Streptococcus agalactiae Mu-20 and construction method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to streptococcus agalactiae Mu-20 and a construction method and application thereof. The fish-derived streptococcus agalactiae which is lack of capsular polysaccharide, free of hyaluronidase activity, free of hemolysis and negative in trehalose fermentation is constructed, the strain number is Mu-20, the fish-derived streptococcus agalactiae is preserved in Guangdong Microbial Culture Collection Center on June 13, 2022, and the preservation number is GDMCC No: 62540; the strain does not carry chloramphenicol resistance marker genes, is high in biological safety, has no risk of virulence enhancement, is high in immunogenicity, and can protect immunized fish from resisting invasion of streptococcus agalactiae, and meanwhile, the attenuated live vaccine is easy to culture, simple to use and low in production cost; the strain provided by the invention can be used for preparing attenuated live vaccines for fish streptococcosis, and has extremely high commercial application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to Streptococcus agalactiae Mu-20, a construction method thereof, and applications thereof. Background Art

[0002] Streptococcus agalactiae is the main pathogen of streptococcosis in fish.

[0003] At present, the prevention and control of streptococcosis in fish mainly rely on chemical and biological agents such as antibiotics, disinfectants, and probiotics. Due to the extensive use of antibiotics during the treatment process, it will not only cause drug residues, resulting in problems with the quality and safety of aquatic products, but also lead to an increase in the drug resistance of pathogenic bacteria, a significant decline in the treatment effect of chemical drugs, and further endanger the healthy aquaculture of fish. The vaccines for streptococcosis in fish mainly include inactivated vaccines, subunit vaccines, and attenuated vaccines. The use of inactivated vaccines is limited due to reasons such as the need for injection, inconvenient operation, and unsatisfactory effects. Subunit vaccines have problems such as high production costs, weak immune responses, and the need for injection immunization, and there are currently no commercial product developments and applications. Attenuated vaccine strains have been widely concerned due to their advantages such as low production costs, good immune effects, convenient use, and the ability to induce maternal immunity. Currently, through the retrieval of academic papers and patents, attenuated vaccine strains of Streptococcus agalactiae obtained by resistance pressure screening, attenuated vaccine strains of Streptococcus agalactiae obtained by genetic engineering modification, and attenuated vaccine strains obtained by natural screening have been found. Therefore, the development of effective attenuated live vaccines has important practical significance for the prevention of streptococcosis in fish. Summary of the Invention

[0004] In order to solve the above problems, one of the objectives of the present invention is to provide an attenuated strain of fish-derived Streptococcus agalactiae with targeted gene deletion, namely Streptococcus agalactiae Mu-20. This strain has low toxicity, strong immunogenicity, and significantly reduced pathogenicity to fish, but can survive in fish for a long time, can stimulate fish to produce antibodies, and has a good protective effect.

[0005] Another objective of the present invention is to provide a construction method for the above-mentioned Streptococcus agalactiae Mu-20.

[0006] Another objective of the present invention is to provide the applications of the above-mentioned Streptococcus agalactiae Mu-20.

[0007] The present invention is achieved through the following technical solutions:

[0008] The Streptococcus agalactiae Mu-20 of the present invention is an attenuated strain of fish-derived Streptococcus agalactiae lacking a capsule, with 20 deleted genes. It was deposited at the Guangdong Provincial Microbial Culture Collection Center on June 13, 2022, with the deposit number GDMCC No: 62540, and the English Latin name is Streptococcus agalactiae.

[0009] The construction method of Streptococcus agalactiae Mu-20 includes the following steps:

[0010] (1) Electrotransform the recombinant plasmid pSET4s-cps into the competent cells of Streptococcus agalactiae strain WC1535 from fish, coat it on a BHI plate containing spectinomycin, and incubate it upside down at 28 ± 2 °C for 30 - 40 h;

[0011] (2) Then pick the obtained monoclonal colonies into BHI liquid medium, culture them at 28 ± 2 °C until the logarithmic growth phase, then dilute them 10,000 - 100,000 times, coat them on a non-resistant BHI plate, and incubate them upside down at 37 °C; obtain Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD.

[0012] Pick monoclonal colonies for colony PCR detection, and the primers are cps-dF and cps-dR.

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

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

[0015] Positive knockout clones can amplify a PCR product with an expected size of 2213 bp. The PCR amplification products of the deletion strain were sequenced and analyzed. The results showed that the cps gene cluster of the mutant strain had a deletion, indicating that the cps deletion mutant strain had been successfully constructed, named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD. Samples of strain WC1535 and strain WC1535△cps were fixed and negatively stained respectively, and the capsules of the strains were observed by transmission electron microscopy. The results showed that compared with the wild strain, the capsule of the mutant strain WC1535△cps was missing.

