FtsZ protein preparation method, product and application

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a method for preparing FtsZ protein, a product and an application. The method for preparing FtsZ protein comprises the following steps: constructing a biomaterial for ftsZ gene expression; inducing the expression of the biomaterial using IPTG to obtain FtsZ protein inclusion bodies; ultrasonically treating the FtsZ protein inclusion bodies, separating and purifying, and obtaining FtsZ protein; the concentration of the IPTG induction is 0.1-0.6 mM; the time of the IPTG induction is 1-7 h. The method provided by the present invention can prepare FtsZ protein with high GTPase activity, and the highest GTPase activity is 0.998 nmol / ml / min, which can be used to screen antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for preparing an FtsZ protein, a product and an application thereof. Background Art

[0002] Pathogenic E. coli can not only cause diarrhea in humans and animals such as livestock and poultry, but also lead to outbreaks of extraintestinal diseases. The number of cases of E. coli diarrhea is estimated to be 1.7 billion per year worldwide, of which about 760,000 children die as a result, and 9.6-11% of patients in hospitals die from E. coli sepsis.

[0003] Long-term irrational use of antibiotics has led to the emergence of high resistance, multi-resistance, pan-resistance and the induction of double infection in bacteria, and there is still no effective treatment. Studies have found that colistin, known as the "last antibiotic", cannot kill Escherichia coli. Therefore, there is an urgent need to develop new antibacterial drugs to fight pathogenic Escherichia coli. Summary of the invention

[0004] In view of the technical problems existing in the background technology, the present invention provides a method for preparing FtsZ protein, a product and an application. The method for preparing FtsZ protein provided by the present invention can produce FtsZ protein with high GTPase activity, the highest GTPase activity is 0.998nmol / ml / min, and can be used to screen antibacterial drugs.

[0005] The invention provides a method for preparing FtsZ protein, comprising the following steps: constructing a biological material for ftsZ gene expression; inducing the expression of the biological material by using IPTG (isopropyl-β-D-thiogalactoside) to obtain FtsZ protein inclusion bodies; ultrasonically treating the FtsZ protein inclusion bodies, separating and purifying, and obtaining FtsZ protein; the concentration of the IPTG induction is 0.1-0.6 mM; and the time of the IPTG induction is 1-7 hours.

[0006] Preferably, the temperature of the ultrasonic treatment is 0-4°C; the total duration of the ultrasonic treatment is 8-12 min; the cycle of the ultrasonic treatment is: ultrasonic treatment for 4-6 s, with an interval of 8-12 s; and the power of the ultrasonic treatment is 80-250 W.

[0007] Preferably, the separation and purification uses Ni 2+ -NTA affinity column and 400-750 mM imidazole eluent; the separation and purification retains proteins with a molecular weight > 14 kDa.

[0008] Preferably, the sequence of the ftsZ gene includes SEQ ID NO.1.

[0009] The present invention provides a FtsZ protein, and the FtsZ protein is prepared by the FtsZ protein preparation method.

[0010] The invention provides a broad-spectrum antibacterial drug screening system, comprising the FtsZ protein.

[0011] The present invention provides a method for preparing the FtsZ protein, or the FtsZ protein, or the application of the broad-spectrum antibacterial drug screening system in screening antibacterial drugs.

[0012] The present invention provides a method for screening a broad-spectrum antibacterial drug, comprising the following steps:

[0013] S1, incubating the FtsZ protein with GTP and the compound to be screened;

[0014] S2. Detect the GTPase activity of the FtsZ protein, and judge the broad-spectrum antibacterial properties of the compounds to be screened based on the GTPase activity of the FtsZ protein: if the GTPase activity of the FtsZ protein decreases, the compounds to be screened have antibacterial effects.

[0015] Preferably, the GTP concentration of the co-incubation is 0.25-4 mM.

[0016] Preferably, the co-incubation time is 20-40 min.

[0017] Beneficial effects:

[0018] The present invention provides a method for preparing FtsZ protein, comprising the following steps: constructing a biomaterial for ftsZ gene expression; using IPTG to induce the expression of the biomaterial to obtain FtsZ protein inclusion bodies; ultrasonically treating the FtsZ protein inclusion bodies, separating and purifying to obtain FtsZ protein; the concentration of the IPTG induction is 0.1-0.6mM; the time of the IPTG induction is 1-7h. The method provided by the present invention can prepare FtsZ protein with high GTPase activity, the highest GTPase activity is 0.998nmol / ml / min, and can be used to screen antibacterial drugs.

