Universal antibacterial drug for reversing drug resistance of bacteria and application thereof

Abstract

The application discloses a kind of general type antibacterial drug of reverse bacteria body drug resistance and its application, and the AntiB-SGN system includes: (1) at least one oligonucleotide probe;(2) AntiB-SGN protein molecule, polynucleotide coding the protein molecule or plasmid containing polynucleotide coding the protein molecule.The application can target to destroy exogenous drug resistance gene obtained by introducing exogenous AntiB-SGN system into prokaryotic cell, restore the sensitivity of bacteria body to antibiotic.And the system of the application is strong in universality, small in size, without sequence preference, simple in component, more conducive to in vivo delivery, has carried out meaningful exploration to solve the problems and drug resistance phenomenon in the development of existing antibacterial drugs, and has good clinical application potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a general type of antibacterial drug for reversing bacterial drug resistance and application thereof. BACKGROUND

[0002] Traditional antibiotics are natural, synthetic or semi-synthetic compound molecules with antibacterial activity, which can effectively control bacterial infection. However, with the abuse of traditional antibiotics and the adaptation of bacteria, bacteria develop drug resistance mechanisms, including but not limited to changing the target, changing the membrane permeability, producing inactivation enzymes, etc. The problem of drug resistance is increasingly serious in hospitals, farms and other places, and has posed a major threat to global public health and environmental safety.

[0003] Although researchers have made efforts to develop new antibiotic compound molecules, the speed of developing new antibiotics is much slower than the speed of bacteria developing drug resistance. With the help of advanced sequencing technology, the genomic nucleic acid sequences of various types of bacteria can be quickly measured, and the root cause of pathogenic bacteria, i.e., "genomic genetic material", can be attacked, which is a new direction for the development of new antibacterial drugs.

[0004] At present, the CRISPR / Cas system has been applied to attack bacterial nucleic acid genetic material, which can destroy drug-resistant genes by targeting, restore the sensitivity of bacteria to antibiotics, limit the transfer and prevalence of harmful genes among microorganisms, and kill bacteria. However, there are still some problems in developing antibacterial drugs based on the CRISPR / Cas system to attack bacterial nucleic acids: (1) The Cas9 or Cas12 protein targeting the genomic DNA is large in size, and the protein itself can reach 200 kDa, the coding mRNA is as long as 4000-nt or more, the pharmaceutical process is complex, the delivery is difficult, and it is not conducive to the safety of in vivo drug use; (2) The Cas13 protein targeting mRNA has a side cutting effect, which will exacerbate off-target problems, and is not conducive to drug safety; (3) The anchoring process is limited by the fixed base pairing mode (PAM / PFS sequence), and cannot achieve arbitrary site attack on bacterial nucleic acid material; (4) The patent rights related to CRISPR are almost all in the hands of foreign companies and experts, which is extremely unfavorable for us to develop and promote various drugs and products based on the CRISPR system.

[0005] Therefore, the present project will develop a small-volume, sequence-unbiased, and highly specific antibacterial drug AntiB-SGN based on attacking bacterial nucleic acid genetic material, which works in prokaryotic cells, targets drug-resistant genes, reverses bacterial drug resistance, and kills bacteria. We hypothesize that this proof-of-concept AntiB-SGN strategy for reversing bacterial drug resistance has potential clinical value and can be used against a variety of known and newly discovered pathogenic bacteria.

[0006] Previously, we have disclosed "a composition and method for arbitrary nucleic acid editing" (202010157724.2), in which patent we disclose the cleavage of nucleic acid material by FEN1-type functional proteins combined with DNA probe compositions, and in Example 8, we disclose the cleavage of nucleic acid material in bacteria. Different from this, in the present invention, we disclose AntiB-SGN, which is a composition formed by another non-FEN1 family functional protein combined with RNA or DNA probe, and uses this composition to cleave endogenous or exogenous nucleic acid material in bacteria.

