperature for 30 min, and the OD640 was measured. Calculate the relative enzyme activity, relative enzyme activity rate = |OD 640 (blank control) - OD 640 (inactivated or non-inactivated culture supernatant)| / OD 640 (blank control) × 100%.

[0049] 3 Virulence determination

[0050] The LD of tilapia was determined using the wild strain WT, Δcps, and ΔcpsΔhylB respectively 50 Measurements. The wild strain WT bacterial liquid was prepared into four concentrations of 1.0×10 6 、1.0×10 7 、1.0×10 8 、1.0×10 9 CFU / mL, and the two bacterial liquids of Δcps and ΔcpsΔhylB were prepared into four concentrations of 5.0×10 9 、1.0×10 10 、2.5×10 10 、5.0×10 10 CFU / mL. 20 tilapia were intraperitoneally injected with 0.1 mL / tail of each concentration of the bacterial liquid, and the control group was injected with the same dose of PBS. During the experiment, the water temperature was maintained at 30 ± 1 °C, and the death numbers were continuously observed for 14 days and the mortality rates of each group were calculated.

[0051] 4. Results

[0052] 4.1 Growth curve

[0053] Compare the growth rates of WT, Δcps, and ΔcpsΔhylB at 28 °C. The strains all entered the logarithmic growth phase at about 4 h of culture and the stationary phase at about 10 h. The growth rates of the three strains were comparable( Figure 3 ).

[0054] 4.2 Detection of the ability to degrade hyaluronic acid

[0055] It is known that hyaluronic acid and serum can produce a turbidity reaction under acidic conditions. If hyaluronic acid is degraded by an enzyme, no turbid reaction phenomenon will occur. The results showed that both the WT strain and the Δcps strain could degrade hyaluronic acid to produce a clear zone, while the ΔcpsΔhylB strain could not degrade hyaluronic acid to produce a clear zone( Figure 4 ). The results of the turbidimetric method are shown in the table. Both the WT strain and the Δcps strain have a certain hyaluronidase activity, and the enzyme activity of ΔcpsΔhylB has been lost( Figure 5 ).

[0056] The results of both methods showed that the WT strain and the Δcps strain could degrade hyaluronic acid and had hyaluronidase activity. ΔcpsΔhylB could hardly degrade hyaluronic acid and had no hyaluronidase activity.

[0057] 4.3 Virulence evaluation

[0058] The LD 50 of the WT group, the Δcps group and the ΔcpsΔhylB group to tilapia were 5 8.0×10 9 CFU / tail, 9 3.2×10

[0059] CFU / tail and 50 7.1×10

[0060]

[0061]

[0062] CFU / tail (Table 1), further indicating that the virulence of ΔcpsΔhylB is extremely low.

[0063] Table 1 LD

[0064] Pick a single colony and inoculate it into a test tube containing 5 mL of BHI liquid medium. Incubate it at 28 °C with shaking at 200 r / min for 10 h.

[0065] Then take 1 mL of the bacterial liquid from the test tube and inoculate it into 100 mL of BHI liquid medium, and incubate it overnight under the same conditions. Centrifuge the bacterial liquid to discard the medium, and resuspend and wash the bacterial cells with PBS.

[0066] 2. Effect of immune concentration on immune effect

[0067] Randomly divide tilapia into 5 groups (4 experimental groups and 1 control group, with two rows in each group and 30 fish in each parallel). The immune bacterial liquid concentrations of the 4 experimental groups are set to be 7 1×108 、 1×10 9 、 1×10 10 CFU / mL, the gavage volume for each fish was 0.1 mL, and the control group was gavaged with an equal volume of 1×PBS buffer solution. During the immunization period, the water temperature was maintained at 30±1°C.

[0068] On the 28th day after gavage immunization, wild strain WT was used for gavage challenge. The challenge concentration was 10 9 CFU / mL, the dose was 0.1 mL / fish, and the water temperature during the challenge period was 34±1°C. The number of deaths was recorded daily for 14 days. Calculate the relative percent survival (RPS).

[0069] RPS = (1 - mortality rate of the immunized group / mortality rate of the control group) × 100%

[0070] 3. Effect of immunization temperature on immunization effect

[0071] The tilapia were randomly divided into 8 groups (4 experimental groups and 4 control groups, with 3 replicates in each group and 30 fish in each replicate). During the immunization period of each large group, the water temperatures were set at 22°C, 26°C, 30°C, and 34°C respectively. Each fish was gavaged with 0.1 mL of bacterial solution with a concentration of 10 9 CFU / mL, and the control group was gavaged with an equal volume of 1×PBS buffer solution. 21 days after immunization, the water temperature of each group was gradually adjusted to 34°C, with a maximum adjustment of 2°C per day.

