ApbC gene knockout vibrio vulnificus strain as well as construction method and application thereof

Abstract

The invention discloses an ApbC gene knockout vibrio vulnificus strain and a construction method and application thereof, the ApbC gene knockout vibrio vulnificus strain is constructed by knocking out an ApbC gene through a homologous recombination method and utilizing conjugational transfer among bacteria, and the construction method comprises the following steps: (1) constructing an ApbC gene deleted overlapping fragment; (2) constructing a recombinant suicide plasmid pDM4-ApbC; (3) binding and transferring bacteria; and (4) screening the gene deletion mutant strain, namely performing a first round of screening through chloramphenicol resistance to obtain a first recombinant bacterial colony, and performing a second round of screening on the first recombinant bacterial colony through a sucrose plate. The construction method is simple and easy to operate, the bacterial colony screening efficiency is high, the synthesis efficiency is high, the constructed ApbC gene knockout vibrio vulnificus strain lacks an ApbC gene segment, and the strain is used for revealing the influence of the ApbC gene on the iron-sulfur utilization of the vibrio vulnificus and further researching the virulence and viability of bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial genetic engineering, and in particular to an ApbC gene knocked-out Vibrio vulnificus strain and a construction method and application thereof. Background Art

[0002] Vibrio vulnificus is a Gram-negative bacterium and one of the most invasive Vibrio species. This opportunistic pathogen proliferates rapidly in warm months and accumulates in large numbers in mollusks, causing rapid and fatal infections in humans. Routes of infection include gastrointestinal symptoms caused by eating contaminated seafood, severe primary sepsis, and wound infections caused by open wounds exposed to contaminated water. The mortality rate of primary sepsis is higher than 50%, and death usually occurs within 72 hours of hospitalization. Therefore, exploring the pathogenic mechanism of Vibrio vulnificus is crucial to reducing the mortality rate of Vibrio vulnificus.

[0003] Most cases of sepsis caused by Vibrio vulnificus are associated with underlying diseases, especially cirrhosis, chronic renal failure, and hemochromatosis, which usually lead to elevated serum iron levels. Therefore, iron overload is considered to be one of the main factors in the progression of the disease. The host immune system plays an important role in enhancing susceptibility to Vibrio vulnificus. Iron in animals promotes the growth of Vibrio vulnificus, increases endotoxin, and induces the secretion of tumor necrosis factor α (TNF-α); at the same time, excess iron reduces the activity of neutrophils. In addition to these virulence factors, Vibrio vulnificus also exhibits cytotoxicity that kills host cells due to its increased growth rate, and iron overload can also affect innate and acquired immune responses.

[0004] Iron and sulfur are essential for almost all bacteria. Iron-sulfur proteins that use Fe-S clusters as cofactors perform a variety of important cellular processes, such as electron transport, metabolic reactions, and gene regulation, and are widely distributed. ApbC is a representative member of a unique class of evolutionarily conserved proteins that can bind transient Fe-S clusters and activate apoproteins to promote Fe-S cluster transfer.

[0005] Therefore, it can be inferred that the ApbC gene plays an important role in the biosynthesis of iron-sulfur proteins in Vibrio vulnificus. The construction of ApbC gene-knockout Vibrio vulnificus is conducive to studying the effects of the ApbC gene on the growth and virulence of Vibrio vulnificus, and further exploring the relationship between the iron-sulfur cluster system and the pathogenic mechanism of Vibrio vulnificus. However, there is currently no record of the construction of ApbC gene-knockout Vibrio vulnificus and the corresponding construction method. Summary of the invention

