Cracking protein gene for cracking shigella and application thereof

Abstract

The invention discloses a cleavage protein gene for cleavage of Shigella and application of the cleavage protein gene, and belongs to the technical field of gene engineering. According to the invention, a gene with a protein coding and splitting function in the Shigella freundii microbacteriophage SGF3 is found for the first time, and codon optimization is carried out on the gene to obtain a nucleotide sequence shown as SEQ ID NO.2. The invention also discloses a preparation method of the Shigella freundii microbacteriophage SGF3. The codon-optimized gene sequence can successfully express the cleavage protein LysSGF3 with biological activity in a prokaryotic expression system (escherichia coli), and the unoptimized original gene sequence cannot be expressed in the prokaryotic system. The lysed protein LysSGF3 prepared by the method disclosed by the invention has better bactericidal activity, has a wider lysis spectrum compared with a bacteriophage, and has a good lysis effect on gram-negative bacteria such as shigella, escherichia coli and edwardsiella and gram-positive bacteria such as staphylococcus aureus and bacillus; good data support is provided for further research and development of medicines for treating bacterial malaria.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a lytic protein gene for lysing Shigella and application thereof. Background Art

[0002] Bacillary dysentery, also known as shigellosis, is an acute intestinal infectious disease characterized by Shigella (Shigella dysenteriae) invading the colon mucosa and epithelial cell inflammation and exudation. The main clinical manifestations are fever, abdominal pain, diarrhea, tenesmus, mucus, pus and blood in the stool, accompanied by systemic toxemia symptoms. In severe cases, it can cause septic shock and (or) toxic encephalopathy. It is prevalent all over the world, especially in developing countries. The incidence of bacillary dysentery in my country has been among the top three infectious diseases for many years. Therefore, how to effectively control the frequent occurrence of bacillary dysentery is an important public health issue currently facing my country.

[0003] Now, extensive bacterial resistance caused by the abuse of antibiotics is very common, which has posed a direct threat to human survival and health. In the United States alone, there are more than 2.8 million cases of drug-resistant infections and 35,000 deaths each year. In 2019, at least 1.2 million people died from bacterial antibiotic-resistant infections worldwide, and it is estimated that by 2050, the total number of deaths from antibiotic-resistant organisms worldwide will reach 10 million. Therefore, it has become a top priority to seek new methods for treating pathogens that can replace antibiotics. In the search for alternative strategies to prevent and control resistant bacterial infections, phage therapy has become a preferred option. However, the use of phages alone may face a series of problems such as too narrow antibacterial spectrum, phage resistance and immunogenicity. The lysis spectrum of lytic proteins is wide, the bactericidal effect is good, and bacteria are not easy to develop resistance to them, so they have great potential for application in antibacterial. Therefore, lytic proteins can well solve the problem of limited application range of phages in terms of bactericidal.

[0004] The applicant previously collected environmental samples from a certain urban sewage treatment plant in Beijing and isolated a phage with Shigella flexneri CGMCC 1.10599 as its host bacteria (112646784A, a Shigella flexneri microphage SGF3 and its application). The phage belongs to the rare Microviridae family and cannot lyse strains other than Shigella. It has host specificity. The functions of three of the seven open reading frames (ORFs) of the protein coding sequences of phage SGF3 are unknown. Therefore, it is of great research value to explore the protease with lytic function in phage SGF3 and to express and purify it in vitro as an antibacterial protein preparation.

[0005] In summary, how to provide a lytic protein that lyses Shigella is of great significance in the field of new drug development for the treatment of bacterial malaria. Summary of the invention

[0006] In view of the above problems, the present invention provides a lytic protein gene for lysing Shigella and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] Application of the 4033-4410 fragment of the whole gene of Shigella flexneri microphage SGF3 in encoding a lytic protein for lysing Shigella.

[0009] As an inventive concept identical to the above technical solution, the present invention also requests protection for a lytic protein gene Lys for lysing Shigella, which is obtained by codon optimization of the 4033-4410 fragment of the complete gene of the Shigella flexneri microphage SGF3 (GenBank accession number MN266305.1), and the specific nucleotide sequence is shown in SEQ ID NO.2.

[0010] Specifically, codon optimization was performed based on the usage frequency of codons in Escherichia coli using the software GeMS.

[0011] As an inventive concept identical to the above technical solution, the present invention also requests protection for a lytic protein LysSGF3, which is encoded by a nucleotide sequence as shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.3.

[0012] As an inventive concept identical to the above technical solution, the present invention also requests protection for a method for preparing a lytic protein LysSGF3, comprising the following steps: (1) constructing a recombinant expression vector pET30a-Lys using the sequence shown in SEQ ID NO.2, and then constructing a recombinant strain through the recombinant expression vector pET30a-Lys for cultivation and propagation; (2) inducing the recombinant strain to express the protein; and (3) isolating and purifying to obtain the lytic protein LysSGF3.