[0016] (3) Electrotransform the recombinant plasmid pSET4s-hylB into the competent cells of Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD from fish, coat it on a BHI plate containing spectinomycin, and incubate it upside down at 28 ± 2 °C for 30 - 40 h;

[0017] (4) Pick the monoclonal colonies obtained from the above culture and transfer them into BHI liquid medium. Culture at 28 ± 2 °C until the logarithmic growth phase, then dilute 10,000 - 100,000 times, spread on a BHI plate without resistance, and culture in an inverted position at 37 °C; obtain the Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB.

[0018] Pick monoclonal colonies for colony PCR detection, and the primers are hylB-dF and hylB-dR.

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

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

[0021] Positive knockout clones can amplify a PCR product with an expected size of 1659 bp. The PCR amplification products of the deletion strain were subjected to sequencing analysis. The results showed that hylB of the mutant strain was deleted, indicating that a mutant strain with hylB deletion had been successfully constructed, named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB.

[0022] (5) Electrotransform the recombinant plasmid pSET4s-cyl into the competent cells of Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB from fish, spread on a BHI plate containing spectinomycin, and culture in an inverted position at 28 ± 2 °C for 30 - 40 h;

[0023] (6) Pick the monoclonal colonies obtained from the above culture and transfer them into BHI liquid medium. Culture at 28 ± 2 °C until the logarithmic growth phase, then dilute 10,000 - 100,000 times, spread on a BHI plate without resistance, and culture in an inverted position at 37 °C; obtain the Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF.

[0024] Single colonies were picked for colony PCR detection, and the primers were cyl-dF and cyl-dR.

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

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

[0027] Positive knockout clones could amplify a PCR product with an expected size of 1716 bp. The PCR amplification products of the deletion strains were subjected to sequencing analysis. It was found that cylE and cylF of the mutant strains were deleted, indicating that the mutant strains with deletions of cylE and cylF 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 electrotransformed into the competent cells of Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF, and then spread on BHI plates containing spectinomycin and incubated at 28 ± 2 °C in an inverted position for 30 - 40 h;

[0029] (8) Single colonies obtained from the above culture were picked into BHI liquid medium and cultured at 28 ± 2 °C until the logarithmic growth phase, and then diluted 10,000 - 100,000 times and spread on BHI plates without resistance and incubated at 37 °C in an inverted position; the Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF△treP was obtained.

[0030] Single colonies were picked for colony PCR detection, and the primers were treP-dF and treP-dR.

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

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

[0033] Positive knockout clones could amplify a PCR product with an expected size of 1847 bp. The PCR amplification products of the deletion strain were sequenced, and it was found that treP of the mutant strain was deleted, indicating that a mutant strain with treP deletion 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△treP. Considering the convenience of writing and communication, this deletion strain was re-numbered as Mu-20, indicating that the deletion strain (Mutant) had 20 gene deletions.

[0034] Specifically, the method for preparing the Streptococcus agalactiae competent cells from fish is as follows:

[0035] (1) Streptococcus agalactiae WC1535 from fish was resuscitated in BHI and cultured overnight at 28 ± 2°C.

[0036] (2) 1 mL of the overnight culture was added to 80 - 120 mL of fresh BHI liquid medium, and cultured with shaking (180 r / min) at 28 ± 2°C for 5 - 6 h. When OD 600 = 0.6, the bacteria were collected.

[0037] (3) The cultured bacteria were centrifuged at 5000 r / min for 8 - 12 min, and the supernatant was discarded.

[0038] (4) The obtained bacteria were gently suspended with 10 - 20 mL of glycerol with a final concentration of 10% - 15%, and centrifuged to remove the supernatant; the Streptococcus agalactiae competent cells from fish were obtained.

[0039] Furthermore, step (4) was repeated twice; the bacteria were gently suspended with 1 mL of 15% glycerol and aliquoted into 1.5 mL centrifuge tubes (100 μL / tube), which were the Streptococcus agalactiae competent cells.

[0040] Preferably, the recombinant plasmids pSET4s-cps, pSET4s-hylB, pSET4s-cyl, and pSET4s-treP were respectively obtained by seamless cloning of the pSET4S plasmid and the cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments, 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, then add them to the Seamless cloning kit ligation system, mix well, and incubate at 50 ± 2 °C for 25 - 35 min to obtain the ligation solution; the volume ratio of the double-digested pSET4S plasmid to 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 of the Seamless cloning kit ligation system to 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), add it to Escherichia coli DH5α competent cells with a volume 10 times that of the ligation solution, incubate on ice for 25 - 35 min, then perform heat shock at 42 °C for 90 s, incubate on ice for 2 - 3 min, and then recover at 37 °C for 25 - 35 min, and spread it on an LB plate containing spectinomycin; screen positive clones by PCR, and verify the positive PCR products by sequencing to obtain the recombinant plasmids named pSET4s-cps, pSET4s-hylB, pSET4s-cyl, and pSET4s-treP.