[0007] In addition, we have previously disclosed "a drug and application for simultaneously targeting RNA and DNA viruses" (202210153943.2), which targets viruses. Different from this, in the present invention, AntiB-SGN targets bacteria. The structures of the two are different, bacteria are single-celled microorganisms with biological structures such as cell walls, while viruses have relatively simple structures and do not have complex structures, mainly consisting of nucleic acids and proteins, so it is difficult to infer from the previous patent that the effect on bacteria is successful. In addition, the "drug and application for simultaneously targeting RNA and DNA viruses" (202210153943.2) patent is also based on a composition of FEN1-type functional proteins combined with DNA probes, while in the present invention, AntiB-SGN is based on a composition of another non-FEN1 family functional protein and RNA probe, so it is difficult to infer from the previous patent that AntiB-SGN has a successful effect on bacteria. SUMMARY

[0008] The purpose of the present invention is to address the deficiencies of existing antibacterial drugs pointed out in the background art, and to provide a small, sequence-unbiased, and highly specific strategy for attacking bacterial nucleic acid genetic material as a supplement, targeting and destroying exogenously acquired drug-resistant genes to restore the sensitivity of bacterial cells to antibiotics.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] An AntiB-SGN system for reversing bacterial drug resistance, the AntiB-SGN system comprising:

[0011] (1) at least one oligonucleotide probe, composed of two parts, one part is a guide sequence that can be complementary to the target drug-resistant gene, and the other part has a nucleic acid secondary structure; the oligonucleotide probe is a DNA probe or an RNA probe;

[0012] (2) AntiB-SGN protein molecule, polynucleotide encoding the protein molecule or plasmid containing the polynucleotide encoding the protein molecule, wherein the AntiB-SGN protein molecule can recognize the nucleic acid secondary structure of the oligonucleotide probe, thereby binding to the oligonucleotide probe, and being guided by the oligonucleotide probe to bind to the target bacterial nucleic acid sequence to destroy the nucleic acid sequence.

[0013] As a preferred embodiment of the present application, the polynucleotide encoding the AntiV-SGN protein molecule is DNA or RNA.

[0014] As a preferred embodiment of the present application, the AntiV-SGN protein molecule is any one of the following:

[0015] (1) a partial functional domain or a full enzyme fragment of Thermus aquaticus DNA polymerase or a mutant thereof;

[0016] (2) a partial functional domain or a full enzyme fragment of Thermus thermophilus DNA polymerase or a mutant thereof;

[0017] (3) a fusion of the full enzyme or fragment in (1) and (2).

[0018] As a preferred embodiment of the present application, the AntiV-SGN protein is a chimeric functional protein formed by fusing each part of Thermus aquaticus DNA polymerase and Thermus thermophilus DNA polymerase, and the coding gene sequence is shown in SEQ ID NO. 1.

[0019] As a further preferred embodiment of the present application, the plasmid containing the polynucleotide encoding the protein molecule is obtained by inserting the chimeric functional protein coding gene shown in SEQ ID NO. 1 into the Bgl II / Xho I enzyme cutting site of pdCas9-bacteria plasmid.

[0020] As a further preferred embodiment of the present application, when the oligonucleotide probe is an RNA probe, it is necessary to insert the chemically synthesized oligonucleotide probe into the plasmid backbone to construct a transcription plasmid.

[0021] The AntiB-SGN system described in the present application is used in the preparation of a drug for reversing or improving bacterial resistance to antibiotics.

[0022] As a preferred embodiment of the present application, the bacteria are pathogenic bacteria selected from at least one of Staphylococcus, Streptococcus, Escherichia, Mycobacterium, Salmonella, Shigella, and Helicobacter; and more preferably, the pathogenic bacteria are Escherichia.

[0023] As a further preferred embodiment of the present application, the drug-resistant antibiotic is selected from β-lactams, tetracyclines, aminoglycosides, macrolides, lincomycins, chloramphenicols, or polypeptide antibiotics; and preferably, the antibiotic is a β-lactam or an aminoglycoside.

[0024] A medicament for treating bacterial infection, which comprises the AntiB-SGN system of the present application, and preferably comprises the AntiB-SGN system of the present application and an antibiotic.