[0019] The present invention provides a method for screening broad-spectrum antibacterial drugs, comprising the following steps: S1, incubating the FtsZ protein with GTP and a compound to be screened; S2, detecting the GTPase activity of the FtsZ protein, and judging the broad-spectrum antibacterial performance of the compound to be screened according to the GTPase activity of the FtsZ protein: if the GTPase activity of the FtsZ protein decreases, the compound to be screened has an antibacterial effect. The present invention can efficiently, simply and economically screen antibacterial drugs based on the FtsZ protein by evaluating the inhibitory effect of the compound to be screened on the GTPase activity of the FtsZ protein. The present invention provides a new idea for the screening of antibacterial drugs, and can be used to find effective new compounds or other antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0021] Figure 1 It is the result of PCR amplification of ftsZ gene in Example 2. Among them, lanes 1, 2, 4, 5, 7, 8: ftsZ gene; lanes 3, 6, 9: negative control; M: DNA marker;

[0022] Figure 2 It is the result of double enzyme digestion of the recombinant plasmid and induced expression of the recombinant FtsZ protein in Example 2. Figure 2 Figure A in the middle shows the double digestion of pMD-FtsZ recombinant plasmid and pET-28a plasmid by Nco I / Xho I; Lane 1: double digestion of pMD-FtsZ recombinant plasmid; Lane 2: double digestion of pET-28a plasmid; M: DNA marker; Figure 2 Figure B in the middle shows the induced expression of FtsZ at different temperatures (16-37°C); M: protein marker; Figure 2 Figure C in the middle shows the expression of FtsZ at different induction times and different IPTG concentrations; CK: pET28a empty vector; PC: bovine serum albumin; M: protein marker.

[0023] Figure 3 is the separation and purification result of the recombinant FtsZ protein in Example 2. Among them, Figure 3 Figure A in the middle indicates that the ultrasonic power is 227.5W; Lane 1: precipitate after ultrasonication; Lane 2: supernatant after ultrasonication; Figure 3 Figure B in the middle shows that the ultrasonic power is 162.5 W; Lane 1: precipitate after ultrasonication; Lane 2: supernatant after ultrasonication; Figure 3 Figure C in the middle shows that the ultrasonic power is 97.5W; Lanes 1 and 3: precipitate after ultrasonication; Lanes 2 and 4: supernatant after ultrasonication; Figure 3Figure D in the middle shows the effect of different imidazole concentrations on the elution of FtsZ; Lane 1: flow-through peak; Lane 2: 250 mM imidazole; Lane 3: 300 mM imidazole; Lane 4: 350 mM imidazole; Lane 5: 400 mM imidazole; Lane 6: 450 mM imidazole; Lane 7: 500 mM imidazole; Lane 8: 550 mM imidazole; Lane 9: 600 mM imidazole; Lane 10: 650 mM imidazole; Lane 11: 700 mM imidazole; Lane 12: 750 mM imidazole; Figure 3 Figure E in the middle shows the effect of different elution times on the elution of FtsZ. Lane 1: precipitate after ultrasound; Lane 2: supernatant after ultrasound; Lane 3: flow-through peak; Lane 4: eluted once with 500mM imidazole; Lane 5: eluted twice with 500mM imidazole; Lane 6: eluted three times with 500mM imidazole; Lane 7: eluted four times with 500mM imidazole; Lane 8: eluted five times with 500mM imidazole; Lane 9: eluted six times with 500mM imidazole; M: protein marker.

[0024] Figure 4 The results of the GTPase activity assay of the recombinant FtsZ protein and the screening of its inhibitors in Example 3 and Example 4 are shown in FIG. Figure 4 Panel A in the middle represents the determination of GTPase activity of FtsZ protein; Figure 4 Panel B in the middle shows the effect of alkaloids on the GTPase activity of FtsZ protein.

[0025] Figure 5 is the result of the interaction between berberine hydrochloride and FtsZ protein in Example 6. Figure 5 Figure A in the middle shows the effect of different concentrations of GTP on FtsZ aggregation; Lane 1: 0 mM GTP; Lane 2: 0.25 mM GTP; Lane 3: 0.5 mM GTP; Lane 4: 1 mM GTP; Lane 5: 2 mM GTP; Lane 6: 4 mM GTP; Lane 7: 8 mM GTP; M: protein marker; Figure 5 Figure B in the middle shows the effect of different concentrations of berberine hydrochloride on FtsZ aggregation; Lanes 1-3: berberine hydrochloride was incubated with FtsZ for 0.5h, and then GTP was added and incubated for 0.5h; Lanes 4-6: berberine hydrochloride was incubated with FtsZ and GTP for 1h; Lanes 1 and 4: 1.56μg / mL berberine hydrochloride; Lanes 2 and 5: 6.25μg / mL berberine hydrochloride; Lanes 3 and 6: 25μg / mL berberine hydrochloride; Lane 7: Control (FtsZ+GTP); 8: Control (FtsZ); M: protein marker; Figure 5 Figures C and D in the middle represent MD analysis of the binding of berberine hydrochloride to FtsZ; Figure D is an enlarged view of Figure C. DETAILED DESCRIPTION

[0026] The invention provides a method for preparing FtsZ protein, comprising the following steps: constructing a biological material for expressing ftsZ gene; and inducing the expression of the biological material by using IPTG.