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

[0044] Extract the pSET4S plasmid, perform double digestion with Hind III and EcoR I, digest at 37 °C for 3 - 5 h, and finally recover the product with 0.8% agarose gel to obtain the double-digested pSET4S plasmid; the digestion system is as follows: 1 μg of pSET4S, 1 U of Hind III, 1 U of EcoR I, 3 μL of 10× Buffer, and make up to 30 μL with ddH2O.

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

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

[0047] The application of Streptococcus agalactiae Mu-20 is to apply the Streptococcus agalactiae Mu-20 to the prevention and control of streptococcosis in fish, especially to the live attenuated vaccine against streptococcosis in fish.

[0048] The mutant strain Mu-20 of the present invention cannot synthesize the capsule normally due to the deletion of the gene cluster related to capsule synthesis, and the capsule is the most important virulence factor of Streptococcus agalactiae. Therefore, this capsule-deficient strain will significantly reduce the pathogenicity of the strain itself. The Mu-20 strain cannot synthesize hyaluronidase due to the deletion of hylB. Since hyaluronidase can degrade hyaluronic acid between tissue cells, Streptococcus agalactiae uses the secreted hyaluronidase to facilitate diffusion between tissue cells, thereby increasing the pathogenicity of the strain. Therefore, the Mu-20 strain has a significantly reduced ability to spread between tissues due to the lack of hyaluronidase activity, further weakening the pathogenicity of Streptococcus agalactiae. The Mu-20 strain does not have hemolytic activity after the deletion of cylE and cylF, because cylE and cylF are involved in the synthesis of rhamnolipids with hemolytic activity. Therefore, Streptococcus agalactiae does not hemolyze on blood agar plates after the deletion of cylE and cylF, further reducing the pathogenicity of the strain. In addition, the Mu-20 strain cannot utilize extracellular trehalose due to the deletion of the trehalose transport-related gene treP, thereby reducing the ability of the strain to utilize carbon sources and affecting the survival ability of the strain in the natural environment, indicating that the Mu-20 strain released into the natural environment will not cause negative impacts on the microecological environment and has higher biosafety.

[0049] Main advantages of the present invention: In the present invention, 20 genes including the capsular polysaccharide gene cluster cps of strain WC1535, the hyaluronidase gene hylB, the hemolysin synthesis-related genes cylE and cylF, and the trehalose utilization-related gene treP are all deleted to construct a mutant strain Mu-20. The virulence of this mutant strain to tilapia and zebrafish is significantly decreased. Since the strain Mu-20 lacks important virulence-related genes and carbon source utilization-related genes of Streptococcus agalactiae, the safety of this strain is high and there is no risk of reversion to high virulence. This strain has high immunogenicity and can protect immunized tilapia from the invasion of Streptococcus agalactiae. Moreover, the large-scale fermentation process is simple and the production cost is low, laying a solid foundation for effectively controlling the occurrence and prevalence of fish streptococcosis in China. The mutant strain Mu-20 is intraperitoneally injected to infect tilapia. This deletion mutant strain can survive in tilapia for more than 11 days on average and can effectively stimulate tilapia to produce immunity for a long time. And when this deletion strain artificially infects tilapia (10g, 50g, and 100g), no tilapia dies and no clinical symptoms are shown. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Pictures of hyaluronic acid degradation of the wild strain WC1535 and the deletion strain Mu-20 of Streptococcus agalactiae. There is a clear zone for the wild strain, and no clear zone for the deletion strain;

[0051] Figure 2 Pictures of the hemolytic activity of the wild strain WC1535 and the deletion strain Mu-20 on Columbia defatted sheep blood agar plates. There is a hemolytic zone for the wild strain; no hemolytic zone for the deletion strain

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

[0053] Figure 4 Pictures of the growth competition test of the deletion strain Mu-20 and other strains of pathogenic bacteria. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention will be further described in detail below in combination with the specific embodiments to help those skilled in the art understand the present invention, but it does not limit the protection scope 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. Cited 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] Based on the genomic sequence reference of Streptococcus agalactiae strain WC1535 (Genbank accession number: CP016501), primers for PCR amplification of the target fragment were designed using Primer 5.0 software with the genomic sequence of this strain as the template. Using cps-dF and cps-dR as primers, the deletion of the cps gene cluster was detected by PCR amplification; using hylB-dF and hylB-dR as primers, the deletion of hylB was detected by PCR amplification; using cyl-dF and cyl-dR as primers, the deletions of cylE and cylF were detected by PCR amplification; using treP-dF and treP-dF as primers, the deletion of treP was detected by PCR amplification.