[0025] Advantages of the present application:

[0026] (1) The present application provides a novel antibacterial drug based on attacking the nucleic acid genetic material of bacteria and its application, which is not sequence-biased, has strong universality, and is suitable for different types of bacteria and different target genes, thereby making up for the shortcomings of traditional antibiotic drugs.

[0027] (2) The present application provides a novel antibacterial drug based on attacking the nucleic acid genetic material of bacteria and its application, which has simple component synthesis and can significantly reduce the cost as a drug. When applied to different types of bacteria and different target genes, only the guide sequence of the probe (only 20-30 bases) needs to be changed to target different types and subtypes of bacterial genomes, thereby incurring only a small economic and time cost and making up for the shortcomings of traditional antibiotic drugs.

[0028] (3) The present application provides a novel antibacterial drug based on attacking the nucleic acid genetic material of bacteria and its application, which has a molecular weight much smaller than most reported Cas proteins, is beneficial to in vivo delivery, and makes up for the shortcomings of the currently developed antibacterial drugs based on the CRISPR / Cas system.

[0029] (4) The present application provides a novel antibacterial drug based on attacking the nucleic acid genetic material of bacteria and its application, which has little growth toxicity and side effects on non-target bacteria (such as beneficial bacteria). BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A schematic diagram of the principle of the present application and the closest prior art.

[0031] Figure 2 A result graph of Example 1 of the present application.

[0032] Figure 3 A result graph of Example 2 of the present application.

[0033] Figure 4 Figure 3 is a result graph of Example 3 of the present application.

[0034] Figure 5 Figure 4 is a result graph of Example 4 of the present application.

[0035] Figure 6 Figure 5 is a result graph of Example 5 of the present application.

[0036] Figure 7 Figure 6 is a result graph of Example 6 of the present application.

[0037] Figure 8 Figure 7 is a result graph of Example 7 of the present application.

[0038] Figure 9 Figure 8 is a result graph of Example 8 of the present application.

[0039] Figure 10 Figure 9 is a result graph of Example 9 of the present application. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

[0041] Example 1 Construction and prokaryotic expression of functional protein in AntiB-SGN

[0042] AntiB-SGN is composed of two components of functional protein expression plasmid and probe expression plasmid. First, the prokaryotic expression plasmid of functional protein is constructed. Through whole gene synthesis, the coding gene sequence (SEQ ID NO. 1) of a chimeric functional protein (referred to as TaqTth protein for short) formed by fusing one part of Thermus aquaticus DNA polymerase and one part of Thermus thermophilus DNA polymerase is obtained.

[0043] Through whole gene synthesis, the coding gene sequence (SEQ ID NO. 2) of Thermus thermophilus DNA polymerase (referred to as TthPol protein for short) is obtained.

[0044] A prokaryotic expression vector backbone pdCas9-bacteria (purchased from Mingling Company) was selected, which contains tetR / A promoter, Cm resistance and p15A replicon. SEQ ID NO. 1 was inserted into the Bgl II / Xho I enzyme cutting site of the plasmid to obtain a recombinant plasmid, named p-aTc-TaqTth Figure 2 A). The ligation product was added into DH5a chemically competent cells by heat shock method, ice bath for 30 min, 42°C for 90 s, placed on ice for 2 min, added with 1 mL LB medium, 37°C, 220 rpm / min for 1 h, about 100 μL bacterial liquid was taken and coated on Cm resistant LB solid medium, 37°C overnight culture. The positive colonies were picked and sent for sequencing, and the correct sequencing was the protein expression plasmid. After induction by adding tetracycline (200 ng / mL), TaqTth protein could be normally expressed in bacteria Figure 2 B), with a molecular weight of about 95 kDa.

[0045] SEQ ID NO. 1 in p-aTc-TaqTth plasmid was replaced by SEQ ID NO. 2 to obtain a recombinant plasmid, named p-aTc-TthPol. The ligation product was added into DH5a chemically competent cells by heat shock method, ice bath for 30 min, 42°C for 90 s, placed on ice for 2 min, added with 1 mL LB medium, 37°C, 220 rpm / min for 1 h, about 100 μL bacterial liquid was taken and coated on Cm resistant LB solid medium, 37°C overnight culture. The positive colonies were picked and sent for sequencing, and the correct sequencing was the protein expression plasmid. After induction by adding tetracycline (200 ng / mL), TaqTth protein could be normally expressed in bacteria, with a molecular weight of about 95 kDa.