[0013] Preferably, the method for constructing the recombinant expression vector in step (1) is as follows: the pET30a plasmid and the Lys gene shown in SEQ ID NO.2 are double-digested with NdeI and XhoI respectively, the double-digested products are connected and transformed into Escherichia coli, positive clones are screened, and plasmids are extracted to obtain the recombinant expression vector pET30a-Lys.

[0014] Preferably, the recombinant strain construction method in step (1) is as follows: the recombinant expression vector pET30a-Lys is transformed into competent Escherichia coli to obtain the recombinant strain.

[0015] As an inventive concept identical to the above technical solution, the present invention also requests protection for the use of the 4033-4410 fragment of the above Shigella flexneri microphage SGF3 full gene or the above Shigella lytic protein gene Lys or the above lytic protein LysSGF3 in the preparation of drugs for treating bacterial malaria.

[0016] It can be seen from the above technical scheme that compared with the prior art, the present invention is the first to discover a gene in the Shigella flexneri microphage SGF3 that has the function of encoding a lytic protein, and has performed codon optimization to obtain a nucleotide sequence as shown in SEQ ID NO.2. Based on the sequence, a recombinant expression vector pET30a-Lys was successfully constructed, and the recombinant expression vector was introduced into Escherichia coli competent cells. After induced expression, a lytic protein LysSGF3 with biological activity was obtained, and the in vitro expression of the protein was achieved, while the unoptimized original gene sequence could not be expressed in the Escherichia coli system.

[0017] The lytic protein LysSGF3 prepared by the method of the present invention has good bactericidal activity and a wider lysis spectrum than bacteriophages. It has a good lysis effect on gram-negative bacteria such as Shigella, Escherichia coli, and Edwardsiella, and gram-positive bacteria such as Staphylococcus aureus and Bacillus. It is not easy to induce bacteria to produce drug resistance, is safe and efficient, and provides good technical support for the research and development of phage-derived antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :The comparison diagram of the gene sequence of hypothetical protein NGUA29_02817 and the bacteriophage Coliphage NC7 genome (GenBank: DQ079886.1) from 662bp to 1039bp in the NCBI database;

[0019] Figure 2 : PCR electrophoresis of the E. coli recombinant in Example 2, 1: D2000 DNA Ladder (M1060-50T, Solarbio); 2: E. coli recombinant;

[0020] Figure 3: SDS-PAGE image of protein expression of recombinant plasmid pET30a-Lys, wherein, M: protein Marker (C540010-0250, Bio-Industry), 1: cleavage protein LysSGF3 cell disruption solution without IPTG, 2: cleavage protein LysSGF3 cell disruption supernatant without IPTG, 3: cleavage protein LysSGF3 cell disruption precipitate without IPTG, 4: cleavage protein LysSGF3 cell disruption solution with IPTG, 5: cleavage protein LysSGF3 cell disruption supernatant with IPTG, 6: cleavage protein LysSGF3 cell disruption precipitate with IPTG;

[0021] Figure 4 : SDS-PAGE image of protein expression of recombinant plasmid pET30a-Lys after purification, wherein, M: protein marker (C540010-0250, Bio-Industry), 1: purified cleavage protein LysSGF3 solution;

[0022] Figure 5 :Construction of SDS-PAGE images of protein expression of recombinant plasmids without codon optimization after IPTG induction under different conditions, where A: After adding IPTG to a final concentration of 0.5mmol / L, shake culture was carried out at 16℃ and 180rpm for 16h, B: After adding IPTG to a final concentration of 0.5mmol / L, shake culture was carried out at 25℃ and 180rpm for 10h, C: After adding IPTG to a final concentration of 0.5mmol / L, shake culture was carried out at 37℃ and 180rpm for 4h, M : Protein Marker (C540010-0250, Bio-Industry), 1: non-codon-optimized recombinant bacterial cell disruption solution without IPTG, 2: non-codon-optimized recombinant bacterial cell disruption supernatant without IPTG, 3: non-codon-optimized recombinant bacterial cell disruption precipitate without IPTG, 4: non-codon-optimized recombinant bacterial cell disruption solution with IPTG, 5: non-codon-optimized recombinant bacterial cell disruption supernatant with IPTG, 6: non-codon-optimized recombinant bacterial cell disruption precipitate with IPTG;

[0023] Figure 6 : The results of the inhibitory effect test of the lytic protein LysSGF3 on the host bacteria Shigella flexneri CGMCC 1.10599, wherein 1: 0.6 mg / ml lytic protein LysSGF3, 2: PBS buffer, 3: 0.1 mg / ml lytic protein LysSGF3, 4: PBS buffer;