[0027] In the present invention, the sequence of the ftsZ gene preferably includes SEQ ID NO.1. The sequence shown in SEQ ID NO.1 is codon-optimized, has a size of 1,170 bp, and has a higher expression level. The biological material for ftsZ gene expression preferably includes an expression vector containing the ftsZ gene and an engineered bacterium; the expression vector preferably includes pET28-ftsZ; the engineered bacterium preferably includes a positive transformant of Escherichia coli BL21 (DE3) transformed with the expression vector.

[0028] In a preferred embodiment of the present invention, the FtsZ protein is expressed by prokaryotic expression of Escherichia coli, and the primer sequence is F: 5′-catg ccatgg gcatgtttgaaccaatggaac-3′ (SEQ ID NO. 2) and R: 5′-ccg ctcgag atcagcttgcttacgcagga-3′ (SEQ ID NO. 3), wherein the underline indicates the restriction site.

[0029] The present invention uses IPTG to induce the expression of the biomaterial to obtain FtsZ protein inclusion bodies. In the present invention, the concentration of the IPTG induction is 0.1-0.6mM, preferably 0.2mM; the time of the IPTG induction is 1-7h, preferably 4-6h, more preferably 5h; the temperature of the IPTG induction is 16-37°C, preferably 37°C.

[0030] The present invention adopts ultrasonic treatment to treat the FtsZ protein inclusion body. In the present invention, the ultrasonic treatment is preferably carried out in an ice bath environment; the temperature of the ice bath environment is preferably 0-4°C; the total duration of the ultrasonic treatment is preferably 8-12 minutes, more preferably 10 minutes; the cycle of the ultrasonic treatment is preferably: ultrasonic 4-6 seconds, interval 8-12 seconds; more preferably: ultrasonic 5 seconds, interval 10 seconds; the power of the ultrasound is preferably 80-250W, more preferably a range value between any two power values ​​of 97.5W, 162.5W and 227.5W, more preferably 97.5W.

[0031] The present invention separates and purifies the FtsZ protein inclusion bodies treated with ultrasound to obtain the FtsZ protein. In the present invention, the separation and purification preferably uses Ni 2+-NTA affinity column and imidazole eluent, preferably eluted multiple times in large volume; the working concentration of the imidazole eluent is preferably 400-750mM, more preferably 450-600mM, more preferably 500mM; the separation and purification of the present invention preferably retains proteins with a molecular weight greater than 14kDa.

[0032] In a preferred embodiment of the present invention, the preparation method of the FtsZ protein includes: synthesizing a 1,170bp ftsZ gene, amplifying it, and inserting it into the cloning vector pMD-19T to obtain a recombinant plasmid pMD-ftsZ; after Xho I / Nco I double digestion, inserting it into the pET-28a expression vector to obtain a recombinant plasmid pET28-ftsZ, and transforming Escherichia coli BL21 (DE3). Positive transformants were screened, and after induction with 0.2mM IPTG, the FtsZ protein was expressed in the form of inclusion bodies in Escherichia coli BL21, and its C-terminus had 6 His. The expressed FtsZ protein was stained with Ni 2+ After purification by -NTA affinity column, recombinant FtsZ protein with a size of about 41 kDa was obtained.

[0033] The present invention can significantly increase the expression of FtsZ protein by inducing biomaterial expression through IPTG; and the FtsZ protein obtained after separation and purification of the expression product has high GTPase activity. The GTPase activity of the FtsZ protein is up to 0.998nmol / mL / min, and can be used to screen antibacterial drugs.

[0034] The present invention provides a FtsZ protein prepared by the FtsZ protein preparation method. The FtsZ protein has the appearance of white powder and a molecular weight of 41.13 kDa.

[0035] The invention provides a broad-spectrum antibacterial drug screening system, which comprises the FtsZ protein.

[0036] The present invention also provides a method for preparing the FtsZ protein, or the use of the FtsZ protein, or the broad-spectrum antibacterial drug screening system in screening antibacterial drugs.

[0037] The present invention selects FtsZ (bacterial filament temperature-sensitive mutant Z) protein as an antibacterial drug target. FtsZ, as a highly conserved prokaryotic protein, is a homolog of eukaryotic tubulin, but has only 7% sequence similarity. FtsZ protein is present in almost all bacteria and is highly conserved. It plays an important role in prokaryotic cell division, but does not exist in eukaryotic organisms. At the same time, FtsZ protein is also a GTPase, which polymerizes into a single chain in a GTP-dependent manner and further assembles into a Z ring inside the bacterial cytoplasmic membrane. FtsZ protein is the first cell division protein molecule to reach the central division site of the cell. In the presence of GTP, it polymerizes into a Z ring and recruits other proteins involved in bacterial cell division (including FtsA, ZapA (YshA), FstL, FtsW, etc.) to form a division complex that can cause cell membrane contraction. Abnormalities in FtsZ assembly and GTPase activity can inhibit the formation of Z rings and cell septa, thereby leading to abnormal bacterial cell division and even cell death. The present invention utilizes the above-mentioned characteristics of the FtsZ protein to screen compounds with broad-spectrum antibacterial effects, and has good application potential.

