Method for carrying out gene knockout and complementation on staphylococcus aureus chp gene

Abstract

The invention discloses a method for carrying out gene knockout and complementation on a staphylococcus aureus chp gene. The gene knockout method comprises the following steps: 1) extracting genome DNA of staphylococcus aureus; 2) designing primers; 3) amplifying upstream and downstream homologous arms of the chp gene and knocking out resistance gene segments; (4) carrying out enzyme digestion on an empty vector and constructing a recombinant plasmid; 5) transforming the recombinant plasmid; 6) screening positive transformants; 7) knocking out the inserted DNA fragment and amplifying; 8) electrotransformation of staphylococcus aureus; 9, positive transformants are screened, and the staphylococcus aureus chp gene knockout strain is obtained.The invention further provides a method for conducting knockout gene back-supplement on the staphylococcus aureus knockout strain. The knockout operation of the chp gene and the back-supplement operation of the knockout strain are successfully achieved, and research on the important virulence factor chp of staphylococcus aureus can be helped.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a method for gene knockout of Staphylococcus aureus based on the principle of homologous recombination and complementation of the knockout strain. Background Art

[0002] Staphylococcus aureus is an important human pathogen that can cause various severe infections, such as pneumonia, sepsis, endocarditis, etc. In recent years, ST59-type Staphylococcus aureus strains have gradually replaced ST239 as the most main ST type in China, and the chp gene is considered to be one of the main contributing factors to the prevalence of ST59-type strains, but the current understanding is still limited. Summary of the Invention

[0003] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for gene knockout and complementation of the chp gene of Staphylococcus aureus.

[0004] The technical solution for the present invention to achieve its purpose is as follows: A method for gene knockout of the chp gene of Staphylococcus aureus, comprising the following steps: 1) Extract the genomic DNA of Staphylococcus aureus; 2) Primer design: Take the upstream and downstream homologous arms of the target gene chp to be knocked out as two fragments, and respectively design primer pairs UP-F shown in SEQ ID NO. 1 and UP-R shown in SEQ ID NO. 2 for amplifying the upstream homologous arm, and primer pairs DN-F shown in SEQ ID NO. 3 and DN-R shown in SEQ ID NO. 4 for amplifying the downstream homologous arm; 3) Amplify the upstream and downstream homologous arms of the target gene and the knockout resistance gene fragment: Using the genomic DNA of Staphylococcus aureus containing the target gene as a template, perform PCR amplification with UP-F and UP-R as primer pairs and DN-F and DN-R as primer pairs respectively to amplify the upstream and downstream homologous arm knockout resistance gene fragments of the target gene; 4) Empty vector digestion and recombinant plasmid construction: Digest the empty vector with a restriction endonuclease to obtain a vector plasmid fragment, and treat the vector plasmid fragment with the upstream and downstream homologous arms of the target gene in a recombinase reaction system to construct a recombinant plasmid; 5) Transformation of the recombinant plasmid: 6) Screening of positive transformants: Plate screening was performed on the recombinant plasmid after the treatment in step 5) to obtain positive transformants; single colonies of positive transformants were selected, colony PCR was performed using UP-F and DN-R as primer pairs, the PCR products were detected by agarose gel electrophoresis, and the monoclonal strains with the target bands were sequenced. Strains with correct sequences were selected to extract plasmids to obtain the gene knockout recombinant plasmid; 7) Amplification of the knockout inserted DNA fragment: Using the gene knockout recombinant plasmid as a template, the knockout inserted DNA fragment was amplified using UP-F and DN-R as primer pairs; 8) Electrotransformation of Staphylococcus aureus: The knockout inserted DNA fragment was inserted into the Staphylococcus aureus genome by electrotransformation to achieve the knockout of the target gene; 9) For the Staphylococcus aureus after the treatment in step 8), colony PCR was used to screen out the positive transformants in which the knockout resistance gene was correctly inserted into the target gene position of the Staphylococcus aureus genome, and the Staphylococcus aureus chp gene knockout strain was obtained.

