Acinetobacter baumannii capsule related gene knockout method and application

Abstract

The invention discloses a method for knocking out acinetobacter baumannii capsule-related genes and application of the acinetobacter baumannii capsule-related genes. According to the gene knockout method, the acinetobacter baumannii capsule-related genes can be specifically knocked out, target genes are knocked out in a traceless mode through a bacterium Rec homologous recombination system, the acinetobacter baumannii with the capsule-related genes knocked out is obtained, important pathogenic factors of the acinetobacter baumannii can be clarified, and the acinetobacter baumannii capsule-related genes can be used for preparing the acinetobacter baumannii capsule-related genes. It is proved that the capsule-related gene plays an important role in the toxicity of the acinetobacter baumannii. The acinetobacter baumannii is LAC-4, and the capsule related gene is a wza gene. The capsular gene of the strain can be effectively knocked out, and the method is simple and easy to popularize.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a method and application of a knockout strain of capsule-related genes of Acinetobacter baumannii based on homologous recombination. Background Art

[0002] In recent years, as a member of the common clinical multidrug-resistant bacteria combination "ESKAPE", the virulence of Acinetobacter baumannii (AB) has been increasing, changing the previous concept that "Acinetobacter baumannii belongs to low-pathogenic environmental bacteria". The AB with enhanced virulence is becoming a pathogen that seriously endangers patient safety. Among the many pathogenic factors of AB, capsule-related virulence genes are a key type. The capsule surrounding the bacterial body can protect the bacteria from external threats and resist host defense; studies have confirmed that the virulence of Klebsiella pneumoniae and other bacteria that have lost their capsule is extremely weakened, but the capsule-related virulence genes - wxya The role and mechanism of the gene in Acinetobacter baumannii need further study.

[0003] Homologous recombination is a genetic engineering technique used to insert, repair or replace DNA sequences. It is achieved through recombination events between homologous genes on the same or different chromosomes. Homologous recombination can be used to selectively knock out a gene in a cell to further study its function. Gene knockout is achieved by introducing homologous fragments to replace part or all of the sequence in the target gene. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application of knocking out genes related to the capsule of Acinetobacter baumannii in view of the deficiencies of the prior art.

[0005] The technical scheme for achieving the purpose of the present invention is as follows: A method for knocking out a capsule-related gene of Acinetobacter baumannii, comprising the following steps: (1) Design primers to amplify the upstream and downstream homology arms of the knockout target gene, and use high-fidelity enzymes to amplify the upstream and downstream homology arms of the target gene; (2) Using overlapping PCR to connect the upstream and downstream homology arms in step (1); (3) Using two restriction endonucleases to treat the plasmid and the connected upstream and downstream homology arms to obtain a linearized plasmid and upstream and downstream homology arms; (4) Using DNA ligase, the linearized plasmid in step (3) is connected to the upstream and downstream homology arms to construct an allele exchange vector for knocking out the capsule-related gene fragment; (5) transferring the allele exchange vector into Escherichia coli S17-1 cells by chemical transformation, and screening donor bacteria carrying the allele exchange vector; (6) The donor bacteria and Acinetobacter baumannii undergo a first homologous recombination by conjugation, inserting the allele from the donor bacteria into Acinetobacter baumannii to obtain a diploid; (7) Sucrose-containing culture medium was used to promote the second homologous recombination of Acinetobacter baumannii, thereby deleting the target sequence and the inserted plasmid backbone sequence to obtain haploid.

[0006] In step (1), the primers of the upstream and downstream homology arms include upstream-F, upstream-R, downstream-F and downstream-R primers, which are located in the upstream region and downstream region of the target knockout sequence, respectively, as shown in SEQ ID NO.1-SEQ ID NO.4.

[0007] In step (3), the plasmid is pMo130 plasmid, and the restriction endonuclease is any two restriction sites in the multiple cloning site region of the plasmid.