[0038] The present invention provides a method for screening a broad-spectrum antibacterial drug, comprising step S1: co-incubating the FtsZ protein with GTP and a compound to be screened; in the present invention, the GTP concentration of the co-incubation is preferably 0.25-4 mM, more preferably 0.5-2 mM, more preferably 1-1.25 mM. The co-incubation time is preferably 20-40 min, more preferably 30 min.

[0039] After co-incubation, the method for screening broad-spectrum antibacterial drugs of the present invention includes step S2: detecting the GTPase activity of the FtsZ protein, and judging the broad-spectrum antibacterial performance of the compound to be screened according to the GTPase activity of the FtsZ protein: if the GTPase activity of the FtsZ protein decreases, the compound to be screened has an antibacterial effect.

[0040] In the present invention, the broad-spectrum antibacterial drug screening method is preferably used for drug screening of bacterial pathogens, especially Gram-negative bacteria (such as Escherichia coli, Salmonella, etc.) infectious diseases. The drug preferably includes alkaloid compounds, especially including one or more of sophoridine, matrine, theophylline, betaine, betonicine, tetrandrine, sanguinarine chloride, sinomenine hydrochloride, nonivamide and berberine hydrochloride.

[0041] In a preferred embodiment of the present invention, it was found through broad-spectrum antibacterial drug screening that: sophoridine has the strongest inhibitory effect on the GTPase activity of recombinant FtsZ protein; berberine hydrochloride prevents the polymerization of recombinant FtsZ protein in a concentration-dependent manner and binds to the FtsZ protein through hydrogen bonding; compared with other alkaloid molecules, sanguinarine exhibits higher antibacterial activity against Escherichia coli and Salmonella.

[0042] The broad-spectrum antibacterial drug screening method provided by the invention takes FtsZ protein as a target and has the advantages of being convenient, rapid, simple to operate and highly efficient.

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used can be obtained from commercial sources.

[0044] Example 1

[0045] The method for screening broad-spectrum antibacterial drugs targeting FtsZ protein comprises the following steps:

[0046] (1) Gene cloning and construction of recombinant vector: The ftsZ gene (1,170 bp) and its primers were synthesized. The primer sequences for PCR were: F: 5′-catgccatgggcatgtttgaaccaatggaac-3′ (SEQ ID NO. 2) and R: 5′-ccgctcgagatcagcttgcttacgcagga-3′ (SEQ ID NO. 3). The gene was amplified and inserted into the cloning and expression vectors, respectively, to obtain the recombinant plasmids pMD-ftsZ and pET28-ftsZ.

[0047] (2) Induction, expression and purification of recombinant FtsZ protein: After IPTG induction of positive transformants, FtsZ was expressed in E. coli BL21 (DE3) in the form of inclusion bodies. After ultrasonication of the inclusion bodies, the expressed FtsZ protein was purified by Ni 2+ -NTA affinity column purification was performed to obtain a recombinant FtsZ protein of approximately 41 kDa with 6 His residues at its C-terminus.

[0048] (3) Determination of GTPase activity of recombinant FtsZ protein: The enzymatic activity of recombinant FtsZ protein was determined by measuring the ability of GTPase to release GTP free phosphate (Pi) using the malachite green phosphate assay.

[0049] (4) Construction of antibacterial drug screening method: The recombinant FtsZ protein was incubated with GTP and the compound to be screened for 30 min. The GTPase activity of the recombinant FtsZ protein was detected and the changes in the effect of the compound on the FtsZ protease activity were observed. When the enzyme activity decreased, the compound to be screened had an antibacterial effect, and this was used to screen antibacterial drugs targeting the FtsZ protein.

[0050] in:

[0051] Step (1) Primers are designed using the ftsZ gene sequence of Escherichia coli CVCC195 (purchased from the China Veterinary Microbial Culture Collection Center), and the gene ftsZ is amplified using synthetic gene DNA as a template. The target gene is identified and recovered by agarose gel electrophoresis, and inserted into the cloning vector pMD-19T to obtain a recombinant plasmid pMD-ftsZ, which is transformed into Escherichia coli DH5α to obtain positive transformants, and the plasmid pMD-ftsZ is extracted. After double digestion with XhoI and NcoI, the target gene ftsZ fragment is obtained and inserted into the expression vector pET28a with the same enzyme digestion. The obtained recombinant plasmid pET28-ftsZ is transformed into the expression strain BL21 (DE3), and the positive transformants are screened by colony PCR.

