CN1268768C - Screen method for antibiotics - Google Patents

Abstract

The invention relates to a method for screening antibacterial drugs. Antibiotics are traditionally screened according to their antibacterial ability. The present invention comprises the following steps: synthetic pathogenic bacteria ribosomal nucleic acid is fixed on carrier A as the target of antibiotic screening; nucleic acid random fragments are fixed on carrier B; material to be screened is dissolved in buffer; Carrier B of the fragment adsorbs and removes non-specific binders in the concentrated solution of the object to be screened; uses carrier A immobilized with the target to capture specific binders in the concentrated solution of the object to be screened; washes off the specific binding substances bound to the target with the eluent Bacteriostasis test was carried out for sexual conjugates; separation and purification of specific conjugates with antibacterial ability were carried out. By adopting the method of the invention, specific antibiotics capable of specifically inhibiting the growth of certain bacteria can be screened out, and broad-spectrum antibiotics capable of inhibiting the growth of various bacteria can also be screened out.

Description

A screening method for antibiotics

technical field

The invention relates to a method for screening antibacterial drugs.

Background technique

With the widespread use of antibiotics, the resistance of microorganisms has gradually increased. According to reports, more than 90% of the currently isolated Staphylococcus aureus can produce β-lactamase, which can resist penicillin drugs. Staphylococcus aureus (MRSA) emerges rapidly, due to the continuous accumulation and transfer of various drug resistances among strains. So far, only glycopeptide antibiotics still maintain activity against MRSA. However, Hiramatsu has isolated a MRSA strain resistant to vancomycin (a glycopeptide antibiotic). Although the mechanism of resistance is not yet clear, once this resistance spreads, the infection of MRSA strains may There is no medicine available.

At the same time, the aggravation of environmental pollution, social and cultural changes have made human immunity continue to decline, new pathogens and conditional pathogens continue to appear, and diseases that were not previously considered to be caused by bacteria, such as stomach cramps, Heart disease etc. have also been found to be caused by bacteria. The increasing drug resistance of this microorganism and the continuous emergence of new pathogenic bacteria have become a serious social problem. To solve this problem, the most effective way is to intensify efforts to screen and develop new antibiotics.

But the development of new drugs has become increasingly difficult. The reason is mainly the lack of new screening targets and effective screening methods. Traditionally, antibiotics are screened based on their antibacterial ability. With the understanding of the mechanism of action of antibiotics, screening methods targeting essential enzymes, receptors, ion channels, etc. have been gradually developed. In recent years, due to the progress of human genome and microbial genome work, new targets continue to emerge, coupled with the establishment of high-throughput screening methods, the rapid development of new drug screening has been achieved.

The ideal target should be a substance that is necessary for cell survival, highly conserved in pathogenic bacteria, and absent or very different in humans. Most of the currently used targets are proteins with clear biochemical functions. Although the screening of such targets is relatively easy, there is a disadvantage that bacteria may develop resistance to the antibiotics found by mutating the site of action (target). Medicinal properties. The work of using RNA as a target to screen antibiotics has attracted more and more attention. Echer believes that in terms of targets, the value of RNA is twice as large as that of the genome. Steven also believes that the development of antibiotic resistance using RNA as a target is relatively slow. In fact, aminoglycoside antibiotics can play a role by binding to the A site of ribosomal 16S rRNA. Studies have shown that the gene (rDNA) encoding 16S rRNA is about 1500 bp in length and is composed of a series of conserved regions and variable regions. These conserved sequences have hardly changed during the evolution of millions of years, such as 1351~ Position 1369 (based on Escherichia coli) is a conserved region of G ^- bacteria, positions 1401-1419 are conserved regions of G ⁺ bacteria, and positions 1167-1189 are conserved in all bacteria. The binding site between streptomycin and rRNA is encoded by the non-conserved region of 16S rDNA, so when the 524th position on the rRNA is mutated from guanine (G) to cytosine (C), streptomycin cannot be well Bind it well, and the bacteria develop resistance to streptomycin. If the conserved sequence of rRNA is used as a target to screen for antibiotics, the generation of such drug resistance will be greatly reduced.

In addition to combining with some small molecular substances, RNA can also combine with proteins (RNA-bindingproteins) and antisense oligonucleotides (antisense oligonucleotides). However, most proteins and antisense oligonucleotides have relatively large molecular weights, and it is difficult to enter the cells of pathogenic microorganisms. If peptides that can specifically bind to RNA can be screened from microbial metabolites, it may become a promising candidate. of antibiotics.