[0006] The purpose of the present invention is to provide an ApbC gene knocked-out Vibrio vulnificus strain and a construction method and application thereof. The strain is obtained by knocking out the ApbC gene through a homologous recombination method and constructing it by using conjugation transfer between bacteria. The construction method is simple, easy to operate, and has high colony screening efficiency and synthesis efficiency. The constructed ApbC gene knocked-out Vibrio vulnificus strain lacks an ApbC gene fragment, and the lack of the ApbC gene fragment can affect the synthesis of Vibrio vulnificus iron-sulfur protein (Fe-S protein) without being lethal. By comparing the ApbC gene knocked-out Vibrio vulnificus strain with a wild strain of Vibrio vulnificus, the influence of the ApbC gene on the biological characteristics of Vibrio vulnificus and the important biological functions of the iron-sulfur cluster system can be more accurately studied, which is used to reveal the influence of the ApbC gene on the iron-sulfur utilization of Vibrio vulnificus and further study the virulence and viability of the bacteria.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, there is provided an ApbC gene knockout Vibrio vulnificus strain, wherein the ApbC gene knockout Vibrio vulnificus strain lacks an ApbC gene fragment; The ApbC gene-knockout Vibrio vulnificus strain is classified and named Vibrio vulnificus △ApbC (Vv-△ApbC), and is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M20232565, and the deposit date being December 15, 2023.

[0008] Furthermore, the strain is deficient in Fe-S protein synthesis.

[0009] The second aspect of the present invention provides a method for constructing a Vibrio vulnificus strain with ApbC gene knockout, comprising the following steps: (1) Construction of overlapping fragments of the ApbC gene deletion In this step, the overlapping fragments of the ApbC gene knockout are amplified by the overlap extension PCR method. The specific steps are as follows: 1.1 According to the target gene sequence, two pairs of primers, ApbC F1 / R1 and ApbC F2 / R2, were designed to amplify the upstream and downstream fragments of the ApbC gene from the Vibrio vulnificus genome. Among them, the primers ApbC F1 and R2 were designed with restriction sites at the 5' end, and the primers ApbC F2 and R1 were designed with 15-20bp overlapping sequences at the 5' end; 1.2 Using the upstream and downstream sequences of the product of step 1.1 as templates, add primers ApbC F1 and ApbC R2 for PCR amplification to obtain overlapping fragments of the ApbC gene deletion.

[0010] (2) Construction of recombinant suicide plasmid pDM4-△ApbC 2.1 The obtained overlapping fragments of the ApbC gene deletion were double-digested according to the restriction sites designed by the primers, and the suicide plasmid pDM4 was also digested with the same restriction endonuclease; 2.2 The overlapping fragments of the ApbC gene deletion after restriction digestion were connected with the suicide plasmid pDM4 to construct the recombinant suicide plasmid pDM4-△ApbC; 2.3 The ligation product was heat-shock transformed into competent E. coli DH5α λpir, and then spread on LB plates containing 10 μg / mL chloramphenicol for screening. The positive clones were amplified by PCR using primers Cat-F and Cat-R and sequenced. The correctly sequenced recombinant plasmid was named pDM4-△ApbC and the strain was preserved. The suicide plasmid pDM4-△ApbC was extracted using a plasmid extraction kit. 2.4 The suicide plasmid pDM4-△ApbC extracted in step 2.3 was introduced into the conjugation donor bacteria E. coli S17-1 λpir by the same heat shock transformation method.

[0011] (3) Bacterial conjugation transfer This step involves direct contact between the donor bacteria and the recipient bacteria, and the recombinant plasmid is introduced into the recipient bacteria to cause homologous recombination. The specific steps are as follows: 3.1 Mix wild-type Vibrio vulnificus and E. coli S17-1 λpir bacterial suspension and centrifuge, resuspend and inoculate on BHI thick blood agar plates, and culture at 30°C for 24 hours; 3.2 Use BHI medium to fully suspend the plaques, centrifuge the suspension and resuspend it; 3.3 Dilute the bacterial solution in multiple ratios, spread on BHI blood agar plates containing 10 μg / mL chloramphenicol and 12.5 μg / mL polymyxin E, and culture at 37°C until colonies appear; 3.4 Pick a single colony and place it in BHI liquid culture medium containing 12.5 μg / mL polymyxin E. Culture it on a shaking table until it is visible to the naked eye. Use primers ApbC F1 and ApbC R2 to perform PCR identification on the bacterial liquid to obtain the first recombinant colony.