[0052] In step (2), after the pET28-ftsZ plasmid is transformed into the Escherichia coli expression strain BL21 (DE3), the FtsZ protein inclusion body is obtained by IPTG induction expression, and the preparation of the recombinant FtsZ protein is completed by ultrasonic lysis of the bacteria, purification, dialysis, lyophilization, and enzyme activity detection. Among them, the IPTG concentration is 0.2mM, the induction time is 5h, and the induction temperature is 37°C. The bacterial solution is stored in an ice bath for ultrasonication, and the optimal ultrasonic power is set to 97.5W. Each cycle works for 5s, the interval is 10s, and the ultrasonic time is 10min. Dialysis is performed in ultrapure water through a dialysis bag with a molecular weight cutoff of 14kDa. The freeze-drying conditions are: -80°C overnight, 110kPa freeze-drying for 72h. The purity of the recombinant FtsZ protein obtained in step (2) is greater than 90%, and the GTPase activity is greater than 40%.

[0053] Step (3) The GTP solution (5 mM) was diluted 2-fold with a buffer solution (containing 50 mM Tris (pH 7.5), 250 mM KCl and 5 mM MgCl2) and added sequentially to a half-volume 96-well transparent plate containing 3 μM FtsZ (20 μL / well). After incubation at 37°C for 30 min, MG acid reagent (5 μL / well) was added to the 96-well plate and incubated at room temperature for 10 min. MG blue reagent was added to the reaction and reacted at room temperature for 20 min. The sample well without GTP solution was used as a negative control. At OD 620nmThe release of free Pi was measured under 4% phosphate and the GTPase activity of the recombinant FtsZ protein (nmol / mL / min) in the enzymatic reaction was calculated based on the phosphate standard curve. When the GTP concentration was 1.25 mM, the enzyme activity of the recombinant FtsZ protein reached the highest level, which was 0.998 nmol / mL / min.

[0054] In step (4), the optimal substrate concentration of GTP in the incubation system is 1-1.25 mM.

[0055] Example 2

[0056] Preparation of recombinant FtsZ protein: The recombinant FtsZ protein was prepared by cloning the ftsZ gene and constructing a recombinant plasmid, inducing expression, optimizing the expression conditions, and then ultrasonically treating the protein. The protein was then purified by Ni column, dialyzed and freeze-dried.

[0057] The ftsZ gene was codon optimized for E. coli and gene synthesis was performed to increase the expression level. The codon-optimized ftsZ gene sequence is SEQ ID NO.1. The specific primer F: 5′-catg ccatgg gcatgtttgaaccaatggaac-3′ (SEQ ID NO.2) and R:5′-ccgc tcgagatcagcttgcttacgcagga-3′(SEQ ID NO.3) was used to amplify the ftsZ gene. The restriction enzyme sites Xho I and Nco I are underlined. The PCR product of the ftsZ gene was purified by a DNA product purification kit, cloned into the pMD-19Tsimple vector, and transformed into the E. coli cloning strain DH5α to obtain positive transformants; the plasmid pMD-ftsZ was extracted according to the instructions of the plasmid extraction kit. The positive pMD-ftsZ plasmid was double-digested with Xho I and Nco I, and the obtained ftsZ DNA fragment was ligated to the pET-28a expression vector digested with the same restriction endonuclease, and ligated with T4 ligase at 16°C or room temperature for 4 hours or overnight, and transformed into DH5α. Plasmid pET28-ftsZ was extracted and identified by DNA sequencing and Xho I / Nco I double digestion method, then transformed into E. coli expression strain BL21 (DE3) and cultured on LB plate (50 μg / mL kanamycin) for 16-18 h (37°C). Positive transformants were screened by colony PCR; a single growing colony was inoculated in LB liquid medium and cultured at 37°C (250 rpm) for 16-18 h. The bacterial liquid was then transferred to LB medium with 50 μg / mL kanamycin and cultured at 37°C (200 rpm) until the optical density at 600 nm (OD) was 0.6. 0.4 mM IPTG was added and induced at 37°C and 250 rpm for 5 h. The culture was centrifuged at 5,000 g / min for 10 min (4°C), and the supernatant and bacteria were collected separately. The expression of FtsZ protein was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0058] The positive transformants were cultured at 37°C (200 rpm) until OD 600nm =0.6, induced at different temperatures (16°C, 25°C and 37°C), different concentrations (0.2mM, 0.4mM and 0.6mM) of IPTG or at different times (1h, 3h, 5h and 7h). At each time point, induced bacterial samples (2mL) were collected and centrifuged at 5,000g / min for 10min at 4°C and analyzed by 12% SDS-PAGE.