[0072] On the 28th day after gavage immunization, wild strain WT was used for gavage challenge. The challenge concentration was 10 9 CFU / mL, the dose was 0.1 mL / fish, and the water temperature during the challenge period was 34±1°C. The number of deaths was recorded daily for 14 days. Calculate the relative percent survival (RPS).

[0073] 4. Results

[0074] The results of the immunization dose and immunization temperature tests showed that the relative protection rate increased with the increase in the concentration of ΔcpsΔhylB bacterial solution. Among them, when the immunization dose was 1×10 9 CFU / fish, the relative protection rate was the highest at 65.91% (Table 2, Figure 6 ). The RPS decreased with the increase in the immunization temperature. Among them, the RPS was the highest at 73.24% when the immunization temperature was 22°C and the lowest at 29.33% at 34°C (Table 3, Figure 7 ). The test results showed that when the immunization dose of the ΔcpsΔhylB strain bacterial solution was between 1×10 6 and 1×10 9When the concentration is CFU / tail, intragastric immunization with the WC1535ΔcpsΔhylB attenuated vaccine can stimulate Oreochromis niloticus to produce corresponding immune responses and protective effects. Moreover, the higher the immunization concentration and the lower the immunization temperature, the better the protective effect.

[0075] Table 2 Immunoprotective effects of different immunization dose groups

[0076]

[0077] Table 3 Immunoprotective effects of different immunization temperature groups

[0078]

[0079] Example 4 Oral immunization effect of sodium alginate (SA) and carboxylated chitosan (CCS) microcapsule live vaccine

[0080] 1. Vaccine preparation

[0081] Sodium alginate (SA), carboxylated chitosan (CCS), monascus pigment and methionine were added to sterilized ultrapure water for dissolution, so that their final mass-volume percentages reached 2%, 1%, 0.03% and 0.05% respectively. The freshly cultured ΔcpsΔhylB was centrifuged to collect the bacterial cells, and the bacterial cells were resuspended with the above solution to make the concentration of the bacteria reach 1×10 9 CFU / mL. The mixed solution was dropped into 3% CaCl2 solution with a syringe for solidification for 30 min. After fishing out and washing with sterilized ultrapure water, it was stored at 4°C. The prepared vaccine was named CCS-SA-ΔcpsΔhylB.

[0082] 2. Immunization experiment

[0083] The experimental fish were randomly divided into two groups (immunization group and control group), with three parallels in each group and 25 fish in each parallel. Each fish in the immunization group was quantitatively fed 0.1 g of CCS-SA-ΔcpsΔhylB, and the next day, the same amount of CCS-SA-ΔcpsΔhylB vaccine was fed continuously. The control group was fed the same amount of empty capsules without encapsulated live bacteria according to the same feeding procedure as above. The water temperature was 30°C during the immunization period. 21 days after immunization, the water temperature of each group was gradually adjusted to 34°C, with a maximum adjustment of 2°C per day. On the 28th day after immunization, the wild strain WT was used for intragastric challenge. The challenge concentration was 10 9 CFU / mL, and the dose was 0.1 mL / tail. The water temperature was 34±1°C during the challenge period. The number of deaths was recorded daily for 14 d. The relative percent survival (RPS) was calculated.

[0084] 3. Results

[0085] The average mortality rate after challenge with CCS-SA-ΔcpsΔhylB was 29.0%, and the average mortality rate of the control group was 60%. The relative protection rate of the immunized group was calculated to be 51.7%.

[0086] The above description of the embodiments is to enable those of ordinary skill in the art to understand and apply the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the embodiments herein, and the improvements and modifications made by those skilled in the art to the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. Streptococcus agalactiae WC1535ΔcpsΔhylB, characterized in that, It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on February 28, 2025, with the deposit number: GDMCC NO.65955, and the address of the deposit unit is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB according to claim 1, characterized in that, The following steps are involved: (1) Construction of the Δcps deletion strain of Streptococcus agalactiae WC1535: construct a recombinant plasmid pSET4S-cps, electro-transform the recombinant plasmid pSET4S-cps into WC1535 competent cells, quickly add BHI medium, transfer the mixed solution to a sterile centrifuge tube for incubation; culture the culture in BHI medium containing spectinomycin for three generations; from the third generation onwards, take the bacterial solution of each generation and dilute it with sterile PBS gradient, then spread it on BHI plates without antibiotics and BHI plates containing spectinomycin for overnight culture. When the number of colonies on the BHI plates without antibiotics is significantly more than that on the BHI plates containing spectinomycin, pick the suspected mutant strain that can grow on the BHI plates without antibiotics and culture it in BHI medium overnight, and perform PCR identification. Send the PCR product of the suspected deletion strain for sequencing to compare whether the target fragment is missing, and retain the Streptococcus agalactiae WC1535Δcps deletion strain with gene deletion after comparison; (2) Construction of the ΔcpsΔhylB deletion strain of Streptococcus agalactiae WC1535: construct the recombinant plasmid pSET4S-hylB, electro-transform the recombinant plasmid pSET4S-hylB into WC1535Δcps competent cells, quickly add BHI medium, and transfer the mixed solution to a sterile centrifuge tube for incubation; culture the culture in BHI medium containing spectinomycin for three generations; from the third generation onwards, take the bacterial solution of each generation and dilute it with sterile PBS gradient dilution and then spread it on BHI plates without antibiotics and BHI plates containing spectinomycin for overnight culture. When the number of colonies on the BHI plates without antibiotics is significantly more than that on the BHI plates containing spectinomycin, pick the suspected mutant strain that can grow on the BHI plates without antibiotics and culture it in BHI medium overnight, and perform PCR identification. The PCR product of the suspected deletion strain is sent for sequencing to compare whether the target fragment is missing, and retain the Streptococcus agalactiae WC1535ΔcpsΔhylB deletion strain with gene deletion after comparison.

3. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB as described in claim 2, characterized in that, The steps of constructing the recombinant plasmid pSET4S-cps include: using the whole genome DNA of the wild strain WC1535 of Streptococcus agalactiae as a template, respectively amplifying the upstream and downstream homologous arm sequences of the capsule synthesis gene cluster; double-digesting pSET4S with EcoRⅠ and HindⅢ restriction endonucleases to linearize it; using a seamless cloning kit to connect the upstream homologous arm and downstream homologous arm of the amplified capsule synthesis gene cluster and the double-digested linearized pSET4S vector, transforming the connected vector into DH5α Escherichia coli competent cells, spreading the culture on an LB plate containing spectinomycin, identifying and screening positive clones by colony PCR, extracting plasmids from positive colonies and sending them for sequencing verification.

4. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB as described in claim 3, characterized in that, The primers for amplifying the upstream and downstream homology arm sequences of the capsule synthesis gene cluster are cps-up-F / R and cps-down-F / R:

5. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB as described in claim 3, characterized in that, The PCR identification primers for the WC1535Δcps deletion strain are cps-dF / cps-dR:

6. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB as described in claim 2, characterized in that, The method for constructing the recombinant plasmid pSET4S-hylB includes: using the whole genome DNA of Streptococcus agalactiae wild strain WC1535 as a template to amplify the upstream and downstream homologous arm sequences of the hyaluronidase-encoding gene hylB; double-digesting pSET4S with EcoRⅠ and HindⅢ restriction endonucleases to linearize it; applying a seamless cloning kit to ligate the upstream homologous arm, downstream homologous arm of the hlyB gene and the double-digested and linearized pSET4S vector, transforming the ligated vector into DH5α Escherichia coli competent cells, spreading the culture on an LB plate containing spectinomycin, identifying positive clones by colony PCR, and sending the plasmid extracted from the positive colonies for sequencing verification.

7. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB according to claim 6, characterized in that, The primers for amplifying the upstream and downstream homologous arm sequences of the hyaluronidase-encoding gene hylB are hylB-up-F / R and hylB-down-F / R:

8. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB as described in claim 6, characterized in that, The PCR identification primers for the WC1535ΔcpsΔhylB deletion strain are hylB-dF / hylB-dR:

9. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB according to claim 2, characterized in that, In step (1), the process of culturing the culture in BHI medium containing spectinomycin is as follows: spreading the culture on an LB plate containing spectinomycin, culturing overnight at 28 °C until single colonies grow, picking single colonies into BHI medium containing spectinomycin, and culturing in an inverted position at 28 °C for 12 h; inoculating the overnight culture into BHI medium containing spectinomycin, culturing at 37 °C with shaking at 180 rpm; inoculating the overnight culture into BHI medium, culturing at 28 °C with shaking at 180 rpm.

10. The construction method of Streptococcus agalactiae WC1535ΔcpsΔhylB according to claim 2, characterized in that, In step (2), the process of culturing the culture in BHI medium containing spectinomycin is as follows: spreading the culture on an LB plate containing spectinomycin, culturing overnight at 28 °C until single colonies grow, picking single colonies into BHI medium containing spectinomycin, and culturing in an inverted position at 28 °C for 12 h; inoculating the overnight culture into BHI medium containing spectinomycin, culturing at 37 °C with shaking at 180 rpm; inoculating the overnight culture into BHI medium, culturing at 28 °C with shaking at 180 rpm.

11. The application of a Streptococcus agalactiae WC1535ΔcpsΔhylB as described in claim 1 in the preparation of an oral live vaccine for tilapia.

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