[0012] (4) Screening of gene deletion mutants This step further screens positive clones by utilizing the sucrose resistance gene of the plasmid. The specific steps are as follows: 4.1 Pick the positive clones and place them in BHI liquid medium overnight. After centrifugation, resuspend them in BHI medium. Dilute the bacterial solution 10, 100, 1000, and 10000 times with PBS, spread them on BH solid plates containing 10wt% sucrose, and culture them in an incubator overnight at 37°C. 4.2 Pick the strains on the BHI solid plate containing 10wt% sucrose, and spot them on the BHI solid plates containing 10μg / mL chloramphenicol and without chloramphenicol, respectively, and culture them in an incubator; the strains that do not grow on the BHI solid plate containing 10μg / mL chloramphenicol and grow on the BHI solid plate without chloramphenicol are suspected gene deletion mutant strains; pick the strains without chloramphenicol resistance, and repeat the above operation to prevent the occurrence of false positives; 4.3 Select the target strain without chloramphenicol resistance, inoculate it into BHI liquid culture medium, use primers MutApbC-F and MutApbC-R for PCR identification and extract the genome for sequencing; save the strain with correct sequencing, and finally obtain the Vibrio vulnificus ApbC gene knockout strain Vv-△ApbC.

[0013] The third aspect of the present invention provides a use of a Vibrio vulnificus strain with an ApbC gene knocked out, wherein the Vibrio vulnificus strain with an ApbC gene knocked out is used to study the function of the ApbC gene in Vibrio vulnificus.

[0014] Specifically, the ApbC gene knockout Vibrio vulnificus strain is used in studying the pathogenic mechanism of Vibrio vulnificus.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention is the first to use polymyxin and chloramphenicol in combination as screening antibiotics. Vibrio vulnificus itself has polymyxin resistance. In the first round of screening, the combined use of polymyxin and chloramphenicol greatly improved the efficiency of the first recombinant colony screening; (2) The fusion fragments of the upstream and downstream homology arms were obtained by overlapping PCR technology and connected to the suicide plasmid pDM4. After heat shock, they were introduced into competent Escherichia coli DH5α λpir for replication to obtain a large number of recombinant plasmids, which improved the efficiency of subsequent conjugation transfer. (3) The present invention knocks out the ApbC gene by homologous recombination and constructs an ApbC gene-knockout Vibrio vulnificus strain by using bacterial conjugation transfer. The construction method is simple, easy to operate, has high colony screening efficiency, and high synthesis efficiency. The constructed ApbC gene-knockout Vibrio vulnificus strain lacks the ApbC gene fragment. Since iron-sulfur proteins are involved in many important life processes of bacteria and are highly conserved in evolution, knocking out the ApbC gene fragment will often lead to bacterial death, making research difficult. The deletion of the ApbC gene fragment can affect the synthesis of iron-sulfur proteins in Vibrio vulnificus without being lethal. By comparing the ApbC gene-knockout Vibrio vulnificus strain with the wild strain of Vibrio vulnificus, the effect of the ApbC gene on the biological characteristics of Vibrio vulnificus and the important biological functions of the iron-sulfur cluster system can be more accurately studied, which is used to reveal the effect of the ApbC gene on the utilization of iron and sulfur by Vibrio vulnificus and further study the virulence and survival ability of the bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0017] Figure 1 This is a schematic diagram of the principle of obtaining overlapping fragments of ApbC gene knockout by two-step amplification based on the overlap extension PCR method.

[0018] Figure 2 Schematic diagram of the principle of homologous recombination.

[0019] Figure 3 This is the map of suicide plasmid pDM4.

[0020] Figure 4 Construction of the ApbC gene knockout Vibrio vulnificus strain, (A) ApbC gene upstream and downstream PCR amplification, M: DL5000 marker; 1: ApbC gene upstream fragment; 2: ApbC gene downstream fragment; (B) ApbC gene upstream and downstream overlap extension PCR amplification, M: DL5000 marker; 1-2: ApbC gene upstream and downstream overlapping fragments; (C) PCR identification of ApbC gene knockout Vibrio vulnificus, M: DL5000 marker; 1: ApbC gene knockout Vibrio vulnificus; 2: wild-type Vibrio vulnificus; 3: negative control; (D) ApbC gene knockout sequencing results.