[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] Entrust Guangzhou Aiji Biotechnology Co., Ltd. to synthesize cps-(up-down), hylB-(up-down), cyl-(up-down) and treP-(up-down) gene fragments respectively according to the provided gene sequences.

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

[0071] 4.1 Double digestion of pSET4S plasmid

[0072] Extract the pSET4S plasmid, perform double digestion with Hind III and EcoR I, and the digestion system is shown in Table 1. Digest at 37°C for 3 h, and recover the digested products by gel extraction.

[0073] Table 1 Double digestion reaction 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 respectively. Take 6 μL of the double-digested pSET4S plasmid and mix it with cps-(up-down), hylB-(up-down), cyl-(up-down) and treP-(up-down) gene fragments (3 μL each), then add 1 μL of the Seamless cloning kit ligation system, mix well and incubate at 50°C for 30 min.

[0077] 4. Transformation and Screening of Recombinant Plasmids pSET4s-cps, pSET4s-hylB, pSET4s-cyl and pSET4s-treP

[0078] Take 10 μL of the ligation solution and add it to 100 μL of Escherichia coli DH5α competent cells. Incubate on ice for 30 min, then heat shock at 42 °C for 90 s, incubate on ice for 2 min, and then recover at 37 °C for 30 min. Spread on an LB plate containing chloramphenicol. Screen positive clones by PCR, and verify the positive PCR products by sequencing. Name the obtained recombinant plasmids pSET4s-cps, pSET4s-hylB, pSET4s-cyl and pSET4s-treP respectively.

[0079] 5. Construction of Multigene-Deleted Streptococcus agalactiae Strain Mu-20

[0080] A method for preparing competent cells of a fish-derived Streptococcus agalactiae WC1535 strain is as follows:

[0081] (1) Resuscitate the fish-derived Streptococcus agalactiae WC1535 in BHI (brain heart infusion medium) and culture overnight at 30 °C;

[0082] (2) Take 1 mL of the overnight culture and add it to 100 mL of fresh BHI liquid medium, and culture with shaking at 30 °C for 5 - 6 h. When OD 600 = 0.6, collect the bacterial cells;

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

[0084] (4) Gently suspend the bacterial cells with 20 mL of 15% glycerol, centrifuge, and discard the supernatant;

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

[0086] (6) Gently suspend the bacterial cells with 1 mL of 15% glycerol, aliquot into 1.5 mL centrifuge tubes, which are the competent cells of Streptococcus agalactiae.

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

[0088] The recombinant plasmid pSET4s-cps was electrotransformed into the competent cells of Streptococcus agalactiae strain WC1535, and then spread on a BHI plate containing spectinomycin and incubated at 28 °C in an inverted position for 36 h. Single colonies were picked and inoculated into BHI liquid medium, cultured at 28 °C until the logarithmic growth phase, then diluted 10,000 - 100,000 times and spread on a BHI plate without resistance and incubated at 37 °C in an inverted position. Single colonies were picked for colony PCR detection, and the primers were cps-dF: 5′-TATGATTAAACGAAACTTCCTC-3′ (SEQ ID NO.1) and cps-dR: 5′-AGCATTCAATAGCAGCACTCC-3′ (SEQ ID NO.2). The positive knockout strain could amplify a PCR product with an expected size of 2213 bp. The positive PCR product was verified by sequencing, and the results showed that the cps gene cluster deletion mutant strain had been successfully constructed, named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD.