[0008] In step (5), pMo130-Tel-F and pMo130-Tel-R primers are designed on both sides outside the plasmid multiple cloning site region for screening donor bacteria carrying the allele exchange vector, wherein: the pMo130-Tel-F and pMo130-Tel-R primer pair is located outside the plasmid restriction site, as shown in SEQ ID NO.5-SEQ ID NO.6.

[0009] In step (6), the donor bacteria and Acinetobacter baumannii are dropped onto an MHA agar plate at a volume ratio of 2:1 and cultured overnight. The bacterial cells are collected and resuspended, and a portion of the bacterial suspension is spread on an agar plate containing potassium tellurite and kanamycin for culture. The purpose of kanamycin is to reversely screen the donor bacteria. PCR amplification is performed using upstream-F / downstream-R or upstream-R / downstream-F primers to screen diploids.

[0010] In step (7), diploid colonies are first selected and continuously subcultured in LB broth medium containing 10% sucrose, and then spread on potassium tellurite / ampicillin sodium double-drug plates, and catechol solution is sprayed on the plates to select white clones. Aim-in-F and aim-in-R primers are designed inside the knockout target gene, and LF and LR primers are designed outside the upstream and downstream regions of the genome sequence for screening haploids, as shown in SEQ ID NO.7-SEQ ID NO.10.

[0011] The Acinetobacter baumannii is LAC-4, and the capsule-related gene is wxya Gene.

[0012] An application of a knockout strain of capsule-related genes of Acinetobacter baumannii based on homologous recombination.

[0013] Compared with the existing research status, the beneficial effects of the present invention are as follows: The invention provides a simple and effective method for constructing capsule-related gene knockout strains for Acinetobacter baumannii, designs primers for amplifying upstream and downstream homologous arms, utilizes high-fidelity enzymes to amplify upstream and downstream homologous arms of target genes, utilizes enzyme cutting technology to construct an allele exchange vector for knocking out target genes, transfers the allele exchange vector into a donor bacterium through chemical transformation, performs a first homologous recombination with a recipient bacterium (Acinetobacter baumannii) through conjugation, inserts the allele from the donor bacterium into the recipient strain, utilizes sucrose culture medium to promote the recipient bacterium to undergo a second homologous recombination, deletes the target sequence and the inserted plasmid backbone sequence, and obtains a haploid. The method is simple, efficient, and easy to operate, and is suitable for knocking out capsule-related genes of Acinetobacter baumannii. The obtained knockout strain is helpful to fill the research gap of the influence of capsule-related genes of Acinetobacter baumannii on bacterial virulence. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a map of the vector containing the allele for knocking out the capsule-related gene fragment constructed in the examples of the present invention.

[0015] Figure 2 This is a graph showing the results of PCR detection by electrophoresis in an embodiment of the present invention.

[0016] Figure 3 This is the survival curve of mice after intraperitoneal bacterial infection in an example of the present invention. DETAILED DESCRIPTION

[0017] The present invention will be described in detail below according to the accompanying drawings and preferred embodiments, and the purpose and effect of the present invention will become more clear. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0018] The present invention provides a simple and effective method for constructing a traceless capsule-related gene fragment knockout strain for Acinetobacter baumannii. Specifically, Acinetobacter baumannii is subjected to an operation comprising the following steps: (1) Primer design Using snapgene software: a. Design primers for the target gene and name them aim-F / aim-R; b. Design primers for the sequences of about 500 bp upstream and downstream of the target gene in the wild type (WT), so as to amplify the upstream and downstream sequences of about 500 bp respectively by PCR. When designing primers, it is necessary to add protective bases (according to the table) and restriction site sequences to the front ends of the upstream F primer and the downstream R primer according to the restriction sites on the selected suicide plasmid, and name them upstream-F / upstream-R and downstream-F / downstream-R respectively; as shown in SEQ ID NO.1- SEQ ID NO.4.

[0019] c. Design primers on the outer sides of the selected restriction site of the plasmid and name them (taking pMo130 as an example) pMo130-Tel-F / pMo130-Tel-R; as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0020] d. Design primers LF and LR on the outer sides of the upstream and downstream of the target gene; as shown in SEQ ID NO.9 and SEQ ID NO.10.