[0021] Figure 5 This is the Sdhb mRNA expression diagram of the wild-type Vibrio vulnificus strain and the Vibrio vulnificus strain with the ApbC gene knocked out.

[0022] Figure 6 This is a comparison chart of the SDH enzyme activity of the wild strain of Vibrio vulnificus and the strain with the ApbC gene knocked out.

[0023] Figure 7 Comparison of the extracellular and extracellular bacterial counts of the wild-type Vibrio vulnificus strain and the ApbC gene-knocked-out Vibrio vulnificus strain. (A) Bacterial counts of the wild-type Vibrio vulnificus strain and the ApbC gene-knocked-out Vibrio vulnificus strain after being cultured in RPMI 1640 medium for 2-4 hours. (B) Bacterial counts of the extracellular suspension of the wild-type Vibrio vulnificus strain and the ApbC gene-knocked-out Vibrio vulnificus strain after infecting J774A.1 macrophages in RPMI 1640 medium for 2-4 hours.

[0024] Figure 8 This is the survival curve of the effects of wild-type Vibrio vulnificus strain and ApbC gene-knockout Vibrio vulnificus strain on animal survival. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The primers used in the following examples are shown in Table 1: Table 1 Primer names and sequences

[0027] The full-length sequence of the ApbC gene in the present invention is shown in SEQ ID NO.5: SEQ ID NO.5:

[0028] Description of microbial preservation: The ApbC gene-knocked Vibrio vulnificus strain of the present invention is classified and named Vibrio vulnificus △ApbC, and was deposited in the China Center for Type Culture Collection on December 15, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCCNO: M 20232565.

[0029] The present invention provides a method for establishing a gene knockout Vibrio vulnificus, the theoretical basis of which is the principle of gene homologous recombination: including single exchange and double exchange, to complete the knockout of the ApbC gene. The specific principle is as follows Figure 2 shown.

[0030] The suicide plasmid used in the present invention is pDM4, which is a kind of R plasmid derived plasmid, has a conjugative transfer gene, and is one of the commonly used tools for constructing bacterial gene deletion mutant strains. pDM4 is derived from the pGP704 plasmid and contains a special replicon (R6K) that can only be activated under the action of the π protein encoded by the pir gene. Therefore, the vector can only replicate, proliferate and survive in the host bacteria containing the pir gene. In addition, the plasmid also contains two genes for screening markers, namely the chloramphenicol Cm resistance gene with positive selection and the sucrose sensitive gene sacB with negative selection. The sacB gene can encode fructan sucrase, which catalyzes the hydrolysis of sucrose into fructan and is lethal to many Gram-negative bacteria. Therefore, when the living environment of Gram-negative bacteria contains sucrose, under the induction of sucrose, the activation of the sacB gene of the plasmid can kill a variety of Gram-negative bacteria, and only when the plasmid containing the sacB gene is lost can the host bacteria survive. See the suicide plasmid map. Figure 3 .

[0031] The method for establishing ApbC gene knockout Vibrio vulnificus specifically comprises the following steps: (a) Construction of overlapping fragments for ApbC gene knockout; (b) Construction of recombinant suicide plasmid pDM4-△ApbC; (c) conjugative transfer of bacteria; (d) Screening of gene deletion mutants.

[0032] Step (a) uses an overlapping PCR-based method to amplify the overlapping fragments of the target gene deletion. The specific principle is shown in Figure 1 , the specific steps are as follows: 1) First PCR: According to the target gene sequence, two pairs of primers, ApbC F1 / R1 and ApbC F2 / R2, were designed. The restriction sites SacI and XhoI were added to the 5' ends of the primers ApbCF1 and R2, and 15-20 bp overlapping sequences were added to the 5' ends of the primers ApbC F2 and R1. The primer sequences are shown in Table 1; The upstream sequence primers ApbC F1 / R1 and the downstream sequence primers ApbC F2 / R2 were used for PCR amplification using Vibrio vulnificus genomic DNA as template to obtain the upstream and downstream fragments of the ApbC gene, see Figure 4 A; PCR conditions were: Initial denaturation: 95°C for 5 min; PCR cycle: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s, and the above steps were repeated 35 times; Final extension: 72°C for 10 min.