In addition to proteins and RNA, DNA also plays a very important role in the life activities of bacteria. However, the work of using DNA as a target to screen antibiotics has not received much attention, mainly because most of the antibiotics found to act on DNA lack selective toxicity. But the potential of DNA as a target warrants attention. Studies have shown that some substances can specifically bind to DNA, for example, the repressor protein in the lactose operon of Escherichia coli can recognize and bind to a 26bp DNA sequence. Carrasco demonstrated that the anticancer antibiotic AT2433-B1 can recognize specific DNA sequences. Charles discovered that the isothiocyanates of kanamycin and neomycin can be covalently attached to specific DNA fragments. Therefore, if the conserved region of rDNA is used as the target to screen for substances that can specifically bind to it, it is possible to develop antibiotics that can specifically inhibit bacterial growth without affecting the function of human DNA.

Since the negative strands of rRNA and rDNA are the same except that U replaces T, the antibiotics obtained by screening their conserved sequences as targets will not only combine with rRNA to affect the assembly of ribosomes, but also destroy the function of ribosomes. In addition, it is also possible to bind to DNA to inhibit replication; and these target regions are highly conserved in bacteria, and drug resistance due to target variation is almost unlikely to occur. The antibiotics thus screened have great application prospects not only in the medical field, but also in food preservation and article preservation.

Contents of the invention

The technical problem solved by the present invention is to provide a screening method for antibacterial drugs based on prokaryotic ribosomal nucleic acid as a target, and to establish a novel antibiotic screening model based on bacterial ribosomal nucleic acid, especially the conserved sequence as a target.

The present invention mainly comprises the following steps: 1) synthesizing the ribosomal nucleic acid of pathogenic bacteria (including 16S rRNA, 16S rDNA, 23S rRNA or 23S rDNA sequence containing at least 18 bases in the complete sequence, especially the conserved fragment thereof) as an antibiotic For the target to be screened, the above target is fixed on the carrier A; 2) the nucleic acid random fragment (containing 8 to 12 bases) is fixed on the carrier B; 3) the substance to be screened is concentrated and then dissolved in the buffer; 4) Use the carrier B immobilized with nucleic acid random fragments to adsorb and remove the non-specific binders in the concentrated solution of the screening object; 5) use the carrier A immobilized with the target to capture the specific binders in the concentrated solution of the screening object; 6) use the elution Washing the specific binders bound to the target, and carrying out the antibacterial test; 7) separating and purifying the specific binders with antibacterial ability to obtain antibiotic candidates.

The synthesis of ribosomal nucleic acid and random fragments of nucleic acid is a mature technology. The bacterial ribosomal nucleic acid and random fragments of nucleic acid required by the present invention can be synthesized in the laboratory or purchased from biotechnology companies.

The present invention can use 16S rRNA or 23S rRNA of a certain specific pathogenic bacteria as a target to screen specific antibiotics that can specifically bind to it, such as 16S rRNA or 23S rRNA of methicillin-resistant Staphylococcus aureus (MRSA) Target screening of obligate antibiotics that have a specific inhibitory effect on such pathogenic bacteria; it is also possible to screen broad-spectrum antibiotics based on the rRNA conserved sequences shared by a certain type of pathogenic bacteria. These conservative sequences can be derived from 16S rRNA (or 16S rDNA ), can also come from 23S rRNA (23SrDNA). Such as AGAGUUUGAUCCUGGCUCAG, GGUUACCUUGUUACGACUU, and AGGAGGUGAUCCAACCGCA from 16S rRNA that are conserved in all bacteria; or from 16S rRNA that is conserved in Gram-positive pathogenic bacteria such as GGCUUCUGCUGUUACAAA, or from 16S rRNA that is conserved in Gram-negative pathogenic bacteria Conserved such as CCCGGCUUCGUAUUCACC, or a sequence from 16S rRNA that is only conserved in a certain type of pathogenic bacteria such as Rickettsia, such as UAAUGCCGGGAACUAUAAGAA; or a sequence from 23S rRNA that is conserved in all bacteria such as UUCGCCUUUCCCUCACGGUACU, etc.