[0089] The competent cells of the Streptococcus agalactiae cps gene cluster deletion strain were prepared in the same way as the wild strain WC1535. The recombinant plasmid pSET4s-hylB was electrotransformed into the competent cells of WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD, and then spread on a BHI plate containing spectinomycin and incubated at 28 °C in an inverted position for 36 h. Single colonies were picked and inoculated into BHI liquid medium, cultured at 28 °C until the logarithmic growth phase, then diluted 10,000 - 100,000 times and spread on a BHI plate without resistance and incubated at 37 °C in an inverted position. The Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB was obtained. Moreover, there was no clear zone on the hyaluronic acid medium for this strain, indicating that this strain had no hyaluronidase activity ( Figure 1)。Single colonies were picked for colony PCR detection. The primers were hylB-dF: 5′-TTCACCTGCCTCCAGTTTACCGC-3′ (SEQ ID NO.3) and hylB-dR: 5′-GTGAAGAACTCGGCTGGGAACC-3′ (SEQ ID NO.4). The positive knockout strain could amplify a PCR product with an expected size of 1659 bp. The positive PCR product was verified by sequencing. The results showed that a mutant strain with the cps gene cluster and hylB deleted had been successfully constructed, and it 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 WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB were prepared. The recombinant plasmid pSET4s-cyl was electrotransformed into the competent cells, and then spread on BHI plates containing spectinomycin and cultured upside down at 28 °C for 36 h. Single colonies were picked into BHI liquid medium and cultured at 28 °C until the logarithmic growth phase, then diluted 10000 - 10000 times and spread on BHI plates without resistance and cultured upside down at 37 °C. The Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF was obtained. Moreover, no hemolysis zone appeared on the Columbia defatted sheep blood agar plate ( Figure 2)。Single colonies were picked for colony PCR detection. The primers were cyl-dF: 5′-ATGCTGATGGAGAAAGGCTGAGTA-3′ (SEQ ID NO.5) and cyl-dR: 5′-CACTTAACTCATCACAGCCACCAC-3′ (SEQ ID NO.6). The positive knockout strains could amplify a PCR product with an expected size of 1798 bp. The positive PCR products were verified by sequencing. The results showed that mutants with deletions of the cps gene cluster, hylB, cylE, and cylF had been successfully constructed and 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 WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF were prepared. The recombinant plasmid pSET4s-treP was electrotransformed into the competent cells, and then spread on BHI plates containing spectinomycin and cultured upside down at 28 °C for 36 h. Single colonies were picked into BHI liquid medium and cultured at 28 °C until the logarithmic growth phase, then diluted 10,000 - 100,000 times and spread on non-resistant BHI plates and cultured upside down at 37 °C. The Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF△treP was obtained. The results of the carbon source utilization test showed that this deletion strain could not utilize trehalose ( Figure 3)。Single colonies were picked for colony PCR detection. The primers were treP-dF: 5′-ATTGTGCGTTCATTACATCCAGAG-3′ (SEQ ID NO.7) and treP-dR: 5′-AGGAATCATCTTGTCCAAAACTA-3′ (SEQ ID NO.8). The positive knockout strains could amplify a PCR product with an expected size of 1847 bp. The positive PCR products were verified by sequencing. The results showed that mutant strains with deletions of the cps gene cluster, hylB, cylE, cylF, and treP had been successfully constructed and were named WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF△treP. Since the naming number of this deletion strain was relatively long, it was re-numbered as Mu-20, that is, the first two English letters of Mutant + the number of deleted genes 20.

[0092] The reaction system for the above PCR detection: 25 μL of 2×PCR Master Mix, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of the target bacterial solution, and finally add double-distilled water H2O to 50 μL.

[0093] The PCR amplification procedure was as follows:

[0094] a. React at 95°C for 4 min, cycle 1 time

[0095] b. React at 94°C for 30 s, cycle 30 times

[0096] c. React at 56°C for 30 s, cycle 30 times

[0097] d. React at 72°C for 150 s, cycle 30 times

[0098] e. React at 72°C for 10 min, cycle 1 time

[0099] f. React at 4°C for 10 min, cycle 1 time

[0100] Finally, 1% agarose gel electrophoresis was used to detect the results of PCR amplification, and the positive PCR products could be subjected to sequencing analysis.

[0101] 6. Competitive experiment between deletion strain Mu-20 and other strains

[0102] To evaluate the difference in the competitive ability of the deletion strain Mu-20 when co-cultured with other strains, the deletion strain Mu-20 was inoculated in equal proportions (1:1) with Streptococcus agalactiae wild strain WC1535, Aeromonas hydrophila GKY1, Aeromonas veronii Ci1701, Edwardsiella tarda GD1701, Escherichia coli ATCC 25922, Plesiomonas shigelloides LS1 and other strains. After the deletion strain Mu-20, Streptococcus agalactiae WC1535, Aeromonas hydrophila GKY1, Aeromonas veronii Ci1701, Edwardsiella tarda GD1701, Escherichia coli ATCC 25922 and Plesiomonas shigelloides LS1 and other strains were activated respectively, they were inoculated into BHI liquid medium and cultured overnight on a shaker at 30 °C, and then the bacterial liquid concentrations were measured respectively. The deletion strain Mu-20 was inoculated into 6 bottles of 0.2×BHI liquid medium (200 mL) respectively, and the final concentration of the bacterial liquid was 1×10 6 CFU / mL. Streptococcus agalactiae WC1535, Aeromonas hydrophila GKY1, Aeromonas veronii Ci1701, Edwardsiella tarda GD1701, Escherichia coli ATCC 25922 and Plesiomonas shigelloides LS1 were inoculated into the above 0.2×BHI liquid medium (200 mL) respectively, so that the final concentrations of the bacterial liquids of WC1535, GKY1, Ci1701, GD1701, ATCC25922 and LS1 were 1×10 6 CFU / mL, that is, the ratio of the deletion strain Mu-20 to its competing strains was 1:1. After inoculation, the bacterial liquid was placed in an incubator at 28 °C and cultured statically.