[0024] Figure 7:The MIC value detection diagram of the lytic protein LysSGF3 against the host bacteria Shigella flexneri CGMCC 1.10599 by 96-well plate method, wherein 1: PBS buffer + bacterial suspension, 2-11: different concentrations of lytic protein LysSGF3 + bacterial suspension, 12: MH medium;

[0025] Figure 8 : MH plate coating test lysis protein LysSGF3 host bacteria Shigella flexneri CGMCC 1.10599 MBC value detection diagram, wherein, 1: PBS buffer + bacterial suspension, 2-11: different concentrations of lysis protein LysSGF3 + bacterial suspension, 12: MH culture medium;

[0026] Fig. 9 : Analysis of the changes in the lysis effect of different concentrations of lysis protein LysSGF3 on the host bacteria Shigella flexneri CGMCC 1.10599 over time;

[0027] Fig.10 :Secondary structure analysis of cleavage protein LysSGF3;

[0028] Fig.11 :Tertiary structure analysis of cleavage protein LysSGF3;

[0029] Fig.12 :Analysis results of transmembrane domain of lytic protein LysSGF3. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0032] The following reagents are illustrative:

[0033] LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L.

[0034] LB liquid medium containing 50 μg / ml kanamycin: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, kanamycin 50 μg / ml.

[0035] MH medium: beef powder 2g / L, soluble starch 1.5g / L, acid hydrolyzed casein 17.5g / L, pH adjusted to neutral (25℃).

[0036] The solid culture medium is prepared by adding 1.5% agar to the corresponding liquid culture medium.

[0037] Example 1 Gene acquisition and codon optimization of lytic protein

[0038] According to the genome annotation of Shigella microphage SGF3 (GenBank accession number MN266305.1) obtained from the literature (“Isolation and characterization of SGF3, novel Microviridaephage infecting Shigella fexneri”), it was found that Shigella microphage SGF3 contains three proteins of unknown function, namely gpC (GenBank accession number QFG06253.1), hypothetical protein NGUA29_02817 (GenBank accession number QFG06255.1) and hypothetical protein KP700603_26274 (GenBank accession number QFG06256.1). The genomes of the three proteins were compared using the NCBI-BLASTn (nr / nt) database, and it was found that the hypothetical protein NGUA29_02817 of phage SGF3 had a 98.68% similarity with the 662 bp to 1039 bp (including the lytic protein) of the phage Coliphage NC7 genome (GenBank: DQ079886.1), see Figure 1 It is speculated that the hypothetical protein NGUA29_02817 (the corresponding open reading frame is ORF6) is a protein with lytic function in SGF3 phage.

[0039] Based on the sequence of the above-mentioned phage SGF3 open reading frame ORF6 (4033bp to 4410bp sequence of the whole genome of phage SGF3, as shown in SEQ ID NO.1), Nanjing GenScript Biotechnology Co., Ltd. used the software GeMS to optimize the codons of the above-mentioned basic sequence according to the usage frequency of codons in Escherichia coli. The gene sequence Lys obtained after optimization is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.3.

[0040] Example 2 Construction of recombinant expression vector pET30a-Lys

[0041] (1) Double enzyme digestion

[0042] The gene sequence Lys shown in SEQ ID NO. 2 and the pET30a vector plasmid were double-digested using restriction endonucleases NdeI and XhoI. Among them, the double enzyme digestion system of Lys (50μL) is: 35μL of Lys gene fragment, 1μL of EcoRⅠ and NotⅠ with a concentration of 15U / μL, 5μL of 10×M buffer, 8μL of ddH2O, and the conditions are: after the reaction system is fully mixed, it is incubated in a water bath at 37℃ for 3h, and then in a water bath at 80℃ for 20min to terminate the reaction; the double enzyme digestion system of pET30a vector plasmid (50μL) is: 8μL of pET-30a(+), 1μL of EcoRⅠ and NotⅠ with a concentration of 15U / μL, 5μL of 10×M buffer, 35μL of ddH2O, and the conditions are: after the reaction system is fully mixed, it is incubated in a water bath at 37℃ for 3h, and then in a water bath at 80℃ for 20min to terminate the reaction.

[0043] (2) T4 connection

[0044] The products of the double-enzyme-cut gene and double-enzyme-cut vector obtained above were connected using T4 DNA ligase. The connection system (20 μL) was: 1 μL of 10×T4 DNA Ligase with a concentration of 5 U / μL, 2 μL of double-enzyme-cut plasmid, 10 μL of double-enzyme-cut gene, 2 μL of 10×buffer, and 5 μL of ddH2O. Operation: Mix the above components and react at 16°C for 16 hours.