[0005] A method for constructing a complementation strain of MRSA ST59 △chp, and the method for obtaining the Staphylococcus aureus chp gene knockout strain is as described in claim 1; The construction of the complementation strain includes the following steps: 1) Using the genomic DNA of MRSA ST59 as a template, the full length of the chp gene plus its promoter region and the relevant binding sites of ribosomes were amplified; 2) Construct the complementation plasmid pLI50-chp; at the same time, the empty plasmid pLI50 was transferred as a negative control.

[0006] 3. An application of the Staphylococcus aureus chp gene knockout strain as described in claim 1.

[0007] The beneficial effects of the present invention are as follows: The method for knocking out the chp gene provided by the present invention can specifically target the chp gene of Staphylococcus aureus to obtain strains with the chp gene cluster knocked out, thereby providing technical support for clarifying the specific function of the important virulence factor - chp of Staphylococcus aureus. Description of the drawings

[0008] Figure 1 It is the plasmid map of the vector plasmid pKOR1 used in the examples of the present invention.

[0009] Figure 2 It is the RT-qPCR verification chart of the chp gene knockout result in the examples of the present invention. Detailed implementation manners

[0010] The present invention will be further described below with reference to the drawings and examples.

[0011] 1. Extraction of genomic DNA from Staphylococcus aureus 1) Resuscitate the strain from the -80 °C refrigerator and activate it by streaking in four zones on a Columbia blood agar plate.

[0012] 2) Pick single colonies from the blood agar plate, suspend them separately in 4 mL of TSB for activation, and culture them overnight with shaking at 37 °C.

[0013] 3) Centrifuge at 12000 g for 2 min and discard the supernatant. At the same time, wash the bacterial cells with sterile deionized water and repeat 3 times to collect the bacterial cells.

[0014] 4) Resuspend the bacteria in 200 μL of sterile deionized water, then add 5 μL of lysostaphin (10 mg / mL), mix well, and incubate in a water bath at 37 °C for more than 2 h until the bacteria are lysed and become clear and transparent.

[0015] 5) Add 20 μL of proteinase K (20 mg / mL), mix well, and incubate in a water bath at 56 °C for 30 min.

[0016] 6) Add 200 μL of absolute ethanol and 200 μL of Buffer BD solution, and mix by shaking.

[0017] 7) Discard the waste liquid, put the adsorption column back into the collection tube, add 600 μL of detergent PW Buffer, and centrifuge at 12000 rpm for 1 min.

[0018] 8) Discard the waste liquid. Add 500 μL of detergent Wash Buffer and centrifuge at 12000 rpm for 1 min.

[0019] 9) Place the adsorption column in a centrifuge tube and centrifuge at 12000 rpm for 2 min.

[0020] 10) Take out the adsorption column and air-dry the residual detergent at room temperature.

[0021] 11) Place the adsorption column into another sterile 1.5 mL EP tube. Add 100 μL of sterile deionized water preheated to 65 °C in the center of the adsorption membrane, let it stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 1 min to elute the DNA.

[0022] 12) Detect the concentration of DNA with NanoDrop2000, label it, and store it at -20 °C in the refrigerator.

[0023] 2. Primer design First, according to the genomic sequences in the sequenced strains, specific primers targeting approximately 1000 bp fragments upstream and downstream of chp were designed respectively. Using the DNA of Staphylococcus aureus genome as a template, the upstream and downstream homologous arm fragments of the chp gene were amplified by PCR. The homologous arm primers are represented by UP-F / UP-R and DN-F / DN-R respectively (SEQ ID NO.1 - SEQ ID NO.4). Among them, it includes the attB site and restriction enzyme sites.

[0024] 3. Amplification of the upstream and downstream homologous arms of the chp gene The PCR reaction used the genomic DNA of Staphylococcus aureus as a template and was amplified successively using PrimeSTAR® Max DNA Polymerase.