[0103] After the deletion strain Mu-20 was co-cultured with the competing strains for 1, 3, 5, 7, 10, 15 d, the above-mentioned cultured bacterial liquids were diluted respectively, and then spread on BHI plates for colony counting. Since the colony morphologies of the deletion strain Mu-20 and Aeromonas hydrophila GKY1, Aeromonas veronii Ci1701, Edwardsiella tarda GD1701, Escherichia coli ATCC 25922 and Plesiomonas shigelloides LS1 strains were significantly different and could be distinguished by naked eye observation, colony counting could be directly carried out. Considering that the colony morphologies of the deletion strain Mu-20 and the Streptococcus agalactiae wild strain were similar and could not be determined by naked eye observation, the PCR detection method was used in this experiment. The bacterial liquids of the deletion strain Mu-20 and the wild strain group were spread on plates, and then colonies (48) were randomly selected for PCR amplification. The PCR amplification primers were cps-dF and cps-dR. When the extension time was 3 min, the deletion strain Mu-20 showed positive fragments in the PCR results, and the wild strain was negative in the PCR results.

[0104] According to the results of the above colony counting, with time as the abscissa and the percentage of the deletion strain Mu-20 / competing strain as the ordinate, the survival trends of the vaccine strain and the competing strain were plotted ( Figure 4 ). As Figure 4 can be seen, when the deletion strain Mu-20 was co-cultured with the competing strain, on the 3rd day, the deletion strain Mu-20 began to decline, and then continued to decline. By the 15th day, the proportions of the deletion strain Mu-20 were 0.16%, 0%, 0.09%, 0%, 0.11%, and 0.24% of the strains LS1, ATCC25922, GYK1, Ci1701, GD1701, and WC1535, respectively.

[0105] After the deletion strain Mu-20 was co-cultured with Aeromonas hydrophila, Aeromonas veronii, Edwardsiella tarda, Escherichia coli, and Plesiomonas shigelloides-like, the abundance of the deletion strain Mu-20 showed a gradually decreasing trend in the later stage of culture. Even at 15 days of co-culture, the deletion strain Mu-20 could not be detected in some experimental groups. The above results indicate that the deletion strain Mu-20 is at a competitive disadvantage when co-cultured with Aeromonas hydrophila, Aeromonas veronii, Edwardsiella tarda, Escherichia coli, and Plesiomonas shigelloides-like, further indicating that the deletion strain Mu-20 has high biosafety during application.

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

[0107] Tilapia (with a body weight of about 20 g) were randomly divided into 4 groups, with 30 tails in each group. The deletion strain Mu-20 and the wild strain WC1535 were resuspended with sterile PBS respectively, and then diluted to 2.0×10 9 CFU / mL. Each tilapia was intraperitoneally injected with 0.1 mL of the bacterial suspension and raised under the condition of water temperature 28±0.5°C. The survival and disease conditions of the tilapia were observed, and the death time and number of tilapia were recorded, and the observation continued for 2 weeks. The results showed that the mortality rate of tilapia injected with the wild strain at a concentration of 2.0×10 9 CFU / mL was 100%; when the deletion strain Mu-20 was intraperitoneally injected at a concentration of 2.0×10 9 CFU / mL, no tilapia died, and no clinical symptoms were observed. The experimental results indicate that when the infection dose is as high as 2.0×10 9 CFU / mL, the deletion strain Mu-20 is non-pathogenic to tilapia.

[0108] 8. Experiment on the survival ability of the deletion strain Mu-20 in tilapia

[0109] The deletion strain Mu-20 cultured to the logarithmic growth phase was diluted with sterile PBS to 1×10 9CFU / mL, intraperitoneally inject tilapia (30 fish, 0.1 mL per fish), and raise them under the condition of water temperature 28 ± 0.5 °C. Dissect 3 tilapia every 2 days, take the spleen and kidney to isolate bacteria, and conduct for 20 consecutive days. The experimental results show that the deletion strain Mu-20 has the longest survival time in the spleen and kidney of tilapia, exceeding 11 days. This indicates that the deletion strain Mu-20 can continuously stimulate the tilapia body to produce antibodies, and this deletion strain has very important application value as a live attenuated vaccine against Streptococcus agalactiae.