[0033] 2) Second PCR: Using the upstream and downstream gel recovery products of 1) as templates, primers ApbC F1 and ApbC R2 were added for PCR amplification to obtain the overlapping fragments of the complete target gene deletion, see Figure 4 B; PCR conditions were: Initial denaturation: 95°C for 5 min; PCR cycle: denaturation at 95°C for 15s, annealing at 60°C for 15s, and extension at 72°C for 1min, and the above steps were repeated 35 times; Final extension: 72°C for 10 min.

[0034] Step (b) comprises the following steps: 1) The overlapping fragments of the target gene knockout were double-digested with SacI and XhoI according to the restriction sites designed by the primers. The suicide plasmid pDM4 was also digested with the same restriction endonuclease and incubated in a water bath at 37°C for 4 hours.

[0035] 2) Use T4 DNA ligase to connect the gel-recovered product after enzyme digestion, i.e., the overlapping fragments of the target gene deletion, to the pDM4 suicide plasmid, and connect at 16°C for 18 hours to construct the gene knockout plasmid pDM4-△ApbC. The molar ratio of the vector to the exogenous DNA fragment is controlled at 1 / 3-1 / 10, and the amount of the vector is between 50-200 ng.

[0036] 3) Take all the ligation products in 2) and add them to the competent cell surface of E. coli DH5α λpir, mix gently, and place on ice for 30 minutes; heat shock with 42℃ hot water for 90 seconds, and quickly place on ice for 2 minutes; under sterile conditions, add 900μL LB medium, shake at 200rpm for 2 hours, centrifuge at 5000rpm for 5 minutes, discard 900μL of supernatant, resuspend bacteria in the remaining medium, and evenly spread on LB plates containing 10μg / mL chloramphenicol; place in a 37℃ incubator for 12-18 hours. Pick a single colony on the chloramphenicol LB plate and place it in LB liquid medium containing 10μg / mL chloramphenicol, shake and culture until turbidity is visible to the naked eye. Use primers Cat-F and Cat-R to amplify and identify the positive clones by PCR and send them for sequencing. The recombinant plasmid with correct sequencing is named pDM4-ApbC and the strain is preserved. The suicide plasmid pDM4-△ApbC is extracted using a plasmid extraction kit.

[0037] 4) The suicide plasmid pDM4-△ApbC extracted in 3) was introduced into the conjugation donor bacterium E. coli S17-1 λpir according to the above method.

[0038] The specific steps of step (c) are as follows: 1) Activate wild-type Vibrio vulnificus and E. coli S17-1 λpir containing suicide plasmid pDM4-△ApbC on blood agar plates and LB solid medium containing 10μg / mL chloramphenicol, respectively. Pick a single colony and culture it in antibiotic-free medium overnight on a shaker. Mix equal volumes of wild-type Vibrio vulnificus and E. coli S17-1 λpir containing a double amount of bacteria, centrifuge at 8000rpm for 2min, resuspend in 100μL BHI medium, and spot on BHI thick blood agar plates, and then culture upright at 30℃ for 24h.

[0039] 2) Fully suspend the bacterial plaques with 1 mL of BHI medium. Centrifuge the suspension at 8000 rpm for 2 min and resuspend it with 1 mL of BHI medium.

[0040] 3) Dilute the bacterial solution with BHI medium at a ratio of 10 -1 , 10 -2 , 10 -3 , 100 μL of the stock solution was spread on a BHI blood plate containing 10 μg / mL chloramphenicol and 12.5 μg / mL polymyxin E, and cultured in a 37°C incubator for 24-48 h until colonies grew, during which the formation of single colonies was observed.

[0041] 4) Pick a single colony and place it in BHI liquid culture medium containing 12.5 μg / mL polymyxin E until it is visible to the naked eye. Use primers ApbC F1 / R2 to perform PCR identification of the bacterial liquid. At the same time, use Martex F / R PCR to amplify the fragment of the specific gene MARTX of Vibrio vulnificus to determine whether the candidate mutant strain is Vibrio vulnificus and obtain the first recombinant colony.

