Process for the preparation of 30s ribosomal protein s1 and its use as an antibacterial potentiator

Abstract

The application provides a preparation method of 30S ribosomal protein S1 and use of the 30S ribosomal protein S1 as an antibacterial synergist, and belongs to the technical field of biological medicines. The 30S ribosomal protein S1 in the application is a bioactive protein produced by Acinetobacter bacteria, and has the effect of enhancing the antibacterial capacity of antibiotics. In the process of being combined with streptomycin sulfate, the 30S ribosomal protein S1 greatly enhances the inhibiting capacity of streptomycin sulfate on avian pathogenic E.coli without showing the effect of inhibiting bacteria itself, which lays a foundation for future practical application, and also plays a reference role for the development of new antibacterial synergists.

Description

Technical Field

[0001] This invention discloses a method for preparing 30S ribosomal protein S1, and also discloses its use as an antibacterial synergist, belonging to the field of biomedical technology. Background Technology

[0002] With the increasing use of antibiotics in recent years, some bacteria have developed resistance under antibiotic pressure, such as methicillin-resistant Staphylococcus aureus (MRSA) and carbapenemase-producing Klebsiella pneumoniae. This poses a significant challenge to the prevention and control of pathogens. Furthermore, there is still a large gap between the development of new antibacterial drugs and their clinical application. Antimicrobial potentiators are considered one of the effective ways to overcome bacterial resistance. They can be used in conjunction with currently ineffective antibiotics to help restore or even enhance the antibiotics' ability to kill pathogens.

[0003] Currently, the most classic antibiotic adjuvant is the sulfonamide potentiator, which inhibits dihydrofolate reductase, preventing dihydrofolate from being reduced to tetrahydrofolate, thereby hindering the synthesis of bacterial nucleic acids and proteins and enhancing the effect of sulfonamide drugs. Of course, new antibacterial potentiators are also emerging. For example, researchers have found that metformin, used to treat diabetes, can enhance the bactericidal effect of doxycycline. Furthermore, bacterial secondary metabolites are the source of many antibiotics, chemotherapy drugs, immunosuppressants, and other drugs, which may contain many potential antibacterial potentiators. Summary of the Invention

[0004] This invention discloses a method for preparing 30S ribosomal protein S1 and its use as an antibacterial synergist. Starting from bacterial metabolites, it seeks synergists that can enhance the effects of existing antibiotics, allowing old drugs to continue to play a role in preventing and controlling pathogens.

[0005] This invention provides a method for preparing 30S ribosomal protein S1, comprising the following steps: 1. Streak Acinetobacter monoclonal antibodies taken from -80℃ onto BHI plates and incubate at 37℃ until single colonies are formed. Then pick single colonies into 3ml of BHI liquid medium and incubate at 37℃ and 200rpm for 20h. Then transfer them to 6L of BHI liquid medium at a ratio of 2% and incubate at 37℃ and 200rpm for 24h. Centrifuge at 10000rpm for 15min and filter through a 0.22μm filter to obtain the fermentation supernatant. 2. Mix the supernatant with acetone at a volume ratio of 1:5, allow to precipitate for 5 hours, centrifuge at 10,000 rpm for 10 min to collect the precipitate, redissolve the precipitate in water, remove residual acetone by rotary evaporation at 45℃, and then freeze-dry to obtain the crude extract. 3. Each time, 180 mg of crude extract was filtered through a dextran gel chromatography column, eluted with ultrapure water at a flow rate of 1 mL / min, and 50 tubes of sample eluent were collected using a 4 mL tube. The absorbance was then measured at 280 nm, and a curve was plotted. Based on the curve trend, the sample eluent was combined into four components: Fr1, Fr2, Fr3, and Fr4. The peak shape and the activity of the combined antibiotics were observed. 4. The gel chromatography precipitate Fr4 was further purified using an XB-C18 preparative high-performance liquid chromatography column. Mobile phase A was distilled water and mobile phase B was methanol. Gradient elution was performed from 0 to 20 min, and the detection wavelength was 280 nm. The elution peak Fr4.2 with a retention time of 5.141 min was obtained. The eluent corresponding to this elution peak was collected. After repeated injections, the antibacterial enhancement target peaks were combined and freeze-dried to obtain 30S ribosomal protein S1.