The bacterium ribosomal nucleic acid sequence that the present invention adopts can be artificially synthesized conserved segment (containing 18 bases at least), or with 27F, 1492R is the 16S rDNA complete sequence synthesized by polymerase chain reaction (PCR) with 27F, 1492R, or The 16S rDNA partial sequence synthesized by PCR with other primers can also be the sequence from 23S rRNA.

The target immobilization material that the present invention adopts can be the nitrocellulose membrane commonly used in southern blotting, PVDF membrane or nylon membrane, can be the glass or silicon chip commonly used in gene chip, can be commonly used in nucleic acid affinity chromatography Cellulose or Sepharose can also be other materials that can adsorb nucleic acid, such as agarose, hydroxyapatite, polypropylene, etc.

The random segment used in the present invention is randomly assembled from four bases, A, T(U), G, and C, and has a length of about 10 bases.

The present invention immobilizes the random nucleic acid fragments above on the carrier B, and first uses these random fragments to remove non-specific binders in the fermentation broth as much as possible; animal cell DNA can also be used instead of nucleic acid random fragments to remove non-specific binders.

The substance to be screened in the present invention can be a microbial fermentation liquid, or an animal and plant tissue extract or an animal and plant cell culture liquid. These substances to be screened should first be concentrated by conventional methods such as extraction and evaporation, on the one hand to increase the concentration of effective substances, on the other hand to remove macromolecular impurities, destroy DNase and RNase to prevent target degradation. Then the concentrate is dissolved in an appropriate amount of phosphate buffer, centrifuged to remove water-insoluble matter (as an antibiotic, it should have sufficient water solubility) to prepare a concentrate of the object to be screened.

The capture of potential antibiotics in the present invention can adopt methods such as filter membrane filtration, liquid phase "hybridization", affinity binding and the like. The so-called filter membrane filtration method is to fix the target on the filter membrane, let the concentrated liquid to be screened flow through the filter membrane, the specific binder will stay on the membrane, and the substances that cannot bind to the target will be filtered out, just like ordinary Filtration is the same; the liquid phase "hybridization" method is to directly incubate the target and the concentrate to be screened under certain conditions to allow the potential antibiotic to bind to the target, and then separate the target-potential antibiotic conjugate; the affinity binding method is The target is first bound to a specific carrier such as cellulose particles, and then incubated with the concentrate to be screened to allow potential antibiotics to bind to the target with affinity, just like the affinity chromatography of mRNA.

The eluent used in the present invention can be a solution of different ionic strength or a solution of different pH. If necessary, nuclease can be used to destroy the target and release the conjugate.

By adopting the present invention, specific antibiotics capable of specifically inhibiting the growth of a certain pathogenic bacterium can be screened out according to the unique ribosomal nucleic acid sequence of the pathogenic bacteria, or specific antibiotics capable of inhibiting various types of bacteria can be screened out according to the conserved ribosomal nucleic acid sequence shared by the pathogenic bacteria. The growth of broad-spectrum antibiotics makes it possible for large-scale, high-throughput screening of new antibiotics with slow development of drug resistance.

Detailed ways

Example 1

The method for the specificity antibiotic of MRSA by membrane filtration method screening comprises the following steps:

1. Cultivate methicillin-resistant Staphylococcus aureus (MRSA), and extract its total DNA with a DNA extraction kit;

2. Primers 27F (5'AGAGTTTGATCCTGGCTCAG 3') and 1492R (5'GGTTACCTTGTTACGACTU 3') for the artificial synthesis of 16S rDNA;

3. Synthesize the complete 16S rDNA sequence of MRSA by PCR; the specific operating conditions are as follows: 0.2mmol/L (each) dNTP, 400nmol/L (each) primer, 5mmol/L MgCl ₂ and 1U Taqplus, with a total volume of 50μL. Reaction conditions: 30 cycles of PCR reaction after denaturation at 94°C for 3 minutes, each cycle including denaturation at 94°C for 1 minute, annealing at 55°C for 45 seconds, and extension at 72°C for 1 minute. At the end of the 30 cycles, an additional 10 minutes was extended at 72°C. After the PCR product was separated by 1.0% agarose gel electrophoresis, it was purified with a PCR purification kit;

4. Heat the entire 16S rDNA sequence of purified MRSA in a water bath at 95°C for 5 minutes, then place it in an ice bath for 5 minutes, then add the denatured 16S rDNA evenly to the nitrocellulose membrane, and fix it by adsorption for 30 minutes , vacuum-dried, and irradiated with ultraviolet light for 30 seconds to make a filter membrane A with a fixed target, and set aside;