[0042] The step (d) further screens positive clones by utilizing the sucrose resistance gene of the plasmid, and the specific steps are as follows: 1) Take the positive bacteria identified by PCR and place them in BHI medium overnight. Centrifuge at 3000g for 7 minutes the next day and resuspend them in 1mL PBS. Dilute the bacterial solution 10 times with PBS. -1 , 10 -2 , 10 -3 , 10 -4 The original solution was spread on a BHI solid plate containing 10 wt % sucrose and cultured in a 37°C incubator overnight.

[0043] 2) Use an autoclaved toothpick to pick up about 100-200 strains on the BHI solid plate containing 10wt% sucrose, and spot them on the BHI solid plates containing 10μg / mL chloramphenicol and without chloramphenicol, respectively, and incubate in an incubator for 8-12 hours. The strains that do not grow on the BHI solid plate containing 10μg / mL chloramphenicol and grow on the BHI solid plate without chloramphenicol are suspected gene deletion mutant strains; pick strains without chloramphenicol resistance and repeat the above operation to prevent false positives.

[0044] 3) Select the target strain without chloramphenicol resistance, inoculate it into BHI liquid culture medium, and culture it in a shaking incubator at 250rpm until it is visible to the naked eye. Use primers ApbC F1 and R2 for colony PCR identification. At the same time, use primers Martex-F / R PCR to amplify the fragment of the specific gene Martex of Vibrio vulnificus to determine whether the candidate mutant strain is Vibrio vulnificus, and extract the genome for sequencing. Figure 4 C, D. Vibrio vulnificus lacking the ApbC fragment (△ApbC Vibrio vulnificus) was successfully constructed and the strain was preserved.

[0045] 1. Verify the mRNA expression of the iron-sulfur protein-related gene succinate dehydrogenase complex iron-sulfur subunit B Sdhb in ApbC-knocked Vibrio vulnificus strains The total RNA extracted from the wild strain of Vibrio vulnificus (WT) and the strain with the ApbC gene knocked out (△ApbC) was reverse transcribed into cDNA as a template, and the expression of the Sdhb gene was detected by qRT-PCR using RT-Sdhb-F and RT-Sdhb-R as primers. 16S rRNA was used as the internal reference gene, and the primers used were RT-16S-F and RT-16S-R. The wild strain of Vibrio vulnificus (WT) was used as the control group. The results of RT-PCR showed that the expression of Sdhb in the strain with the ApbC gene knocked out (△ApbC) was decreased, as shown in Figure 2. Figure 5 , further indicating that the ApbC gene knockout Vibrio vulnificus strain (△ApbC) was successfully constructed.

[0046] 2. Comparison of the biological characteristics of the wild-type Vibrio vulnificus strain (WT) and the ApbC gene knockout strain (△ApbC) The succinate dehydrogenase (SDH) activity detection kit [Boxbio] was used to determine the succinate dehydrogenase activity of the wild-type Vibrio vulnificus strain (WT) and the ApbC gene knockout Vibrio vulnificus strain (△ApbC). The results showed that the SDH activity of the ApbC gene knockout Vibrio vulnificus strain (△ApbC) was significantly lower than that of the wild-type (WT). Figure 6 .

[0047] The specific experimental steps are as follows: 1. Preparation of crude enzyme solution: Centrifuge and collect for about 10 10 Place the bacteria in a centrifuge tube, add 1 mL of reagent 1 and 10 μL of reagent 2, perform ultrasonic treatment in an ice bath (ultrasonic conditions: power 200 W, ultrasonic 3 s, interval 7 s, total time 5 min), centrifuge at 11000 g for 10 min at 4 °C, take the supernatant as the crude enzyme solution, and place it on ice for testing; 2. Determination: 1) Preheat the microplate reader for more than 30 minutes, adjust the wavelength to 600nm, and set the zero value to distilled water; 2) Add the following reagents in sequence to the 96-well plate. See Table 2 for the reagent addition and order: Table 2