[0046] Example 2 Construction of probe expression plasmid in AntiB-SGN

[0047] AntiB-SGN is composed of two components of functional protein expression plasmid and probe expression plasmid. On the basis of Example 1, the functional probe prokaryotic expression plasmid was constructed.

[0048] The probe in AntiB-SGN can be a DNA probe or an RNA probe. The composition of the probe is shown in Table 1. Figure 3-A, consisting of two parts, stem-loop region and guide region, respectively, where stem-loop can be a fixed structure and sequence (5'-AAAGTCGGCCGAAAGGCCGACTTTTTT), and guide region is a region that is reverse complementary to the target. If it is an RNA probe, then a chemically synthesized oligonucleotide needs to be inserted between the Spe I and Eco RI enzyme cutting sites of plasmid backbone pTargetF (purchased from Mingling Company), which contains J23119 promoter, Spe resistance and ColE1 replicon, to construct transcription plasmid p-J23119-target (see Figure 3 -B), for transcription of the probe.

[0049] Example 3 DNA probe mediated AntiB-SGN inhibition of expression of exogenous target protein in bacteria

[0050] To investigate whether AntiB-SGN can inhibit the expression of target genes and proteins in bacteria, green fluorescent protein (EGFP) was used as a target for preliminary investigation, and the schematic diagram is shown in Figure 4 -A.

[0051] The functional protein expression plasmid p-aTc-TaqTth or p-aTc-TthPol (Cm resistance) in AntiB-SGN and commercially purchased EGFP expression plasmid pLac-EGFP (Amp resistance) were transformed into 50 μL of BL21 (DE3) chemically competent cells by heat shock, 1 mL of LB liquid medium without antibiotics was added, and the culture was incubated at 37°C with 220 rpm / min for 1 h. About 100 μL of bacterial solution was spread on Cm / Amp double antibiotic LB solid medium, and the culture was incubated at 37°C overnight. Positive clones were picked to obtain BL21 (DE3) strains containing TaqTth-EGFP or TthPol-EGFP plasmids.

[0052] Since the DNA probe is a single-stranded DNA molecule, it cannot exist in bacteria for a long time, and it will degrade after a period of time, and it cannot be increased or inherited by replication, so it can only be delivered by electroporation. The TaqTth-EGFP or TthPol-EGFP strain is made into an electrochemically competent cell, and the specific steps are as follows: 37°C, 220rpm / min shaking overnight, 1% transfer, shaking to logarithmic growth phase, taking 1mL of bacterial solution into a 1.5mL EP tube, ice bath for 10min. Centrifuge with a 50mL centrifuge tube, 4°C, 3000g, centrifuge for 10min, discard the supernatant. Add 10mL of pre-cooled ultrapure water, resuspend on ice, add 40mL of pre-cooled ultrapure water, mix well, 4°C, 3000g, centrifuge for 10min, discard the supernatant; Add 20mL of 10% glycerol and mix well, 4°C, 3000g, centrifuge for 10min, discard the supernatant; Repeat once, add 20mL of 10% glycerol and mix well, 4°C, 3000g, centrifuge for 10min, discard the supernatant; Finally, add 2mL of 10% glycerol and mix well, aliquot to obtain electrochemically competent cells containing TaqTth-EGFP or TthPol-EGFP plasmid, and store in a minus eighty-degree freezer.

[0053] Design a probe targeting EGFP coding gene or mRNA (EGFP-1), and the electrotransformation system is 100μL electrotransformation competent cells and 500pmol DNA probe added to a 2-mm electrotransformation cup, the reaction conditions are 2.5kV, 25μF, 500Ω, and then recover for 1 hour, add tetracycline inducer (tet, 200ng / mL) to induce AntiB-SGN expression, and target to inhibit the expression of EGFP.