[0025] The PCR amplification conditions are as follows: Step 1 (1 cycle) Step 2 98°C, 10 min 98°C, 10 s 55°C, 5 s 72°C, 5 s / kb (30 cycles) Step 3 (1 cycle) Step 4 72°C, 10 min 10°C, end.

[0026] 4. After electrophoresis separation of the PCR products, they were recovered 1) Add an appropriate amount of Loading Buffer to the PCR products, add all the products to the grooves, and select a suitable DNA marker.

[0027] 2) Set the voltage to 100V and the time to 40 min. Start running the electrophoresis.

[0028] 3) After the electrophoresis is completed, under the ultraviolet gel cutting instrument, quickly cut off the complete target gel strip, turn off the ultraviolet gel cutting instrument, crush the gel strip, put it into a clean 1.5 mL EPP tube. Record the weight, and add 200 μL of NTI sol solution to every 0.1 g of gel block. Then put it into a 50°C metal bath for 10 min to fully dissolve the gel block.

[0029] 4) Add it to the adsorption column, let it stand for 2 min, then centrifuge at 12000 rpm for 1 min and discard the waste liquid.

[0030] 5) Add 700 mL of NT3 solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid. 6) Repeat step 5.

[0031] 7) Centrifuge again for 2 min and discard the waste liquid.

[0032] 8) Let it stand to dry.

[0033] 9) Transfer the adsorption column to a new sterile 1.5 mL EP tube, add 30 μL of sterile deionized water at 65 °C to the center of the adsorption membrane, and centrifuge at 12,000 rpm for 1 min to elute the DNA.

[0034] 10) Detect the DNA purity and concentration with NanoDrop2000, label it and store it at -20 °C in the refrigerator.

[0035] 5. Digestion of the PCR recovery product Use EcoR I and HindIII (rapid restriction endonucleases) to perform digestion reactions on the purified PCR recovery product respectively.

[0036] 6. Purification after digestion of the PCR recovery product 1) Mix the digested reaction product (240 μL) evenly with 5 volumes (1.2 mL) of the binding solution PB Buffer.

[0037] 2) Add it to the adsorption column in batches, let it stand for 2 min, then centrifuge at 12,000 rpm for 1 min and discard the waste liquid.

[0038] 3) Add 500 μL of PW Buffer to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0039] 4) Repeat step 3).

[0040] 5) Centrifuge again for 2 min and discard the waste liquid.

[0041] 6) Let it stand at room temperature to dry for 3 min.

[0042] 7) Transfer the adsorption column to a new sterile 1.5 mL EP tube, add 40 μL of sterile deionized water at 70 °C to the center of the adsorption membrane, and centrifuge at 12,000 rpm for 1 min to elute the DNA.

[0043] Detect the DNA purity and concentration with NanoDrop2000, label it and store it at -20 °C in the refrigerator.

[0044] 7. Ligation of the upstream and downstream of the digested PCR recovery product The PCR products after digestion were ligated with T4 DNA ligase.

[0045] 8. PCR Amplification Verification of the Ligation Products Since it is single digestion, the ligated products need to be verified by PCR, and then the target products are purified. The purified products are the upstream and downstream homologous arms of the gene to be knocked out.

[0046] Using UP-F and DN-R as primers, PCR amplification reaction was carried out with PrimeSTARR Max DNA Polymerase.

[0047] After electrophoresis of the amplified products, the target bands were cut and the PCR products were recovered. The purified products were sent to the company for first-generation sequencing identification.

[0048] 9. Extraction of Plasmid pKOR1 Plasmid pKOR1 is a temperature-sensitive shuttle plasmid (as Figure 1 shown), showing chloramphenicol resistance in positive bacteria and ampicillin resistance in negative bacteria, and usually stored in Escherichia coli.