[0048] 3) Absorbance measurement: Mix the components and start timing. Measure the absorbance at 600 nm after 20 seconds, which is recorded as A1 measurement and A1 blank. Measure the absorbance at 600 nm after about 80 seconds, which is recorded as A2 measurement and A2 blank. Calculate △A measurement = A1 measurement - A2 measurement, △A blank = A1 blank - A2 blank, △A = △A measurement - △A blank; Calculation: SDH (U / mg prot) = ; Where V is the total volume of the reaction system, 2×10 4 L; ɛ is the molar extinction coefficient of 2,6-dichloroindoxyl, 2.1×10 4 L / mol / cm; is the light path of 96-well plate, 0.5 cm; Vsample is the volume of crude enzyme solution added to the reaction system, 0.01 mL; Vsampletotal is the volume of crude enzyme solution, 1.01 mL; Cpr is the sample protein concentration, mg / mL; T is the reaction time, which shall be subject to actual conditions).

[0049] After the wild-type Vibrio vulnificus strain (WT) and the ApbC gene-knocked-out Vibrio vulnificus strain (△ApbC) infected J774A.1 macrophages in RPMI 1640 medium for 2-4 hours, the growth rate of the ApbC gene-knocked-out Vibrio vulnificus strain (△ApbC) in the culture medium was significantly lower than that of the wild-type Vibrio vulnificus strain (WT). Therefore, the bacterial count of the ApbC gene-knocked-out Vibrio vulnificus strain (△ApbC) in the extracellular suspension after infection was significantly lower than that of the wild-type Vibrio vulnificus strain (WT), as shown in Figure 2. Figure 7 .

[0050] The specific experimental steps are as follows: 1. Cell line plating: After counting with an automatic cell counter, take a 6-well cell culture plate and set up the corresponding treatment groups (WT blank group, △ApbC blank group, 2MOI WT stimulated cell group, 2MOI △ApbC stimulated cell group) according to the experimental needs. Add 2×10 6 J774A.1 cells were plated and 2 mL of antibiotic-free culture medium was added to each well. The cells were placed in a 5% CO2 incubator for 1 h to allow them to adhere to the wall. 2. According to the experimental grouping, add 2MOI ApbC gene knockout Vibrio vulnificus bacterial solution to the experimental group wells for stimulation for 4 hours. After the time is up, collect the extracellular suspension, perform multiple dilutions, and count the bacteria on the plate.

[0051] There was no significant difference in the effects of the wild-type Vibrio vulnificus strain (WT) and the ApbC gene knockout Vibrio vulnificus strain (△ApbC) on the survival of iron-overloaded mice. Figure 8 .

[0052] The specific experimental steps are as follows: 1. Concentration of wild-type and knockout strains of Vibrio vulnificus: After counting colonies, adjust the bacterial solution to 1×10 5 CFU / mL, for later use; 2. C57BL / 6J female mice were divided into WT+ iron-overloaded mice group and △ApbC+ iron-overloaded mice group, 3 mice in each group; 3. Each mouse was intraperitoneally injected with 100 μg / g of iron dextran, and 2 hours later, 200 μL of two Vibrio vulnificus bacteria of corresponding concentrations were intraperitoneally injected again; 4. Every hour, take out the mice and fix them in the fixator. Lubricate the rectal thermometer probe with glycerol to detect the rectal temperature at that time point and record it. At the same time, observe the clinical status of the mice and score them according to the clinical score of sepsis. During this period, observe the status of the mice in each group at any time and record the time of death of the mice.

[0053] The present invention can more accurately study the influence of the ApbC gene and iron-sulfur protein on the biological characteristics of Vibrio vulnificus by constructing a Vibrio vulnificus ApbC gene knockout strain.

[0054] The ApbC gene knocked-out Vibrio vulnificus strain can be used to construct other gene deletion strains of the serotype or other serotype deletion strains.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Vibrio vulnificus strain with ApbC gene knocked out, It is characterized in that The ApbC gene knockout Vibrio vulnificus strain lacks an ApbC gene fragment; The ApbC gene-knockout Vibrio vulnificus strain is classified and named Vibrio vulnificus △ApbC, and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20232565 and a deposit date of December 15, 2023.

2. The ApbC gene knockout Vibrio vulnificus strain according to claim 1, It is characterized in that The strain is deficient in Fe-S protein synthesis.