[0110] 9. Immunoprotection experiment of deletion strain Mu-20 on tilapia

[0111] Tilapia (body weight about 16.8 ± 4.2 g) were randomly divided into 2 groups, with 100 fish in each group. The deletion strain Mu-20 was cultured in liquid BHI medium until the stationary growth phase, then resuspended with sterile PBS and diluted to 1.0×10 9 CFU / mL. The tilapia in the immunization group were intraperitoneally injected with 0.1 mL of the Mu-20 bacterial suspension, and the tilapia in the control group were intraperitoneally injected with 0.1 mL of sterile PBS buffer, and raised under the condition of water temperature 25.0 ± 1.0 °C for 28 consecutive days. Then a challenge test was carried out. 90 tilapia were selected from each group for artificial infection. The immunization group and the control group were intraperitoneally injected with the wild strain WC1535 respectively, and the bacterial solution concentration was 3.5×10 8 CFU / mL. Calculate the mortality rates of tilapia in the immunization group and the control group respectively, and then calculate the relative percent survival (RPS). The results show that the RPS after immunizing tilapia with the deletion strain Mu-20 is 92.4%, indicating that the deletion strain Mu-20 has a good immunoprotective effect on tilapia.

[0112] In the present invention, the gene-synthesized cps-(up-down) gene fragment sequence:

[0113]

[0114] Gene-synthesized hylB-(up-down) gene fragment sequence:

[0115]

[0116]

[0117] Gene-synthesized treP-(up-down) gene fragment sequence:

[0118]

[0119] The above description of the embodiments is provided 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 all improvements and modifications made by those skilled in the art based on the disclosure of the present invention should fall within the protection scope of the present invention.

Claims

1. Streptococcus agalactiae Mu-20, characterized in that, It is a weakly virulent strain of Streptococcus agalactiae from fish with a capsule deletion. It has 20 deleted genes and was deposited in the Guangdong Microbial Culture Collection Center on June 13, 2022, with the deposit number GDMCC No: 62540 and the Latin name Streptococcus agalactiae in English.

2. The construction method of Streptococcus agalactiae Mu-20 according to claim 1, characterized in that, It includes the following steps: (1) Electroporate the recombinant plasmid pSET4s-cps into the competent cells of Streptococcus agalactiae strain WC1535 from fish, coat it on a BHI plate containing spectinomycin, and incubate it upside down at 28 ± 2 °C for 30 - 40 h; (2) Then pick the monoclonal colonies obtained from the culture and transfer them into a BHI liquid medium, culture them at 28 ± 2 °C until the logarithmic growth phase, then dilute them 10,000 - 100,000 times, coat them on a non-resistant BHI plate, and incubate them upside down at 37 °C; obtain Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD; (3) Electroporate the recombinant plasmid pSET4s-hylB into the competent cells of Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD from fish, coat it on a BHI plate containing spectinomycin, and incubate it upside down at 28 ± 2 °C for 30 - 40 h; (4) Pick the monoclonal colonies obtained from the culture in step (3) and transfer them into a BHI liquid medium, culture them at 28 ± 2 °C until the logarithmic growth phase, then dilute them 10,000 - 100,000 times, coat them on a non-resistant BHI plate, and incubate them upside down at 37 °C; obtain the said Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB; (5) Electroporate the recombinant plasmid pSET4s-cyl into the competent cells of Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB from fish, coat it on a BHI plate containing spectinomycin, and incubate it upside down at 28 ± 2 °C for 30 - 40 h; (6) Pick the monoclonal colonies obtained from the culture in the above step (5) and inoculate them into BHI liquid medium. Culture at 28 ± 2 °C until the logarithmic growth phase, then dilute 10,000 - 100,000 times, spread on a BHI plate without resistance, and incubate in an inverted position at 37 °C; obtain the Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF; (7) Electrotransform the recombinant plasmid pSET4s-treP into the competent cells of Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF isolated from fish. Spread on a BHI plate containing spectinomycin and incubate in an inverted position at 28 ± 2 °C for 30 - 40 h; (8) Pick the monoclonal colonies obtained from the culture in the above step (7) and inoculate them into BHI liquid medium. Culture at 28 ± 2 °C until the logarithmic growth phase, then dilute 10,000 - 100,000 times, spread on a BHI plate without resistance, and incubate in an inverted position at 37 °C; obtain the Streptococcus agalactiae WC1535△cpsA△cpsB△cpsC△cpsD△cpsE△cpsF△cpsG△cpsH△cpsI△cpsJ△cpsK△cpsL△neuA△neuB△neuC△neuD△hylB△cylE△cylF△treP, namely, the Mu-20 strain.