[0049] 1) Take out the Escherichia coli carrying plasmid pKOR1 from the -80 °C refrigerator, streak it on an LB plate containing 100 ug / mL, and culture it at 30 °C for 24 h.

[0050] 2) Pick a single colony, resuspend it in 4 mL of LB liquid medium containing 100 ug / mL, and culture it overnight at 30 °C with shaking at 220 rpm.

[0051] 3) Centrifuge at 5000g for 10 min at 4 °C, and discard the supernatant.

[0052] 4) Add 250 uL of P1 solution in the kit to completely suspend the bacteria.

[0053] 5) Then add 250 uL of P2 solution and gently invert it up and down 6 times.

[0054] 6) Add 350 uL of P3 solution and immediately gently invert it 6 times.

[0055] 7) Centrifuge at 13800g for 10 min, pipette the supernatant into the adsorption column, let it stand for 3 min, and centrifuge again for 1 min, then discard the supernatant.

[0056] 8) Add 500 uL of PW buffer to the adsorption column and centrifuge at 13800g for 30 s.

[0057] 9) Repeat step 8).

[0058] 10) Centrifuge again for 2 min and discard the waste liquid.

[0059] 11) Air dry at room temperature for 2 min and air dry the washing solution as much as possible.

[0060] 12) Add 100 μL of preheated sterile deionized water to the membrane in the adsorption column and centrifuge at 12,000 g for 2 min.

[0061] 13) Detect the DNA purity and concentration with NanoDrop 2000, label it and store it at -20 °C in the refrigerator.

[0062] 10. B-P reaction between the upstream and downstream homologous arm fragments and the plasmid pKOR1

[0063] 11. Preparation of Escherichia coli competent DH5a cells 1) Take out the Escherichia coli DH5a strain from the -80 °C refrigerator and activate it on an LB plate.

[0064] 2) Pick a single colony, resuspend it in 4 mL of fresh LB liquid medium and culture it overnight at 37 °C.

[0065] 3) Take 500 μL of the fresh bacterial solution and add it to a 100 mL fresh sterile LB liquid culture flask that has been autoclaved at a ratio of 1:200. Culture it at 37 °C with shaking at 220 rpm for about 2 h 30 min until the OD600 reaches about 0.5.

[0066] 4) Place the culture flask with an OD600 of 0.5 on ice and let it stand for 30 min.

[0067] 5) Aliquot it into two pre-cooled 50 mL sterile centrifuge tubes, centrifuge at 4000 g at 4 °C for 20 min, and pour off the supernatant of the bacterial solution.

[0068] 6) Add 25 mL of pre-cooled CaCl 2 solution to the centrifuge tube, resuspend the cells, and centrifuge at 4000 g at 4 °C for 10 min.

[0069] 7) Repeat resuspension with 25 mL of pre-cooled CaCl2 solution and centrifuge 2 times.

[0070] 8) Pour off the supernatant, then add 25 mL of pre-cooled sterile deionized water, resuspend the cells, and centrifuge at 4000 g at 4 °C for 10 min.

[0071] 9) Repeat the operation in step 8) 2 times.

[0072] 10) Pour off the supernatant, add 1 mL of 70% glycerol broth, and resuspend.

[0073] 11) Aliquot into sterile and enzyme-free 1.5 mL EP tubes at a specification of 100 uL of bacterial solution per tube and store at -80 °C.

[0074] 12. Heat shock transformation of B-P reaction products 1) Take out the DH5a competent cells from the -80 °C refrigerator and melt them on ice for 25 min.

[0075] 2) Add the system after the BP reaction to the melted competent cells, place it on ice, and let it stand for about 30 min to make the product and the competent cells fully fit; and turn on the water bath at 42 °C for standby.

[0076] 3) Perform heat transformation at 42 °C in a water bath for 90 s. Immediately insert it into ice for 5 min after the transformation to allow the cell wall of the competent cells to contract.

[0077] 4) Add 1 mL of fresh sterile LB culture medium, culture at 30 °C and 220 rpm for 1 h.