[0021] e. Design primers of about 500 bp in size inside the target gene and name them aim-in-F / aim-in-R, as shown in SEQ ID NO.7 and SEQ ID NO.8.

[0022] (2) Use the primer pairs upstream-F / upstream-R and downstream-F / downstream-R to amplify the upstream and downstream of the target gene of the WT strain by colony PCR. a.PCR system (total amount 50ul): 2 × Rapid Taq Master Mix: 25ul Primer: 1ul each DNA template: 2ul ddH2O: 21ul b.PCR conditions: 94℃ 3min 94℃ 30s 54℃ 30s 72℃ 1min 72℃ 5min 4℃; c. PCR set for 30 cycles; d. Detect the amplified target band by agarose gel electrophoresis.

[0023] (3) Use a DNA Fragment Purification Kit to purify the PCR product from step (2), measure its DNA concentration using nano drop2000 software, and annotate it.

[0024] (4) Use overlap PCR to connect the upstream and downstream products purified in the third step.

[0025] There are two steps: a.①PCR system (total amount 25ul): 2 × High-Fidelity Enzyme: 12.5ul DNA template: Calculate the volume to be added based on the concentration of the product from the previous step and the total amount of 100 ng ddH2O: make up to 25ul; ②PCR conditions: 94℃ 5min 94℃ 30s 55℃ 30s 72℃ 40s 72℃ 5min 4℃ ; ③PCR set 10 cycles b. Continue to add to the PCR product in step ①: ① High-fidelity enzyme 12.5ul 1ul each of primers upstream-F and downstream-R ddH2O:10.5ul ②PCR conditions: 94℃ 5min 94℃ 30s 54℃ 30s 72℃ 40s 72℃ 5min 4℃; ③PCR set for 30 cycles c. Detect the bands by agarose gel electrophoresis. The correct band size should be about 1000bp (500bp upstream + 500bp downstream).

[0026] (5) Gel excision and recovery of the overlap PCR products: a. Make a large-pore agarose gel, add 200-300ul of overlap PCR product into it, and use gel recovery kit to excise and recover the gel (refer to gel recovery kit for specific steps) b. The gel excision products can be sent for next generation sequencing to ensure the correctness of the sequence.

[0027] (6) Construction of vector: ① According to the selected suicide plasmid (above) and restriction enzyme cutting site (above), the plasmid and the above gel recovery product are digested. For example: select PMO130 plasmid (potassium tellurite, kanamycin resistance) (attached Figure 1 ), double digest the plasmid and linear DNA with Not I and BamH I restriction endonucleases: a. Linear DNA digestion system: Buffer 5ul, DNA (total amount 1000ng / concentration), Not I and BamH I, 1ul each, ddH2O: make up to 50ul; b. Plasmid DNA digestion system: Buffer 5ul, DNA (total amount 1000ng / concentration) Not I and BamH I, 1ul each, ddH2O: make up to 50ul; c. The above reaction system was gently mixed and centrifuged instantaneously, and then digested at 30℃ or 37℃ for 10 min.

[0028] ②Purify the DNA after enzyme digestion and determine the concentration.

[0029] ③Use T4 ligase to connect the digested plasmid and linear DNA fragment: a. Reaction system: T4 DNA ligase Buffer 2ul, Plasmid after enzyme digestion (50ng / concentration), Linear DNA after enzyme digestion (37.5ng / concentration), T4 DNA ligase: 1ul, ddH2O: make up to 20ul; b. Reaction conditions: 16℃ for 1~5h.

[0030] (7) Purify the ligation product and introduce it into E. coli S17-1 competent cells: The s17-1 competent cells were dissolved on ice, and the ligation product was added to 100ul s17-1 competent cells (gently blown), placed on ice for 30min, heat-shocked at 42℃ for 45s, and then quickly transferred to ice for 2min. After adding 900ul SOC culture medium and mixing, the cells were cultured at 37℃ and 180r / min for 1h. The products were then spread on 6ug / ml potassium tellurite plates and cultured at 37℃ for >18h to screen the successfully transformed clones.