[0059] Isolate the expressed target protein: add 2 mL of lysis buffer (containing 1 μL 1,000 U / mL DNase I and 2 μL 50 mg / mL lysozyme) to 100 mg of cell pellet and homogenize on a sonicator (JY92-IIN) at 4°C for 10 min (5 s per cycle and 10 s interval) at powers of 227.5 W, 162.5 W, and 97.5 W. Keep the bacterial solution in an ice bath during the sonication. Centrifuge the lysed bacterial solution at 4°C (10,000 g, 15 min) and collect the supernatant and precipitate. Analyze the recombinant protein by 12% SDS-PAGE and lab image software.

[0060] Recombinant FtsZ protein was isolated and purified using the His-tagged inclusion body protein purification kit (CWBIO). The bacterial pellet was resuspended in binding buffer and then centrifuged at 4°C (10,000 g, 20 min). The supernatant was separated, filtered with a filter membrane (pore size 0.22 μm), and then loaded onto Ni 2+ -NTA affinity column. FtsZ protein was eluted with different concentrations of imidazole solution (250-750mM); after dialysis with ultrapure water on a magnetic stirrer (MS-01H) for 24-48h, it was freeze-dried in a vacuum freeze dryer (LGJ-18) for 72h. The concentration of recombinant FtsZ protein was determined by BCA protein detection kit.

[0061] The PCR amplification results of the ftsZ gene are shown in Figure 1 .

[0062] The results of double restriction enzyme digestion of the recombinant plasmid and induced expression of the recombinant FtsZ protein can be found in Figure 2 . Figure 2 Figure A in the middle shows: pMD-FtsZ recombinant plasmid and pET-28a plasmid were double-digested by NcoI / Xho I; Lane 1: Double-digestion of pMD-FtsZ recombinant plasmid; Lane 2: Double-digestion of pET-28a plasmid; M: DNA marker; Figure 2 Figure B in the middle shows the induced expression of FtsZ at different temperatures (16-37°C); M: protein marker; Figure 2 Figure C in the middle shows the expression of FtsZ at different induction times (1-7h) and different IPTG concentrations (0.2-0.6mM); CK: pET28a empty vector; PC: bovine serum albumin; M: protein marker.

[0063] The results of the isolation and purification of recombinant FtsZ protein can be found in Figure 3 . Figure 3 Figure A in the middle indicates that the ultrasonic power is 227.5W; Lane 1: precipitate after ultrasonication; Lane 2: supernatant after ultrasonication; Figure 3Figure B in the middle shows that the ultrasonic power is 162.5 W; Lane 1: precipitate after ultrasonication; Lane 2: supernatant after ultrasonication; Figure 3 Figure C in the middle shows that the ultrasonic power is 97.5W; Lanes 1 and 3: precipitate after ultrasonication; Lanes 2 and 4: supernatant after ultrasonication; Figure 3 Figure D in the middle shows the effect of different imidazole concentrations on the elution of FtsZ; Lane 1: flow-through peak; Lane 2: 250 mM imidazole; Lane 3: 300 mM imidazole; Lane 4: 350 mM imidazole; Lane 5: 400 mM imidazole; Lane 6: 450 mM imidazole; Lane 7: 500 mM imidazole; Lane 8: 550 mM imidazole; Lane 9: 600 mM imidazole; Lane 10: 650 mM imidazole; Lane 11: 700 mM imidazole; Lane 12: 750 mM imidazole; Figure 3 Figure E in the middle shows the effect of different elution times on the elution of FtsZ. Lane 1: precipitate after ultrasound; Lane 2: supernatant after ultrasound; Lane 3: flow-through peak; Lane 4: eluted once with 500mM imidazole; Lane 5: eluted twice with 500mM imidazole; Lane 6: eluted three times with 500mM imidazole; Lane 7: eluted four times with 500mM imidazole; Lane 8: eluted five times with 500mM imidazole; Lane 9: eluted six times with 500mM imidazole; M: protein marker.

[0064] Figure 1 and Figure 2 Figure A in the middle shows that the size of the ftsZ gene fragment is about 1200 bp.

[0065] Figure 2 Figures B and C in the middle show that the optimal induction temperature is 37°C and the induction time is 5h; the optimal IPTG induction concentration is 0.2mM; and the molecular weight of the recombinant FtsZ protein is approximately 41kDa.

[0066] Figure 3 The results showed that the optimum ultrasonic power for bacterial lysis was 97.5 W; the optimum concentration of imidazole was 500 mM during Ni column elution, and large-volume multiple elutions were performed.

[0067] Embodiment 3:

[0068] GTPase activity of recombinant FtsZ protein: The GTPase activity of recombinant FtsZ protein was determined by malachite green phosphate assay, which is determined by measuring the ability of GTPase to release GTP free phosphate (Pi).