[0078] 5) Centrifuge at 4 °C and 12000 rpm for 2 min.

[0079] 6) Discard the supernatant, add 100 ug of LB liquid medium with 100 ug / mL ampicillin resistance, and resuspend.

[0080] 7) Aspirate all the bacterial solution and evenly spread it on an LB plate with 100 ug / mL ampicillin resistance, and culture at 30 °C.

[0081] 13. PCR identification of the positive clone plasmid with successful heat transformation Use the successfully ligated PCR product as the DNA template for the positive control, and sterile deionized water as the DNA template for the negative control. Pick the suspected positive clones grown on the LB plate with 100 ug / mL ampicillin resistance and culture them in LB liquid medium with 100 ug / mL ampicillin resistance for 16 h. The obtained bacterial solution is used as the sample to be tested. According to the gene, select the corresponding primers and conditions for PCR amplification reaction. After preliminary verification by electrophoresis, then perform bidirectional confirmation of the suspected positive PCR products by first-generation sequencing. Confirm the true DH5a-pKOR1-Achp positive clones and then extract the positive homologous arm plasmids.

[0082] 14. Extraction of positive clone plasmids Extract the plasmid of DH5a-pKOR1-△chp and store it in the -20 °C refrigerator.

[0083] 15. Heat transformation of pKOR1-△chp into competent cells DC10B The pKOR1-△chp was thermally transformed into DC10B for modification, and only the modified plasmid could be electrotransformed into competent Staphylococcus aureus.

[0084] 16. Midiprep of the positive plasmid Since the amount of plasmid for electrotransformation needs to be 5 - 10 μg and the concentration of the positive plasmid is relatively high, a plasmid miniprep and midiprep kit was used for extraction. The method is as follows: 1) Streak the identified positive clone strain on an LB plate with 100 μg / mL ampicillin resistance and culture at 30°C for 24 h.

[0085] 2) Pick a single colony and suspend it in 100 mL of LB broth with 100 μg / mL ampicillin resistance, then culture at 30°C for 16 - 18 h.

[0086] Transfer it into two 50 mL centrifuge tubes, centrifuge at 4000 g for 20 min at 4°C, pour off the supernatant, wash the cells once with normal saline, centrifuge at 4000 g for 20 min at 4°C, and pour off the supernatant to retain the cell pellet.

[0087] 3) Add 500 μL of P1 Buffer and mix well to lyse the cells.

[0088] 4) Add 500 μL of Buffer and mix well until it turns blue.

[0089] 5) Add 850 μL of P3 Buffer until the lysate turns white after lysis.

[0090] 6) Centrifuge at 12800 rpm for 10 min at 4°C.

[0091] 7) Add 500 μL of BL buffer to the adsorption column, centrifuge at 12800 rpm for 1 min, and discard the supernatant.

[0092] 8) Add the supernatant from step 6) to the adsorption column and let it stand for 5 min.

[0093] 9) Centrifuge at 12800 rpm for 2 min and pour off the waste liquid.

[0094] 10) Add 600 μL of PW Buffer to the adsorption column, centrifuge at 12800 rpm for 2 min, and discard the supernatant.

[0095] 11) Add 600 μL of Wash Buffer to the adsorption column, centrifuge at 12800 rpm for 2 min, and discard the supernatant.

[0096] 12) Centrifuge again for 2 min, take out the adsorption column and place it on a clean piece of paper to air dry for 2 min.

[0097] 13) Add 100 μL of sterile deionized water preheated at 65 °C to elute the plasmid DNA.

[0098] 14) Detect the purity and concentration of the DNA using NanoDrop2000, label it, and store it at -20 °C in the refrigerator.

[0099] 17. Preparation of competent cells of Staphylococcus aureus 1) Take out Staphylococcus aureus from -80 °C and streak it on a blood agar plate for activation.