[0031] (8) Select the clones grown on the drug plate, streak the plate and perform colony PCR using two pairs of primers: pMo130-Tel-F / pMo130-Tel-R and upstream-F and downstream-R.

[0032] a. The PCR system and conditions are the same as step (2).

[0033] b. Perform agarose gel electrophoresis, detect bands, and perform first-generation sequencing on the PCR product to ensure the sequence is correct. The sequence can then be imported into snapgene software to draw a constructed vector map, named pMo130-aim-up / down.

[0034] (9) Use plasmid conjugation to allow the constructed plasmid to enter WT and undergo a single exchange with WT.

[0035] a. The donor bacteria is: s17-1 containing pMo130-aim-up / down.

[0036] b. The recipient bacteria is the WT strain.

[0037] c. Pick the WT strain and inoculate it into 2 ml LB medium and the s17-1 monoclonal strain and inoculate it into 1 ml LB medium (containing 6 μg / mL potassium tellurite), culture overnight, centrifuge at 5000 r / min for 3 min to remove the supernatant, wash the bacteria twice with 1.5 mL LB, and finally mix the two bacteria with LB and resuspend them. The resuspended liquid is titrated on the surface of the LB plate, wait for it to penetrate and dry, and culture it in an incubator for 16-24 hours.

[0038] d. Collect the bacteria from the surface of the plate, resuspend the bacteria in 100uL PBS, and spread them on LB double-antibody plates (30μg / mL potassium tellurite, 25μg / mL ampicillin sodium). Incubate the double-antibody plates at 37°C for 24h, and pick clones for PCR identification.

[0039] e. PCR primers: the LF and LR primer pairs mentioned above, the PCR system and conditions are as described above.

[0040] f. PCR-correct colonies, suicide plasmid inserted into the genome of Acinetobacter baumannii by single crossover.

[0041] (10) The transformants with correct insertion were continuously subcultured in LB containing 10% sucrose at a ratio of 1:100 (24 hours for one generation). After each generation was cultured for 24 hours, LB plates were coated. After incubation in an incubator, the grown colonies were sprayed with catechol solution. The white clones may be mutants or strains that have reverted to the wild type. The white clones and WT were identified by PCR using three sets of primers. The strain with successful knockout should be: a. aim-in-F / R primer pair product: Knockout strain, no band; WT, 500 bp; Plasmid primers pMo130-Tel-F / pMo130-Tel-R: Knockout strain, no band; WT, no band.

[0042] The s17-1 containing the constructed vector had the corresponding band.

[0043] upstream-F and downstream-R (attached Figure 2 ): In the knockout strain, the band size is only the sum of the length of the upstream and downstream fragments; WT, complete length (upstream + target gene + downstream).

[0044] LF and LR: For knockout strains, the band size is the sequence length of WT minus the length of the knockout gene; WT, complete length.

[0045] d. Perform first-generation sequencing on the PCR product and compare the sequence with WT. The correct knockout strain should confirm the target gene sequence.

[0046] (11) Toxicity verification a. Columbia blood agar plates were inoculated with target bacteria in four zones.

[0047] b. Pick a single colony and add it to sterile saline, adjust the turbidity to 0.5 McFarland turbidity, dilute it 10 times, and inject 100 μL of the solution into female BAlB / c mice about 5 weeks old through the peritoneal cavity. Each group of bacteria contains 10 mice.

[0048] c. After injection, the mice were placed in a room temperature environment, given normal feed and water, and the death of the mice was observed after 24 hours.

[0049] Figure 3 The lethality of the bacteria to mice before and after the knockout of the strain is given. Figure 3 The results show that the lethality of the strain to mice was reduced after knockout, indicating that the knockout was successful.

[0050] Those skilled in the art can understand that the above are only preferred examples of the invention and are not intended to limit the invention. Although the invention is described in detail with reference to the above examples, those skilled in the art can still modify the technical solutions recorded in the above examples or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, etc. made within the spirit and principle of the invention shall be included in the protection scope of the invention.