[0006] This invention also provides a method for recombinant expression of 30S ribosomal protein S1, comprising the following steps: 1. Primer design: F: 5'-CTGGAATTCATGACCGAATCTTTTGCAGCCCTC-3'; R:5'-CTGCGGCCGTTATTTCATTTGCGCTTTAATCAAGTCACC-3'; 2. After large-scale PCR amplification of the target gene, column recovery is performed; 3. After enzyme digestion and ligation, the amplified target gene is inserted into the expression vector pET-28a to obtain the recombinant expression vector; 4. The recombinant expression vector was transformed into Escherichia coli BL21(DE3) to obtain the expression strain; 5. Incubate the expression strain at 37℃ and 180 rpm in a shaker until OD600 = 0.7-0.8, then add isopropyl-β-D-thiogalactoside to a final concentration of 200 μmol / L, induce at 16℃ and 160 rpm for 24 h, then centrifuge at 10000 rpm for 10 minutes, collect the cells, wash the cells with PBS, and sonicate to disrupt them. 6. Purification was performed using a nickel column. The elution was carried out with 20 mM, 100 mM, 200 mM, 400 mM, and 500 mM imidazole, respectively. The elution fraction of 100 mM imidazole was collected, the imidazole was removed, and then the product was lyophilized to obtain the recombinant expression of 30S ribosomal protein S1.

[0007] The present invention also provides a 30S ribosomal protein S1 with antibacterial synergistic effect, which is a protein produced by Acinetobacter monotyphi that can enhance the effect of antibiotics, and the amino acid sequence is shown in SEQ ID NO. 1.

[0008] This invention discloses that when 30S ribosomal protein S1 is combined with streptomycin sulfate, it enhances the inhibitory effect of streptomycin sulfate on pathogenic Escherichia coli O78 in birds.

[0009] The positive effects of this invention are as follows: it provides the ability of 30S ribosomal protein S1 to enhance the effect of antibiotics, which can alleviate bacterial resistance and restore the antibacterial effect of originally ineffective antibiotics. Experiments on avian pathogenic Escherichia coli O78 show that 30S ribosomal protein S1 can effectively enhance the killing power of streptomycin sulfate against this bacterium. It can be used in the fields of medicine, food, pets, and public health protection. Attached Figure Description

[0010] Figure 1 This is a diagram illustrating the verification of the antibacterial synergistic activity of the acetone precipitate during the purification process of 30S ribosomal protein S1 in this invention. Figure 2 This is a preparative high-performance liquid chromatogram of the purification process of 30S ribosomal protein S1 in this invention; Figure 3 The analytical high-performance liquid chromatography (HPLC) chromatogram and purity of component Fr4.1 obtained during the purification of 30S ribosomal protein S1 in this invention; Figure 4 The analytical high-performance liquid chromatography (HPLC) chromatogram and purity of the 30S ribosomal protein S1 (Fr4.2) of this invention are shown below. Figure 5 This invention provides a gel filtration chromatography detection curve and verification of the antibacterial synergistic activity of the eluted components during the purification of 30S ribosomal protein S1. Figure 6 The synergistic effect of the eluent components from preparative high-performance liquid chromatography during the purification of 30S ribosomal protein S1 in this invention on streptomycin sulfate is shown. Figure 7 This is an agarose gel electrophoresis diagram of the target gene of the 30S ribosomal protein S1 in this invention; Figure 8 This is an SDS-PAGE image of the 30S ribosomal protein S1 of this invention. Figure 9 This invention demonstrates the synergistic effect of recombinantly expressed 30S ribosomal protein S1 on streptomycin sulfate. Detailed Implementation

[0011] The present invention is further illustrated below with specific embodiments. These embodiments are only a part of, not all, of the present invention, and are not intended to limit the invention to the scope of these embodiments. Experimental methods not specifically described in the following embodiments were performed according to conventional methods or the product manual.