[0100] 2) Pick a single colony and resuspend it in 4 mL of sterile TSB liquid medium, and culture it overnight at 37 °C with 220 rpm.

[0101] 3) Inoculate the overnight culture at a ratio of 1:200, culture it at 37 °C with 220 rpm for 3 h to make the OD600 around 0.6. Then place it on ice for 30 min to slow down the bacterial growth.

[0102] 4) All the following operations need to be carried out on ice: Aliquot the bacterial suspension into pre-cooled sterile centrifuge tubes, centrifuge at 4000 g at 4 °C for 20 min to collect the bacterial cells.

[0103] 5) Resuspend and wash with sterile deionized water (pre-cooled), and centrifuge at 4 °C, 4000 rpm for 10 min, and repeat the washing 3 times.

[0104] 6) Resuspend and wash with 10% glycerol solution (pre-cooled), centrifuge for 10 min, and repeat the washing 2 times.

[0105] 7) Add 1 mL of 50% glycerol (pre-cooled) to resuspend the bacterial suspension, aliquot it into sterile 1.5 mL EPP tubes at a specification of 100 μL per tube, and store it at -80 °C in the refrigerator for standby.

[0106] 18. Electrotransformation of the homologous recombination plasmid pKOR1-Δchp into competent cells of Staphylococcus aureus 1) Take out the electrotransformation-competent cells of Staphylococcus aureus stored in the -80 °C refrigerator, and place them on ice for 25 min to slowly thaw.

[0107] 2) Add 10 μg of the homologous recombination plasmid pKOR1-Δchp to the electrotransformation-competent cells and incubate for 30 min for conjugation.

[0108] 3) Take out a brand-new sterile electroporation cuvette, place it in the -80 °C refrigerator for pre-cooling for 1 h for standby.

[0109] 4) Add the conjugated homologous recombination plasmid pKOR1-Δchp of the above-mentioned competent cells to the electroporation cuvette and perform electroporation (voltage 2.6 V) for 4 min.

[0110] 5) Add 1 mL of pre-warmed B2 resuscitation liquid medium at 30°C and mix well with the competent cells after electroporation. Quickly transfer the mixture to a sterile 1.5 mL EP tube and incubate at 30°C with shaking at 220 rpm for 3 h.

[0111] 6) Centrifuge at 4000 g for 5 min at 4°C. Aspirate 900 μL of the supernatant, resuspend the remaining 100 μL of the bacterial suspension, and spread it on a TSA plate containing 10 μg / mL chloramphenicol resistance. Incubate the plate in an incubator at 30°C for 48 h.

[0112] 19. Extraction and Identification of Recombinant Plasmid pKOR1-△chp in Staphylococcus aureus Since Staphylococcus aureus is a Gram-positive bacterium with a thick cell wall, it is not possible to perform colony PCR, which is prone to false negatives. Therefore, for the identification of recombinant plasmid pKOR1-△chp in Staphylococcus aureus, the plasmid needs to be extracted first and then subjected to PCR reaction. The plasmid extraction of Staphylococcus aureus requires prior cell wall disruption, and the specific method is as follows: 1) Take out the positive clone carrying the plasmid from the -80°C refrigerator and streak it on a TSA plate containing 10 μg / mL chloramphenicol resistance. Incubate at 30°C for 48 h.

[0113] 2) Pick a single colony and resuspend it in 4 mL of TSB liquid medium containing 10 μg / mL chloramphenicol resistance. Incubate at 30°C with shaking at 220 rpm overnight.

[0114] 3) Centrifuge at 4000 g for 10 min at 4°C.

[0115] 4) Add 250 μL of Solution P1 in the kit to completely suspend the bacteria. Since the cell wall of Staphylococcus aureus is thick, cell wall disruption is required before plasmid extraction. Add 5 μL of lysostaphin and incubate in a water bath at 37°C for 2 h until the bacterial suspension becomes completely clear.

[0116] 5) Then add 250 μL of Solution P2 and gently invert the tube up and down 6 times.