[0054] The expression of fluorescent protein was analyzed by flow cytometry. After 1, 3, and 6 hours of action, 1mL of bacterial solution was taken, centrifuged at 4000rpm for 5min, the supernatant was discarded, 1mL of PBS was added for resuspension, centrifuged and discarded the supernatant, 1mL of PBS was added for resuspension, the suspension was sieved, and flow cytometry was performed. The inhibition effect of TaqTth on the expression of exogenous EGFP in bacteria is shown in Figure 4 -B. When the functional protein is expressed and the probe EGFP-1 exists, the EGFP fluorescence is obviously weakened compared with the control group, and the highest reduction is 71.4%. It shows that AntiB-SGN can inhibit the expression of target protein in bacteria. The inhibition effect of TthPol on the expression of exogenous EGFP in bacteria is: when the functional protein is expressed and the probe EGFP-1 exists, the EGFP fluorescence is obviously weakened compared with the control group, but when there is no EGFP-1, the EGFP fluorescence is also obviously weakened, which shows that the controllability and specificity of TthPol are not good, which is not as good as TaqTth. Therefore, in the subsequent examples, AntiV-SGN expressing TaqTth is used for research.

[0055]

[0056] Example 4 Inhibition of expression of foreign target protein in bacteria by RNA probe mediated AntiB-SGN

[0057] In the above examples, we have verified that DNA probe mediated AntiB-SGN can inhibit the expression of target protein EGFP in bacteria. Next, we will examine whether RNA probe mediated AntiB-SGN also works, as shown in the schematic diagram Figure 5 -A.

[0058] The protein expression plasmid p-aTc-TaqTth (TaqTth as an example, Cm resistance), EGFP plasmid pLac-EGFP (Amp resistance) and probe plasmid (Spe resistance, probe sequence see EGFP-2) in AntiB-SGN were transformed into 50 μL of BL21 (DE3) chemically competent cells by heat shock, 1 mL of LB liquid medium without antibiotics was added, and the culture was incubated at 37°C for 1 h at 220 rpm / min. About 100 μL of bacterial solution was spread on Cm / Amp / Spe triple antibiotic LB solid medium and incubated at 37°C overnight. Positive clones were picked to obtain BL21 (DE3) strains containing TaqTth-probe-EGFP plasmids.

[0059] Tetracycline (tet, 200 ng / mL) was added to induce AntiB-SGN expression. The expression of fluorescent protein was analyzed by flow cytometry. 1 mL of bacterial solution was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, 1 mL of PBS was added for resuspension, the supernatant was discarded again, 1 mL of PBS was added for resuspension, the suspension was sieved, and flow cytometry was performed. The inhibition effect of foreign EGFP expression in bacteria is shown in Figure 5 -B, the experimental group expressed protein and probe, compared with the control group, the fluorescence was significantly weakened, and the highest reduction was 91.2%.

[0060] As can be seen from Examples 3 and 4, the reaction efficiency mediated by RNA probe is higher than that mediated by DNA probe. The possible reason is that DNA probe is a single-stranded DNA molecule and cannot be amplified and inherited by replication; while RNA probe exists in the form of plasmid and can be amplified and inherited by replication.

[0061]

[0062] Example 5 AntiB-SGN reverses the drug resistance of drug-resistant bacteria to aminoglycoside antibiotic kanamycin

[0063] In the above examples, we have verified that AntiB-SGN can inhibit the expression of exogenous target proteins in bacteria. Next, we will investigate whether AntiB-SGN can also inhibit the expression of resistance proteins by targeting resistance plasmids, restoring the sensitivity of bacteria to the corresponding resistance. Since in reality, most bacteria acquire resistance by infecting exogenous drug-resistant plasmids, we will use kanamycin resistance plasmids to simulate this phenomenon. In order to obtain better efficiency, we will use RNA probe-mediated AntiB-SGN for experiments. The schematic diagram is shown in Figure 6 -A.