[0012] Example 1: Further purification of gel chromatography compound Fr4 using an XB-C18 preparative high performance liquid chromatography column. 1. Preparation of mobile phase: Filter distilled water using a vacuum filtration device, and then remove air bubbles by sonication (sonication for 10 minutes, power 100, frequency 50). When using methanol, air bubbles also need to be removed by sonication. If the mobile phase is not moving, sonication is not necessary; otherwise, sonication is necessary again. 2. Sample preparation: Dissolve the Fr4 sample in ddH2O and filter it through a 0.22μm filter; 3. Insert the pump head of the chromatograph into each of the ultrasonically treated solutions, with pump A for the aqueous phase and pump B for the organic phase; 4. Turn on the chromatograph in the following order: turn on the computer, then turn on the three parts of the chromatograph from top to bottom (two control units and one detection unit), then open the software and proceed with the subsequent operations; 5. Exhausting: Set the A / B exhaust program to 2mL / min and 5min. Open the exhaust valves A and B, and press the PURGE button on the pump panel to exhaust one by one. After exhausting, press the PUMP / STOP button to close the exhaust valves. 6. Flush closed pipelines (100% methanol for 5 minutes); 7. Column equilibration: Methanol from 100% to 20% (3 mL / min, one gradient for 4 min), then from 20% to 100%. If the baseline has stabilized, the above equilibration procedure can be terminated or interrupted in a timely manner, or the equilibration time can be extended. 8. Once the baseline is stable, you can begin preparing for sample loading. The detection wavelength is 280 nm, and the sample loading volume is 10 mL per loading. 9. Set a 20-minute elution program and manually collect the sample. Immediately after the detector peak appears, collect the corresponding eluent for Fr4.2 with a retention time of 5.141 min. After repeated injections to collect the target peak eluent, combine the antibacterial enhancement target peaks and freeze-dry to obtain 30S ribosomal protein S1.

[0013] Example 2 Recombinant expression of 30S ribosomal protein S1 1. Primer design: Primers were designed based on the gene sequence of the 30S ribosomal protein S1. EcoRI and EagI restriction enzyme sites were added upstream and downstream, respectively, and the protective base CTG was added before the restriction enzyme sites to obtain the primers as follows: F:5'-CTGGAATTCATGACCGAATTCTTTTGCAGCCCTC-3'; R:5'-CTGCGGCCGTTATTTCATTTGCGCTTTAATCAAGTCACC-3'.

[0014] 2. Using a 50 μL system, amplify the target fragment with PCR in 4 tubes, for a total of 200 μL. After the reaction, take 5 μL from each tube for 1.5% agarose gel electrophoresis. After the target band is detected, use a column PCR product purification kit to recover the PCR amplification product and measure its concentration. After column recovery, take another 2 μL of the recovered product for 1.5% agarose gel electrophoresis to check if the column recovery was successful. 3. Extraction of pET-28a plasmid: Take the bacterial culture frozen at -80°C and inoculate it at 1% into 3 mL LB liquid medium containing 1 / 1000 kanamycin (50 mg / mL), for a total of 4 tubes; incubate overnight at 37°C with a shaker at 200 rpm; after removal, centrifuge at 8000 g for 5 min to collect the bacterial cells into two 1.5 mL tubes, resuspend and wash twice with sterile autoclaved distilled water, retaining only the bacterial cells; fully resuspend the bacterial cells with 320 μL TE buffer, vortex and mix, then add 80 μL lysozyme (concentration of 100 mg / mL), mix thoroughly, and incubate at 37°C for 1 h; invert and mix once every 10 min; after removal, follow the steps of the plasmid extraction kit; finally, elute with 30 μL of RNAfree H2O preheated at 65°C; detect the pET-28a plasmid concentration, and store at -20°C for later use; 4. Double digestion of plasmid and target fragment: The recovered target fragment and the extracted pET-28a vector were placed separately in a reaction system containing EcoRI and EagI restriction endonucleases and digested in a 37℃ water bath for 12 h. The band size was observed by 1.5% agarose gel electrophoresis to see if it was consistent with the expectation. Then the digested products were recovered by column PCR product purification kit. 5. Construction of ligation between target gene and expression vector digestion products: A 20 μL ligation reaction system was constructed on ice. The system consisted of 2 μL T4 DNA Ligase Buffer (10X), 1 μL T4 DNA Ligase, 7 μL target gene fragment (206 ng), 5 μL pET-28a column-recovered fragment (105 ng), and 5 μL RNA-free H2O. The ligation was then carried out overnight at 16°C. 6. Transform the recombinant expression vector into *E. coli* BL21(DE3) to obtain the expression strain: Cool on ice, transform 10 μL of the reaction product into 50 μL of competent cells, gently tap to mix, and incubate on ice for 30 minutes; heat shock at 42°C for 32 seconds, then immediately place on ice for 2 minutes; add 900 μL of LB medium equilibrated to room temperature, and incubate at 200 rpm and 37°C for 3 hours; plate the bacterial culture, incubate overnight, centrifuge and plate the entire bacterial culture onto a kanamycin plate, and incubate overnight; pick a single colony and inoculate it into liquid medium containing 1 / 1000 50 mg / mL kanamycin (final concentration 50 µg / mL), incubate at 200 rpm and 37°C for about 4 hours, and then perform positive clone detection; 7. Induction of expression: The expression strain was cultured at 37℃ and 180 rpm in a shaker until OD600 = 0.7-0.8. Isopropyl-β-D-thiogalactoside was then added to a final concentration of 200 μmol / L. The culture was induced at 16℃ and 160 rpm for 24 h. The cells were then collected by centrifugation at 10000 rpm for 10 minutes, washed with PBS, and sonicated.