[0117] 6) Add 350 μL of Solution P3 and immediately gently invert the tube 6 times.

[0118] 7) Centrifuge at 13800 g for 10 min, transfer the supernatant to the adsorption column, let it stand for 3 min, and then centrifuge for 1 min again. Discard the supernatant.

[0119] 8) Add 500 μL of PW buffer to the adsorption column and centrifuge at 13800 g for 30 s.

[0120] 9) Repeat step 8).

[0121] 10) Centrifuge again for 2 min and discard the waste liquid.

[0122] 11) Air dry at room temperature for 2 min to air dry the washing solution as much as possible.

[0123] 12) Add 100 μL of preheated sterile deionized water to the membrane in the adsorption column, centrifuge at 12,000 g for 2 min.

[0124] 13) Using UP-F and DN-R as primers, the successfully ligated DNA as the positive control, and sterile deionized water as the negative control, perform PCR amplification. The obtained PCR products are identified by agarose gel electrophoresis. Those with bands consistent with the positive control are positive clones. Then send the positive PCR products for sequencing and comparison and identification again.

[0125] 20. Screening of MRSA ST59 △chp knockout mutant 1) Inoculate the identified positive clones into TSA medium with 10 μg / mL chloramphenicol resistance and culture overnight at 30 °C.

[0126] 2) Pick a single colony and inoculate it into 4 mL of TSB with 10 μg / mL chloramphenicol resistance, and culture overnight at 30 °C with shaking at 220 rpm.

[0127] 3) According to a ratio of 1:1000, pipette the bacterial solution and inoculate it into 4 mL of TSB with 10 μg / mL chloramphenicol resistance, and culture overnight at 30 °C with shaking at 220 rpm.

[0128] 4) According to a ratio of 1:1000, pipette the bacterial solution and inoculate it into 4 mL of TSB with 5 μg / mL chloramphenicol resistance, and culture overnight at 30 °C with shaking at 220 rpm.

[0129] 5) According to a ratio of 1:1000, pipette the bacterial solution and inoculate it into 4 mL of TSB with 2.5 μg / mL chloramphenicol resistance, and culture overnight at 30 °C with shaking at 220 rpm.

[0130] 6) According to a ratio of 1:1000, pipette the bacterial solution and inoculate it into 4 mL of TSB with 10 μg / mL chloramphenicol resistance, and culture overnight at 42 °C with shaking at 220 rpm.

[0131] 7) Streak the fresh bacteria grown at 42 °C in four zones on a TSA plate with 10 μg / mL chloramphenicol resistance and incubate overnight in an incubator at 42 °C.

[0132] 8) Pick a single colony on the plate, add it to a liquid TSB medium without antibiotics, and culture overnight at 37 °C, and passage 5 times.

[0133] 9) Dilute the overnight culture of the fifth generation of bacteria with sterile deionized water by 10^6, aspirate 100 μL of the bacterial solution and spread it on a TSA plate containing 50 ng / mL anhydrotetracycline, and incubate overnight at 37°C.

[0134] 10) Pick 400 monoclonal colonies on the plate, first streak them on a TSA plate containing 10 μg / mL chloramphenicol resistance with a sterile pipette tip, and then streak them on a TSA plate without antibiotics, and incubate overnight at 37°C.

[0135] 11) Result interpretation: Colonies that can only grow on the plate without antibiotics but not on the plate containing chloramphenicol resistance are suspected mutant strains with successful knockout. First, preserve them for verification.

[0136] 21. Verification of the suspected knockout strain at the genomic level 1) Use the genomic DNA of the suspected knockout strain as a template, and perform PCR amplification with UP-F and DN-R as primers.

[0137] 2) Select a suitable marker. After electrophoresis, the suspected mutant strain and the positive control in the PCR result show bands of the same size, while the band amplified from the genomic reference control of the wild strain will be larger than the target fragments amplified by the mutant strain and the positive control. The amplified DNA fragment of the knockout strain is sent to the company for sequencing for further confirmation.