5. Use a nucleic acid synthesizer to artificially synthesize nucleic acid random fragments containing 12 bases, evenly add to the nylon membrane, absorb and fix for 30 minutes, vacuum dry, and irradiate with ultraviolet rays for 30 seconds. Make filter membrane B fixed with nucleic acid random fragments, set aside;

6. Screen microbial strains with antibacterial activity, shake the flask for fermentation, collect the fermentation broth, extract with ethyl acetate, collect the organic phase, evaporate the ethyl acetate on a rotary evaporator, and use twice the volume of pH7.0 for the residue Dissolve in phosphate buffer, centrifuge to remove the precipitate, add the supernatant to the Buchner funnel, in which the above-mentioned nylon membrane (filter B) immobilized with random fragments of nucleic acid is placed as a "filter paper" in advance to remove non-specific binding The filtrate slowly flows through the above-mentioned nitrocellulose membrane (filter A) with the target immobilized, so that the specific binder can fully bind to the target;

7. Wash with an acidic eluent (0.1mol/L glycine-HCl buffer, pH3.0), and recover the washed specific conjugate (as a drug, the stronger the binding force, the better, so you can use pH6. 8 phosphate buffer to wash away weaker binding components);

8. Conduct antibacterial and antibacterial tests and isolate and purify related compounds with antibacterial activity;

9. Determine the chemical structure of the purified antibiotic.

Example 2

The method for liquid phase "hybridization" test screening broad-spectrum antibacterial drug comprises the following steps:

1. Entrust relevant biotechnology companies to synthesize the conserved fragment GCGAUUUCCGAACGGGGAAACCC of the 6th segment of 23S rRNA of pathogenic bacteria;

2. Screen microbial strains with antibacterial activity, shake the flask for fermentation, filter the fermentation product, extract the filtrate with ethyl acetate, collect the organic phase, evaporate the ethyl acetate on a rotary evaporator, and use twice the volume of the pH7 Dissolve in .0 phosphate buffer, centrifuge at 5000g for 10 minutes, remove the precipitate, and transfer the supernatant to a clean petri dish;

3. Entrust a relevant biotechnology company to synthesize a random fragment of nucleic acid containing 10 bases, add it to the above-mentioned petri dish, and shake slowly for 30 minutes on a constant temperature shaker at 30°C;

4. Dip a piece of DE-81 filter membrane (diameter slightly smaller than the inner diameter of the culture dish) into the culture dish, and shake slowly on a constant temperature shaker at 30°C for 30 minutes. Since the DE-81 filter membrane can strongly adsorb and retain nucleic acids, those Substances that can randomly bind to nucleic acids are also removed;

5. Add the conserved sequence of the 6th segment of the synthesized 23S rRNA to the petri dish, and shake slowly on a constant temperature shaker at 30°C for 60 minutes;

6. Dip a new clean DE-81 filter membrane (diameter slightly smaller than the inner diameter of the petri dish) into the petri dish, shake slowly on a constant temperature shaker at 30°C for 30 minutes, those that can specifically bind to the target oligonucleotide fragments The substance is transferred to the membrane;

7. Wash the above membrane with high ionic strength eluent (1mol/L NaCl, 0.1mol/L EDTA, 20mmol/L Tris, pH8.0), and recover the specific binder;

8. Conduct antibacterial and antibacterial tests to judge the antibacterial activity;

9. Separation and purification of related substances on a large scale to determine the structure of the substance.

Example 3

The method for affinity binding test screening broad-spectrum antimicrobial drug comprises the following steps:

1. Artificially synthesize the conserved sequence GCAUCAGGUCAAGUCAUC of the 16S rRNA of pathogenic bacteria;

2. Dissolve the synthesized conserved fragment in an appropriate amount of deionized water to make a 0.1mmol/L solution, then evenly add it to the nylon membrane A, fix it by adsorption for 30 minutes, dry it at room temperature, and dry it in a vacuum oven at 80°C for 30 minutes. Make the filter membrane A with the target immobilized and set aside;

3. Artificially synthesize random fragments of nucleic acid with a length of 8 bases, dissolve them in the same way as above, and evenly add them to nylon membrane B, absorb and fix for 30 minutes, dry at room temperature, and dry-bake in a vacuum oven at 80°C for 30 minutes to make nucleic acid immobilized Filter B of random fragments, spare;