[0015] 8. Purification was performed using a nickel column. The elution was carried out with 20 mM, 100 mM, 200 mM, 400 mM and 500 mM imidazole, respectively. The elution fraction of 100 mM imidazole was collected, the imidazole was removed, and then the recombinant 30S ribosomal protein S1 was obtained by freeze-drying. SDS-PAGE analysis was performed simultaneously.

[0016] As can be seen from the above embodiments, the present invention details a method for further purification of 30S ribosomal protein S1 by high performance liquid chromatography, and also provides a prokaryotic expression method for 30S ribosomal protein S1. However, it can also be seen that some modifications can be made to the present invention. Therefore, any modifications or improvements made without departing from the spirit and principles of the present invention are within the scope of protection claimed by the present invention.

[0017] Experimental Example 1: Identification of 30S ribosomal protein S1 First, Fr4.2, purified from preparative high-performance liquid chromatography (using an XB-C18 column), was analyzed (its corresponding peak is shown in the image). Figure 2 Purity was determined using analytical high-performance liquid chromatography (HPLC) with a HILIC column. A single peak appeared at 2.859 min on the chromatogram (see...). Figure 4 And its purity is approximately 98.47% (see...). Figure 4Therefore, this sample can be used for subsequent identification; Fr4.2 was sent to the company for MALDI-TOF / TOF mass spectrometry identification, which identified the peptide sequences VKGGFTVDIGPVR, AFLPGSSLVDTRPIR, NNVVVSR, IKHPSEVVEVGQEVTVK, VLKFDR, VSLGLK, QLGEDPWLAIMSR, ISLGIK, KGDTVEAVILSVDAEGNR, QLNSDPFNDFLAANER, LGDEIEATLK, ASEINRDRVEDATK, SRSINLSIK, SINLSIK, AKDEAEEKEAVANLR, and TIGDLIK. Then, a library search was performed, and it was found that it matched a 30S ribosomal protein S1, and the size of this protein was found to be 61 kDa (see [link to database]). Figure 8 In addition, the amino acid sequence of the protein is shown in SEQ ID NO.1.