Claims

1. A method for knocking out capsule-related genes of Acinetobacter baumannii, characterized in that: The following steps are involved: (1) Design primers to amplify the upstream and downstream homology arms of the knockout target gene, and use high-fidelity enzymes to amplify the upstream and downstream homology arms of the target gene; (2) Using overlapping PCR to connect the upstream and downstream homology arms in step (1); (3) Using two restriction endonucleases to treat the plasmid and the connected upstream and downstream homology arms to obtain a linearized plasmid and upstream and downstream homology arms; (4) Using DNA ligase, the linearized plasmid in step (3) is connected to the upstream and downstream homology arms to construct an allele exchange vector for knocking out the capsule-related gene fragment; (5) transferring the allele exchange vector into Escherichia coli S17-1 cells by chemical transformation, and screening donor bacteria carrying the allele exchange vector; (6) The donor bacteria and Acinetobacter baumannii undergo a first homologous recombination by conjugation, inserting the allele from the donor bacteria into Acinetobacter baumannii to obtain a diploid; (7) Sucrose-containing culture medium was used to promote the second homologous recombination of Acinetobacter baumannii, thereby deleting the target sequence and the inserted plasmid backbone sequence to obtain haploid.

2. The method for producing capsule-related gene knockout strains of Acinetobacter baumannii based on homologous recombination according to claim 1, characterized in that: In step (1), the primers of the upstream and downstream homology arms include upstream-F, upstream-R, downstream-F and downstream-R primers, which are located in the upstream region and downstream region of the target knockout sequence, respectively, as shown in SEQ ID NO.1-SEQ ID NO.

4.

3. The method for knocking out capsule-related genes of Acinetobacter baumannii based on homologous recombination according to claim 1, characterized in that: In step (3), the plasmid is pMo130 plasmid, and the restriction endonuclease is any two restriction sites in the multiple cloning site region of the plasmid.

4. The method for producing a knockout strain of capsule-related genes based on homologous recombination of Acinetobacter baumannii according to claim 1, characterized in that: In step (5), pMo130-Tel-F and pMo130-Tel-R primers are designed on both sides outside the plasmid multiple cloning site region for screening donor bacteria carrying the allele exchange vector, wherein: the pMo130-Tel-F and pMo130-Tel-R primer pair is located outside the plasmid restriction site, as shown in SEQ ID NO.5-SEQ ID NO.

6.

5. The method for knocking out capsule-related genes of Acinetobacter baumannii based on homologous recombination according to claim 1, characterized in that: In step (6), the donor bacteria and Acinetobacter baumannii are dropped onto an MHA agar plate at a volume ratio of 2:1 and cultured overnight. The bacterial cells are collected and resuspended, and a portion of the bacterial suspension is spread on an agar plate containing potassium tellurite and kanamycin for culture. The purpose of kanamycin is to reversely screen the donor bacteria. PCR amplification is performed using upstream-F / downstream-R or upstream-R / downstream-F primers to screen diploids.

6. The method of producing a knockout strain of capsule-related genes based on homologous recombination of Acinetobacter baumannii according to claim 1, characterized in that: In step (7), diploid colonies are first selected and continuously subcultured in LB broth medium containing 10% sucrose, and then spread on potassium tellurite / ampicillin sodium double-drug plates, and catechol solution is sprayed on the plates to select white clones. Aim-in-F and aim-in-R primers are designed inside the knockout target gene, and LF and LR primers are designed outside the upstream and downstream regions of the genome sequence for screening haploids, as shown in SEQ ID NO.7-SEQ ID NO.

10.

7. The method for constructing a Pseudomonas traceless long gene fragment knockout strain based on homologous recombination according to claim 1, characterized in that: The Acinetobacter baumannii is LAC-4, and the capsule-related gene is wxya Gene.

8. An application of the capsule-related gene knockout strain of Acinetobacter baumannii based on homologous recombination as claimed in claim 2.

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