[0069] The steps include: diluting the GTP stock solution (5mM) by 2 times with a buffer solution (containing 50mM Tris, pH 7.5, 250mM KCl and 5mM MgCl2) and adding them sequentially to a half-volume 96-well transparent plate containing 3μM recombinant FtsZ protein (20μL / well). After incubation at 37°C for 30min, MG acid reagent (5μL / well) was added to the 96-well plate and incubated at room temperature for 10min. MG blue reagent was added to the reaction solution and incubated at room temperature for 20min. At OD 620nm The release of free Pi was determined under 4% CO 2 , and the GTPase activity of the recombinant FtsZ protein (nmol / mL / min) in the enzymatic reaction was calculated based on the phosphate standard curve. The solution without GTP was used as a negative control. The experiment was repeated three times.

[0070] GTPase activity results refer to Figure 4 Middle A. The results showed that the recombinant FtsZ protein had GTPase activity, with a maximum activity of 0.998 nmol / mL / min.

[0071] Embodiment 4:

[0072] Determination of the effect of alkaloid compounds on the GTPase activity of recombinant FtsZ protein: The recombinant FtsZ protein was incubated with different alkaloid compounds (including sophoridine, matrine, theophylline, betaine, stachydrine, tetrandrine, sanguinarine chloride, sinomenine hydrochloride, capsaicin and berberine hydrochloride) at 37°C for 30 minutes.

[0073] The steps include: adding compounds of different concentrations (3.13-50 μg / mL) to a half-volume 96-well transparent plate containing 3 μM FtsZ (10 μL / well) and 1.25 mM GTP (15 μL / well). After incubation at 37°C for 30 minutes, MG acid reagent is added to the 96-well plate (5 μL / well) and reacted at room temperature for 10 minutes. After incubation with MG blue reagent at room temperature for 20 minutes, OD 620nm The effect of the compound on the GTPase activity of the recombinant FtsZ protein was evaluated by using the PCR product. The sample without GTP solution was used as a negative control. The experiment was repeated three times.

[0074] The results of the GTPase activity effect experiment can be found in Figure 4 Figure B. The results showed that all 10 alkaloid compounds had inhibitory effects on the GTPase activity of the recombinant FtsZ protein. Among them, sophoridine had the strongest inhibitory effect on GTPase activity, with an inhibition rate of more than 60% at a concentration of 3.13 μg / mL; tetrandrine had the lowest inhibitory effect on GTPase activity.

[0075] Embodiment 5:

[0076] Determination of the antibacterial activity of alkaloid compounds: The determination method is the microbroth dilution method to determine the antibacterial activity of alkaloids. Pick a single colony of the test strain and activate it overnight, transfer 1% to MHB medium, and culture at 37°C until the logarithmic growth phase. Add bacteria (1×10 5 CFU / mL), then add a series of two-fold dilutions of the diluted peptide (0.005-10.24 mg / mL) and place it in a 37°C incubator for incubation for 18-24 hours. No bacteria (culture medium) and no alkaloids (bacterial solution) are used as blank controls and negative controls, respectively. Three replicates are set for each well, and the minimum inhibitory concentration (MIC) is used as the lowest peptide concentration at which no bacterial growth is observed. Take 10 μL of the mixed bacterial solution in MHB culture medium, spread it evenly, and culture it at 37°C for 18-24 hours. The lowest concentration at which no bacteria grow is the minimum bactericidal concentration (MBC). The test results of the antibacterial effect are shown in Table 1:

[0077] Table 1 Results of MIC and MBC values ​​of alkaloid compounds against three types of pathogens

[0078]

[0079]

[0080] Note: - Not determined.

[0081] As shown in Table 1, compared with other alkaloid molecules, sanguinarine chloride showed higher antibacterial activity against Escherichia coli, Salmonella and Staphylococcus aureus, with MIC and MBC values ​​of 0.04-0.16 mg / mL and 0.16-0.32 mg / mL, respectively; followed by tetrandrine, berberine chloride and sophoridine; matrine and sinomenine were active against individual strains; betaine, stachydrine and capsaicin were inactive against Escherichia coli, Salmonella and Staphylococcus aureus.

[0082] Embodiment 6:

[0083] Determination of the interaction between berberine hydrochloride and FtsZ protein: Recombinant FtsZ protein (128 μg / mL) was dissolved in MES buffer (50 mM Tris pH 7.5, 5 mM MgCl2 and 250 mM KCl). Two methods were used to detect the effect of different concentrations of berberine hydrochloride (1.56, 6.25 and 25 μg / mL) on the aggregation of recombinant FtsZ protein. The reaction system was: 10 μL FtsZ (128 μg / mL), 1 μL GTP and 5 μL berberine hydrochloride. The two detection methods are as follows:

[0084] i) adding berberine hydrochloride to the recombinant FtsZ protein solution and incubating for 30 minutes; adding GTP solution (0.25-8 mM) and reacting for 30 minutes.