[0018] Experimental Example 2: Purification of ribosomal protein S1 with antibacterial synergistic effect using activity-guided purification. The activity of the crude extract precipitated from Acinetobacter monocytogenes supernatant at a volume ratio of 5:1 (redissolved in 1 / 10 volume of ddH2O of the culture supernatant) was verified. The extract was divided into a control group, an acetone-precipitate group (Acetone-Pel), a neomycin sulfate group (NEOS), and a combined group of neomycin sulfate and acetone-precipitate (NEOS+Acetone-Pel). The final concentration of neomycin sulfate in all groups was 160 μg / mL. To better reflect the actual antibacterial effect, the results of this experiment do not include the 8 mm diameter of the Oxford cup itself. Figure 1 As shown, after 12 hours of incubation, the NEOS+Acetone-Pel group was compared with the NEOS group. It can be seen that Acetone-Pel has an antibacterial synergistic effect on NEOS, enhancing its killing effect on pathogenic Escherichia coli in birds. At the same time, comparing Acetone-Pel with the Control group, it was found that Acetone-Pel has no antibacterial effect. This indicates that the crude extract can enhance the antibacterial effect of NEOS without having antibacterial effect itself. Therefore, this crude extract was selected for the next step of dextran gel filtration chromatography. The crude acetone extract was subjected to dextran gel filtration chromatography to obtain Fr1, Fr2, Fr3, and Fr4 (lyophilized and reconstituted in 1 / 10 ddH2O). Activity was then validated, with neomycin sulfate (NEOS) reaching a final concentration of 160 μg / mL and streptomycin sulfate reaching a final concentration of 800 μg / mL. The indicator bacterium used was avian pathogenic Escherichia coli O78. Figure 5As shown in the inhibition diagram, compared with the NEOS group alone, Fr4 enhanced the antibacterial effect of NEOS. Since neomycin sulfate is an aminoglycoside antibiotic, streptomycin sulfate (STRS), also an aminoglycoside, was used for activity verification. The results showed that both Fr3 and Fr4 enhanced the antibacterial activity of STRS, while Fr1 and Fr2 did not significantly enhance STRS. Furthermore, comparing Fr1, Fr2, Fr3, and Fr4 individually with the control group, none of them showed antibacterial activity. Both Fr3 and Fr4 enhanced STRS, and because the peak of Fr4 was more singular than that of Fr3 (see...),... Figure 5 (Gel filtration curve of glucan), therefore Fr4 was selected for the next purification step.

[0019] The Fr4 obtained by dextran gel filtration chromatography was finally purified using preparative high-performance liquid chromatography (using an XB-C18 column). Figure 2 As shown, components Fr4.1 and Fr4.2 were obtained (lyophilized and then reconstituted in 1 / 10 ddH2O), and their activity was then verified. The final concentration of streptomycin sulfate was 800 μg / mL. The results were obtained from... Figure 6 As shown, both Fr4.2 and Fr4.1 have the ability to enhance the efficacy of neomycin sulfate (STRS), with Fr4.2 being more effective than Fr4.1. At the same time, neither Fr4.1 nor Fr4.2 showed any antibacterial effect on their own.

[0020] Then, the purity of Fr4.1 and Fr4.2 was determined using analytical high-performance liquid chromatography (using a HILIC column). Figure 3 The HPLC chromatogram of Fr4.1 shows a main peak at 2.914 min, but also a noticeable smaller peak at 4.357 min. Furthermore, the purity of the substance corresponding to the main peak at 2.914 min is only 70.64%, which does not meet the identification criteria. Conversely, the purity of the substance at the 2.914 min main peak is not met. Figure 4It was found that Fr4.2 showed a single peak at 2.859 min on the chromatogram, with a purity of approximately 98.47%. Therefore, this sample could be used for subsequent identification. Fr4.2 was sent to the company for MALDI-TOF / TOF mass spectrometry identification, and the peptide sequences VKGGFTVDIGPVR, AFLPGSSLVDTRPIR, NNVVVSR, IKHPSEVVEVGQEVTVK, VLKFDR, VSLGLK, QLGEDPWLAIMSR, ISLGIK, KGDTVEAVILSVDAEGNR, QLNSDPFNDFLAANER, LGDEIEATLK, ASEINRDRVEDATK, SRSINLSIK, SINLSIK, AKDEAEEKEAVANLR, and TIGDLIK were identified. Then, a library search was performed, and it was found that it matched a 30S ribosomal protein S1, and the size of this protein was known to be 61 kDa. The amino acid sequence of this protein is shown in SEQ ID NO. 1.

[0021] In summary, an activity-guided purification method was used to identify and purify a ribosomal protein S1 from the supernatant of a bacterial strain. This protein can act as an antibacterial synergist, enhancing the bactericidal ability of antibiotics against pathogens without exhibiting antibacterial activity itself. This is expected to help with future practical applications.

Claims

1. The use of 30S ribosomal protein S1 in the preparation of neomycin sulfate or streptomycin sulfate antibacterial synergists, characterized in that: The amino acid sequence of the 30S ribosomal protein S1 is shown in SEQ ID NO. 1; The bacteria in question is avian pathogenic Escherichia coli O78.

Images