[0138] 22. Construction of the complemented strain of MRSA ST59 △chp Using the genomic DNA of MRSA ST59 as a template, amplify the full length of the chp gene plus its promoter region and the relevant binding sites of the ribosome, and then construct the complemented plasmid pLI50-chp. The specific steps and methods for the construction and electroporation of the shuttle plasmid pLI50 and the complemented plasmid pLI50-chp are as described above. At the same time, the empty plasmid pLI50 is used as a negative control. Figure 2 This is the figure for verifying the chp gene knockout result by RT-qPCR in the embodiment of the present invention.

[0139] Those of ordinary skill in the art can understand that the above are only preferred examples of the invention and are not used to limit the invention. Although the invention has been described in detail with reference to the foregoing examples, for those skilled in the art, they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, etc. made within the spirit and principle of the invention shall be included within the protection scope of the invention.

Claims

1. A method for knocking out the chp gene of Staphylococcus aureus, characterized in that: The following steps are involved: 1) Extracting genomic DNA of Staphylococcus aureus; 2) Primer design: The upstream and downstream homology arms of the target gene chp to be knocked out were taken as two fragments, and the primer pair UP-F shown in SEQ ID NO. 1 and UP-R shown in SEQ ID NO. 2 for amplifying the upstream homology arm and the primer pair DN-F shown in SEQ ID NO. 3 and DN-R shown in SEQ ID NO. 4 for amplifying the downstream homology arm were designed respectively; 3) Amplify the upstream and downstream homologous arms of the target gene and the knockout resistance gene fragment: Use the genomic DNA of Staphylococcus aureus containing the target gene as a template, use UP-F and UP-R as primer pairs, and DN-F and DN-R as primer pairs to perform PCR amplification respectively, and amplify the upstream and downstream homologous arms of the target gene and the knockout resistance gene fragment; 4) Enzyme digestion of empty vector and construction of recombinant plasmid: the empty vector is digested with restriction endonucleases to obtain vector plasmid fragments, and the vector plasmid fragments and the upstream and downstream homologous arms of the target gene are treated in a recombinase reaction system to construct a recombinant plasmid; 5) Transformation with recombinant plasmid: 6) Screening of positive transformants: The recombinant plasmid treated in step 5) is screened on a plate to obtain positive transformants; monoclonal colonies of positive transformants are selected, colony PCR is performed using UP-F and DN-R as primer pairs, and monoclonal strains with target bands are obtained after agarose gel electrophoresis of the PCR products for sequencing, and strains with correct sequences are selected to extract plasmids to obtain gene knockout recombinant plasmids; 7) Amplification of knockout insert DNA fragment: Using the gene knockout recombinant plasmid as a template and UP-F and DN-R as primer pairs, amplify the knockout insert DNA fragment; 8) Electrotransformation of Staphylococcus aureus: The knockout insert DNA fragment is inserted into the genome of Staphylococcus aureus using electrotransformation to achieve knockout of the target gene; 9) Using colony PCR to screen the Staphylococcus aureus treated in step 8) for positive transformants in which the knockout resistance gene is correctly inserted into the target gene position of the Staphylococcus aureus genome, thereby obtaining a Staphylococcus aureus chp gene knockout strain.

2. A method for constructing a complemented strain of MRSA ST59 Δchp, characterized in that: The method for obtaining the chp gene knockout strain of Staphylococcus aureus as described in claim 1; The construction of the complement strain includes the following steps: 1) Using MRSA ST59 genomic DNA as a template, amplify the full length of the chp gene plus its promoter region and the relevant ribosome binding site; 2) Construct the complementing plasmid pLI50-chp; at the same time, transfer the empty plasmid pLI50 as a negative control.

3. Use of the Staphylococcus aureus chp gene knockout strain as claimed in claim 1.

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