[0085] ii) Incubate berberine hydrochloride with FtsZ and GTP for 1 hour, and analyze the effect of berberine hydrochloride on the polymerization reaction of recombinant FtsZ protein by 12% SDS-PAGE.

[0086] Test results see Figure 5 .in, Figure 5 Figure A in the middle shows the effect of different concentrations of GTP on FtsZ aggregation; Lane 1: 0 mM GTP; Lane 2: 0.25 mM GTP; Lane 3: 0.5 mM GTP; Lane 4: 1 mM GTP; Lane 5: 2 mM GTP; Lane 6: 4 mM GTP; Lane 7: 8 mM GTP; M: protein marker; Figure 5 Figure B in the middle shows the effect of different concentrations of berberine hydrochloride on FtsZ aggregation; Lanes 1-3: berberine hydrochloride was incubated with FtsZ for 0.5h, and then GTP was added and incubated for 0.5h; Lanes 4-6: berberine hydrochloride was co-incubated with FtsZ and GTP for 1h; Lanes 1 and 4: 1.56μg / mL berberine hydrochloride; Lanes 2 and 5: 6.25μg / mL berberine hydrochloride; Lanes 3 and 6: 25μg / mL berberine hydrochloride; Lane 7: Control (FtsZ+GTP); 8: Control (FtsZ); M: protein marker. Figure 5 The results in Figure A show that FtsZ aggregation is slightly enhanced with the increase of GTP concentration (0.25-8mM); 1mM GTP was selected for the aggregation reaction of FtsZ. Figure 5 The results in Figure B show that 1.56μg / mL, 6.25μg / mL and 25μg / mL berberine hydrochloride all inhibited the polymerization of recombinant FtsZ protein; 1.56μg / mL berberine hydrochloride had the strongest effect on FtsZ aggregation when directly co-incubated for 1h.

[0087] The binding of berberine hydrochloride (ligand) to FtsZ (receptor) was analyzed by using AutoDock software and molecular docking (MD) simulation was performed. The results are shown in Figure 5 Figures C and D in the middle. The results showed that berberine hydrochloride binds to the FtsZ protein molecule (Asp253) through hydrogen bonding.

[0088] The above-mentioned embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of the invention patent protection. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing FtsZ protein, characterized in that: The steps include: building ftZ A biomaterial for gene expression; using IPTG to induce the expression of the biomaterial to obtain FtsZ protein inclusion bodies; ultrasonically treating the FtsZ protein inclusion bodies, separating and purifying, and obtaining FtsZ protein; the concentration of the IPTG induction is 0.1-0.6mM; the time of the IPTG induction is 4-5h; the temperature of the IPTG induction is 37°C; The use ftZ The biomaterial for gene expression is a material containing ftZ Gene expression vector and engineered bacteria; the engineered bacteria are positive transformants of Escherichia coli BL21-DE3 transformed by the expression vector; The FtsZ protein was expressed in E. coli BL21-DE3 in the form of inclusion bodies, and its C-terminus had six His residues. 2+ -NTA affinity column purification; Said ftZ The sequence of the gene is shown in SEQ ID NO.

1.

2. The method for preparing FtsZ protein according to claim 1, characterized in that: The temperature of the ultrasonic treatment is 0-4°C; the total duration of the ultrasonic treatment is 8-12 minutes; the cycle of the ultrasonic treatment is: ultrasonic treatment for 4-6 seconds and an interval of 8-12 seconds; the power of the ultrasonic treatment is 80-250W.

3. The method for preparing FtsZ protein according to claim 1 or 2, characterized in that: The separation and purification uses Ni 2+ -NTA affinity column and 400-750 mM imidazole eluent; the separation and purification retains proteins with a molecular weight > 14 kDa.

4. Use of the method for preparing the FtsZ protein according to any one of claims 1 to 3 in screening antibacterial drugs.

5. A method for screening a broad-spectrum antibacterial drug, characterized in that: The steps include: S1. incubating the FtsZ protein prepared by the FtsZ protein preparation method according to any one of claims 1 to 3 with GTP and a compound to be screened; S2. Detect the GTPase activity of the FtsZ protein, and judge the broad-spectrum antibacterial properties of the compounds to be screened based on the GTPase activity of the FtsZ protein: if the GTPase activity of the FtsZ protein decreases, the compounds to be screened have antibacterial effects.

6. The method for screening broad-spectrum antibacterial drugs according to claim 5, characterized in that: The final concentration of GTP in the co-incubation is 0.25-4 mM.

7. The method for screening broad-spectrum antibacterial drugs according to claim 5, characterized in that: The co-incubation time is 20-40 min.

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