3. A method for constructing a Vibrio vulnificus strain with ApbC gene knockout, It is characterized in that The steps include: S1. Design primers according to the ApbC gene sequence in Vibrio vulnificus, amplify the upstream and downstream sequences of the ApbC gene respectively, and obtain the upstream and downstream fragments of the ApbC gene; then use the homologous fragments between the primers to perform overlap extension PCR to obtain the overlapping fragments of the ApbC gene deletion; S2, inserting the overlapping fragments of the ApbC gene deletion obtained in step S1 into the suicide plasmid to obtain a recombinant suicide plasmid; wherein the suicide plasmid is pDM4, which carries a chloramphenicol resistance gene and a levansucrase gene; S3, transforming the recombinant suicide plasmid obtained in step S2 into competent cells of Escherichia coli DH5α λpir to obtain a large amount of recombinant suicide plasmid; and then transforming the extracted recombinant suicide plasmid into competent cells of donor bacteria Escherichia coli S17-1 λpir; S4. Introducing the recombinant plasmid of the Escherichia coli S17-1 λpir transformed in step S3 into Vibrio vulnificus by inter-bacterial conjugation transfer to cause homologous recombination, and obtaining Vibrio vulnificus with the ApbC gene knocked out; wherein, the method comprises performing a first round of screening by chloramphenicol resistance to obtain first recombinant colonies, and performing a second round of screening on the first recombinant colonies by sucrose resistance to obtain the Vibrio vulnificus with the ApbC gene knocked out.

4. The method for constructing the ApbC gene knockout Vibrio vulnificus strain according to claim 3, It is characterized in that In step S1, the upstream sequence of the ApbC gene carries a restriction site SacI, and the downstream sequence of the ApbC gene carries a restriction site XhoI; Amplifying the upstream sequence of the ApbC gene using a pair of primers with sequences as shown in SEQ ID No: 1 and SEQ ID No: 2; A pair of primers with sequences as shown in SEQ ID No:3 and SEQ ID No:4 were used to amplify the downstream sequence of the ApbC gene.

5. The method for constructing the ApbC gene knockout Vibrio vulnificus strain according to claim 4, It is characterized in that In step S2, the suicide plasmid contains the same restriction site as the overlapping fragment of the ApbC gene deletion, and the overlapping fragment of the ApbC gene deletion after double restriction digestion is connected to the suicide plasmid to obtain a recombinant suicide plasmid.

6. The method for constructing the ApbC gene knockout Vibrio vulnificus strain according to claim 3, It is characterized in that In the step S3, heat shock perforation is used to perform the transformation of the recombinant suicide plasmid.

7. The method for constructing the ApbC gene knockout Vibrio vulnificus strain according to claim 3, It is characterized in that In step S4, a first round of screening is performed using chloramphenicol resistance and polymyxin resistance of Vibrio vulnificus itself to obtain the first recombinant colony.

8. The method for constructing the ApbC gene knockout Vibrio vulnificus strain according to claim 7, It is characterized in that The first round of screening was performed using BHI blood plates containing 10 μg / mL chloramphenicol and 12.5 μg / mL polymyxin E.

9. The method for constructing the ApbC gene knockout Vibrio vulnificus strain according to claim 7 or 8, It is characterized in that The specific process of the second round of screening is as follows: 1) Using a BHI solid plate containing 10 wt% sucrose to screen chloramphenicol-polymyxin-resistant colonies, and obtaining colonies growing on the BHI solid plate containing 10 wt% sucrose; 2) The colonies grown on the BHI solid plate containing 10 wt% sucrose were screened by a BHI solid plate containing 10 μg / mL chloramphenicol and a BHI solid plate without chloramphenicol, respectively, and the colonies that did not grow on the BHI solid plate containing 10 μg / mL chloramphenicol and grew on the BHI solid plate without chloramphenicol were the ApbC gene-knockout Vibrio vulnificus.

10. Application of a Vibrio vulnificus strain with ApbC gene knocked out, It is characterized in that The ApbC gene knockout Vibrio vulnificus strain as described in claim 1 or 2 is used to study the function of the ApbC gene in Vibrio vulnificus.

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