[0064] The protein expression plasmid p-aTc-TaqTth (TaqTth as an example, Cm resistance), kanamycin resistance plasmid pLac-EGFP (Kan resistance), and probe plasmid (Spe resistance) in AntiB-SGN were transformed into 50 μL of BL21 E. coli chemically competent cells by heat shock, 1 mL of LB liquid medium without antibiotics was added, and the culture was incubated at 37°C, 220 rpm / min for 1 h. About 100 μL of bacterial solution was spread on Cm / Kan / Spe triple-resistant LB solid medium, and incubated at 37°C overnight. Positive clones were picked to obtain BL21 strains containing TaqTth-probe-Kan plasmids.

[0065] Without inducing AntiB-SGN, the strain can be resistant to kanamycin due to the presence of the Kan resistance plasmid, and can grow normally on Kan-resistant plates. When AntiB-SGN is induced, AntiB-SGN targets and destroys the Kan resistance protein gene, inhibiting the expression of the Kan resistance protein, and the bacteria restore sensitivity to kanamycin, reversing the drug resistance.

[0066] The BL21 strain containing the TaqTth-probe-Kan plasmid was recovered and spread on plates without tetracycline inducer and plates with tetracycline inducer, respectively, with an unrelated probe (NT) group as a control. As shown in Figure 6 -B, the relative colony count of the target probe group (T) decreased by an average of 31% compared to the NT group on plates with tetracycline inducer. This indicates that AntiB-SGN can restore a certain degree of antibiotic sensitivity to drug-resistant bacteria.

[0067]

[0068] Example 6 AntiB-SGN restores the sensitivity of drug-resistant bacteria to β-lactam antibiotic ampicillin

[0069] In the above examples, we have verified that AntiB-SGN plays a role in restoring the sensitivity to β-lactam antibiotic ampicillin in bacteria by destroying the expression of kanamycin resistance protein in bacteria. Next, we will investigate whether AntiB-SGN can also restore the sensitivity to β-lactam antibiotic ampicillin in ampicillin-resistant bacteria.

[0070] The protein expression plasmid p-aTc-TaqTth (Cm resistance, taking TaqTth as an example), ampicillin resistance plasmid pRSETB-EGFP (Amp resistance), and probe plasmid (Spe resistance) in AntiB-SGN were transformed into 50 μL of BL21 chemically competent cells by heat shock, 1 mL of LB liquid medium without antibiotics was added, and the cells were cultured at 37°C and 220 rpm / min for 1 h. About 100 μL of bacterial solution was spread on Cm / Amp / Spe triple-resistant LB solid medium, and the cells were cultured overnight at 37°C. Positive clones were picked to obtain a BL21 strain containing TaqTth-probe-Amp plasmid.

[0071] Without inducing AntiB-SGN with an inducer, the strain can be resistant to ampicillin due to the Amp resistance plasmid, and can grow normally on an Amp-resistant plate. When AntiB-SGN is induced with a tetracycline inducer, AntiB-SGN targets the destruction of the Amp resistance protein gene and inhibits the expression of the Amp resistance protein, and the bacteria restore the sensitivity to ampicillin and reverse the drug resistance.

[0072] The BL21 E. coli strain containing the TaqTth-probe-Amp plasmid was recovered and spread on plates without tetracycline inducer and plates with tetracycline inducer, respectively, with an unrelated probe (NT) group as a control. As shown in Figure 7 The relative number of colonies of the targeting probe group (T) did not decrease significantly compared with the NT group on the plate without tetracycline inducer, while the relative number of colonies of the targeting probe group (T) decreased by an average of 66% compared with the NT group on the plate with tetracycline inducer. This indicates that AntiB-SGN can restore a certain degree of antibiotic sensitivity in drug-resistant bacteria.

[0073]

[0074] Example 7 Inhibition of expression of endogenous mutation-caused drug resistance genes by AntiB-SGN in bacteria

[0075] In the above examples, we have verified that AntiB-SGN can inhibit the expression of resistance proteins in bacteria, making the bacteria regain sensitivity to the corresponding resistance. Next, we will investigate whether AntiB-SGN can also inhibit the expression of endogenous mutant drug resistance genes. To achieve better efficiency, we will use RNA probe-mediated AntiB-SGN for the experiment.