3. The construction method of Streptococcus agalactiae Mu-20 according to claim 1, characterized in that, After step (2), it further includes the step of picking the monoclonal colonies obtained in step (1) for colony PCR detection. The primers are: cps-dF: 5′-TATGATTAAACGAAACTTCCTC-3′ (SEQ ID NO.1); cps-dR: 5′-AGCATTCAATAGCAGCACTCC-3′ (SEQ ID NO.2).

4. The construction method of Streptococcus agalactiae Mu-20 according to claim 1, characterized in that, After step (4), pick the monoclonal colonies obtained in step (3) for colony PCR detection. The primers are: hylB-dF: 5′-TTCACCTGCCTCCAGTTTACCGC-3′ (SEQ ID NO.3); hylB-dR: 5′-GTGAAGAACTCGGCTGGGAACC-3′ (SEQ ID NO.4).

5. The construction method of Streptococcus agalactiae Mu-20 according to claim 1, characterized in that, After step (6), pick the monoclonal colonies obtained in step (5) for colony PCR detection. The primers are: cyl-dF: 5′-ATGCTGATGGAGAAAGGCTGAGTA-3′ (SEQ ID NO.5); cyl-dR: 5′-CACTTAACTCATCACAGCCACCAC-3′ (SEQ ID NO.6).

6. The construction method of Streptococcus agalactiae Mu-20 according to claim 1, characterized in that, After step (8), pick monoclonal colonies obtained in step (7) for colony PCR detection. The primers are: treP-dF: 5′-ATTGTGCGTTCATTACATCCAGAG-3′ (SEQ ID NO.7); treP-dR: 5′-AGGAATCATCTTGTCCAAAACTA-3′ (SEQ ID NO.8).

7. The construction method of Streptococcus agalactiae Mu-20 according to claim 1, characterized in that, The preparation of the fish-derived Streptococcus agalactiae competent cells includes the following steps: (1) Resuscitate the fish-derived Streptococcus agalactiae WC1535 in BHI and culture overnight at 28 ± 2°C; (2) Add 1 mL of the overnight culture into 80 - 120 mL of fresh BHI liquid medium, and culture it with shaking at 28 ± 2 °C for 5 - 6 h. When OD 600 = 0.6, collect the bacterial cells; (3) Centrifuge the enlarged culture broth at 5000 r / min for 8 - 12 min, and discard the supernatant; (4) Gently suspend the obtained bacterial cells with 10 - 20 mL of glycerol with a final concentration of 10% - 15%, and centrifuge to remove the supernatant; thus obtaining the fish-derived Streptococcus agalactiae competent cells.

8. The construction method of Streptococcus agalactiae Mu-20 according to claim 1, characterized in that, The recombinant plasmids pSET4s-cps, pSET4s-hylB, pSET4s-cyl, and pSET4s-treP are respectively obtained by seamless cloning of the pSET4S plasmid and the cps-(up-down), hylB-(up-down), cyl-(up-down), and treP-(up-down) gene fragments, including the following steps: (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, then add them to the Seamless cloning kit ligation system, mix well, and incubate at 50 ± 2°C for 25 - 35 min to obtain a 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; (2) Take the ligation solution obtained in step (1), add it to Escherichia coli DH5α competent cells with a volume 10 times that of the ligation solution, incubate on ice for 25 - 35 min, then heat shock at 42°C for 90 s, incubate on ice for 2 - 3 min, and then resuscitate at 37°C for 25 - 35 min, and spread on an LB plate containing spectinomycin; screen positive clones by PCR, and verify the positive PCR products by sequencing, thus obtaining the recombinant plasmids named pSET4s-cps, pSET4s-hylB, pSET4s-cyl, and pSET4s-treP.

9. The construction method of Streptococcus agalactiae Mu-20 according to claim 8, characterized in that, The construction of the double-digested pSET4S plasmid includes the following steps: Extract the pSET4S plasmid, perform double digestion with Hind III and EcoR I at 37°C for 3 - 5 h, and finally recover the product using 0.8% agarose gel to obtain the double-digested pSET4S plasmid; wherein the digestion system is: 1 μg of pSET4S, 1 U of HindIII, 1 U of EcoR I, 3 μL of 10× Buffer, and ddH2O is supplemented to 30 μL.

10. The application of Streptococcus agalactiae Mu-20 according to claim 1, wherein, Apply the Streptococcus agalactiae Mu-20 to the prevention and control of fish streptococcosis, especially for the preparation of a live attenuated vaccine against fish streptococcosis.

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