4. Screen the Chinese herbal medicines with antibacterial activity, grind them and extract them with 80% acetone aqueous solution, filter with ordinary filter paper, add the filtrate to a rotary evaporator, and evaporate the acetone to obtain the extract concentrate;

5. Put filter B in a clean petri dish, add the extraction concentrate, shake slowly on a shaker at 30°C for 30 minutes, and remove filter B;

6. Put filter membrane A in a petri dish, shake slowly on a shaker at 30°C for 60 minutes, and take filter membrane A;

7. Wash in sections with Tris-HCl eluent with pH 8.0, 6.0, 5.0, 4.0 to recover the specific binder on filter membrane A;

8. Further conduct antibacterial and antibacterial tests and separate and purify the collected materials.

Example 4

The method for screening the specificity antibiotic of anti-clostridium tetani by affinity binding test comprises the following steps:

1. Artificially synthesized a specific fragment of Clostridium tetani 16S rRNA ACCCUAGAGCAUAAGGGGCAUGAUGAUUUG;

2. Connect the synthesized fragments to cellulose (can be processed by relevant biotechnology companies) to make a stationary phase for target-cellulose affinity chromatography, for future use;

3. Extract calf thymus DNA with phenol-chloroform extraction, treat with alkaline denaturation solution (final concentration: 0.4mol/LNaOH, 40mmol/LEDTA) at room temperature for 10 minutes to denature the DNA, and then evenly add it to the nitrocellulose membrane. Adsorbed and fixed for 30 minutes, dried at room temperature, and dried in a vacuum oven at 80°C for 30 minutes to make a filter membrane immobilized with animal cell DNA for later use;

4. Screen the microbial strains with antibacterial activity, shake the flask for fermentation, collect and concentrate the fermentation broth, dissolve it in an appropriate amount of pH7.0 phosphate buffer, and filter;

5. After rinsing the filter membrane with pH 7.0 phosphate buffer, put it in a clean petri dish, add the above filtrate, and shake slowly on a shaker at 30°C for 30 minutes to allow the substances that can bind to animal cell DNA to adsorb to the filter. On the membrane, remove the filter membrane;

6. Add target-cellulose particles (stationary phase) to the filtrate, mix well, shake slowly on a shaker at 30°C for 30 minutes;

7. Add the above mixture to the chromatography column, and discard the unbound solution;

8. Use elution buffers of different ionic strengths (Tris-HCl buffer containing 0.2, 0.6, 1.0 and 1.4 mol/L NaCl respectively, pH 7.0) as mobile phase segment chromatography, and collect the chromatographic solution;

9. Conduct antibacterial and antibacterial tests;

10. Large-scale separation and purification of ingredients with antibacterial activity to determine the structure of the substance.

Claims (9)

1. A screening method for antibiotics, characterized in that the method comprises the following steps: 1) using bacterial ribosomal nucleic acid as a target, immobilizing it on carrier A; 2) immobilizing random nucleic acid fragments on the carrier B; 3) Dissolving the substance to be screened in the buffer after concentration; 4) Adsorption and removal of non-specific binders in the concentrated solution of the object to be screened with the carrier B immobilized with random nucleic acid fragments; 5) Capture the specific binder in the concentrated solution of the object to be screened with the carrier A immobilized with the target; 6) wash off the specific binding substance bound to the target with the eluent, and carry out the antibacterial test; 7) Separating and purifying the specific binders with antibacterial ability to obtain antibiotic candidates. 2. The method according to claim 1, wherein said bacterial ribosomal nucleic acid is one of 16S rRNA, 16S rDNA, 23S rRNA and 23S rDNA. 3. The method according to claim 1, characterized in that said bacterial ribosomal nucleic acid is a sequence containing at least 18 bases in the whole sequence. 4. The method according to claim 1, characterized in that the length of said random fragment of nucleic acid is 8-12 bases. 5. The method according to claim 1, characterized in that the carrier is one of nitrocellulose membrane, nylon membrane, glass, silicon wafer, cellulose, agarose gel, hydroxyapatite, polypropylene kind. 6. The method according to claim 1, characterized in that said buffer is neutral phosphate buffer. 7. The method according to claim 1, characterized in that the capture of the specific binding substance adopts one of membrane filtration method, liquid phase "hybridization" method and affinity binding method. 8. The method according to claim 1, characterized in that said eluents are solutions of different ionic strengths. 9. The method according to claim 1, characterized in that the eluents are solutions with different pH values.