[0076] The chemical structure of sulfonamide compounds is similar to that of para-aminobenzoic acid (PABA), which can compete with PABA for dihydrofolate synthetase, thereby affecting the synthesis of dihydrofolate, achieving the purpose of inhibiting the growth and reproduction of bacteria. The drug resistance of various bacteria to sulfonamides has been shown to be caused by mutations in sulA (a chromosomal gene encoding dihydrofolate synthetase). Therefore, we designed a probe targeting the sulA coding sequence to investigate the inhibitory effect of AntiB-SGN on sulA, thereby restoring the sensitivity of bacteria to sulfonamides. The results are shown in Figure 8 As shown in the table, AntiB-SGN reduced the mRNA abundance of sulA by 38%, with a p-value less than 0.05, which was significantly different from the irrelevant probe group.

[0077]

[0078] Example 8 Toxicity of AntiB-SGN to bacterial growth

[0079] Whether the expression of AntiB-SGN in the bacterial body has certain toxic side effects on the growth of non-target bacteria is a problem we need to investigate next. Taking the growth of bacteria with introduced non-target probes as an example, as shown in Figure 9 The "empty plasmid + irrelevant probe" group marked by the blue curve is taken as the benchmark, and whether only TaqTth protein is expressed (red curve), only the targeting probe is transcribed (purple curve), or both (green curve), AntiB-SGN does not affect the growth curve of the bacteria, indicating that AntiB-SGN has little toxic side effects.

[0080] Example 9 AntiB-SGN has no fixed base pairing requirement for target base sequence

[0081] The CRISPR system has a PAM or PFS fixed base pairing pattern requirement for the target, which limits the range of anchored targets. The AntiB-SGN system has no fixed base pairing requirement for the target sequence, and in theory can anchor any sequence. To prove this point, we performed sequence analysis on all the probes used in the present application, as shown in Figure 10 As shown in the table, there is no bias in the frequency of occurrence of A, T, C, and G.

Claims

1. The application of the AntiB-SGN system for reversing bacterial resistance in the preparation of drugs to reverse bacterial resistance to antibiotics, characterized in that, The AntiB-SGN system includes: (1) At least one oligonucleotide probe, consisting of two parts, one part being a guide sequence that is complementary to the target drug resistance gene, and the other part having a nucleic acid secondary structure; the oligonucleotide probe is a DNA probe or an RNA probe; (2) The AntiV-SGN protein molecule, the polynucleotide encoding the protein molecule, or the plasmid containing the polynucleotide encoding the protein molecule, wherein the AntiV-SGN protein molecule can recognize the nucleic acid secondary structure of the oligonucleotide probe, thereby binding to the oligonucleotide probe, and then being guided by the oligonucleotide probe to bind to the nucleic acid sequence of the target bacteria and destroy the nucleic acid sequence; the AntiV-SGN protein is Thermus aquaticus DNA polymerase and Thermus thermophilus A chimeric functional protein formed by the fusion of different parts of DNA polymerase, the gene sequence of which is shown in SEQ ID NO.1; The bacteria mentioned are Escherichia coli, and the antibiotic mentioned is kanamycin or ampicillin.

2. The application according to claim 1, characterized in that, The polynucleotide encoding the AntiV-SGN protein molecule is DNA or RNA.

3. The application according to claim 1, characterized in that, The plasmid containing the polynucleotide encoding the AntiV-SGN protein molecule was obtained by inserting the chimeric functional protein encoding gene shown in SEQ ID NO.1 between the Bgl II / Xho I restriction sites of the pdCas9-bacteria plasmid.

4. The application according to claim 1, characterized in that, When the oligonucleotide probe is an RNA probe, the chemically synthesized oligonucleotide probe needs to be inserted into the plasmid backbone to construct a transcription plasmid.

5. A drug for treating bacterial infections, characterized in that, The drug contains the AntiB-SGN system as described in any one of claims 1-4.

6. The drug according to claim 5, characterized in that, It includes the AntiB-SGN system and antibiotic as described in any one of claims 1-4.

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