[0045] (3) Conversion

[0046] Take 5 μL of the ligation product of step (2) and add it to 50 μL of Escherichia coli DH5α competent cells and ice bath for 30 minutes. After taking it out, heat shock it in a 42°C water bath for 90 seconds. After quickly taking it out, ice bath it for 2 minutes, add 300 μL of LB liquid culture medium and shake culture it at 37°C and 180 rpm for 60 minutes. Then, spread the culture on an LB solid culture medium plate containing 50 μg / ml kanamycin and culture it at 37°C for 8 to 12 hours. Randomly pick several single colonies from the plate and inoculate them into LB liquid culture medium containing 50 μg / ml kanamycin. Shake and culture it at 37°C and 180 rpm for 10 to 16 hours to obtain a bacterial solution for use.

[0047] The above bacterial solution was subjected to PCR amplification using the sequence shown in SEQ ID NO.4 (5'-TTTTCATATGATGCGGCATGCGCTCTGCGC-3') as the upstream primer and the sequence shown in SEQ ID NO.5 (5'-TTTCTCGAGAACGGCCTGTCTCATCATGG-3') as the downstream primer. The PCR amplification system (25 μL) was: 12.5 μL of 2×T5 SuperPCR Mix (Colony), 1 μL of each upstream and downstream primer, 1 μL of bacterial solution, and ddH2O to 25 μL; the PCR amplification program was: 94°C pre-denaturation for 5 min; 95°C denaturation for 1 min, 70°C annealing for 1 min, 72°C extension for 1 min, for a total of 34 cycles; and finally 72°C extension for 10 min. 1.5% agarose gel electrophoresis was performed to identify the expected size bands, such as Figure 2 As shown, the target band appeared at 378 bp, and its size was consistent with the length of the target gene. It was predicted that the recombinant expression vector pET30a-Lys was successfully constructed.

[0048] After the recombinant expression vector pET30a-Lys was successfully constructed, a plasmid extraction kit (Omega Bio-Tek, Guangzhou Cuilin Biotechnology Co., Ltd.) was used to extract the plasmid from the bacterial solution (refer to the corresponding instructions for operation and conditions) and stored at -20°C. The recombinant plasmid was sent to Qingke Biological Sequencing Company for sequencing, and the sequencing results and the target gene were BLAST-aligned using the NCBI database. The plasmid with the correct alignment was the recombinant expression vector pET30a-Lys.

[0049] Example 3 Induced expression and purification of cleavage protein LysSGF3

[0050] (1) Induction of cleavage protein LysSGF3

[0051] Take 5 μL of the recombinant plasmid pET30a-Lys obtained in Example 2 and add it to 50 μL of Escherichia coli BL21 (DE3) competent medium, ice bath for 30 minutes, take it out and heat shock it in a 42°C water bath for 90 seconds, quickly take it out, ice bath for 2 minutes, add 500 μL of LB liquid culture medium, shake and culture at 37°C and 180rpm for 40 minutes, then spread the culture on LB solid culture medium containing 50 μg / ml kanamycin, and invert and culture at 37°C for 8 to 12 hours; randomly pick several single colonies on the plate and inoculate them into LB liquid culture medium containing 50 μg / ml kanamycin, and shake and activate culture at 37°C and 180rpm for 10 to 16 hours.

[0052] The above bacterial liquid was inoculated into LB liquid culture medium containing 50 μg / ml kanamycin at an inoculum size of 1%, and cultured at 37°C and 180 rpm for 3 h. IPTG reagent was added to a final concentration of 0.5 mmol / L. After cultured at 37°C and 180 rpm for 4 h, the culture was centrifuged at 4000 g, the supernatant was discarded, the bacterial precipitate was washed three times with sterile PBS solution (pH 7.2, 10 mmol / L), resuspended in 5 mL of sterile PBS solution, and then ultrasonicated (ultrasonic power of 100 W, working for 5 s, interval of 5 s, 280 cycles), the ultrasonicated lysis liquid was collected, and the precipitate and supernatant were collected respectively after centrifugation at 4°C and 11000 rpm for 30 min. The precipitate was the IPTG-induced lysis protein LysSGF3 cell lysis liquid, and the supernatant obtained was the lysis protein LysSGF3.

[0053] The above bacterial liquid was inoculated into LB liquid culture medium containing 50 μg / ml kanamycin at an inoculum size of 1%, and cultured at 37°C and 180 rpm for 7 h. The culture was centrifuged at 4000 g and the supernatant was discarded. The bacterial precipitate was washed three times with sterile PBS solution (pH 7.2, 10 mmol / L), and resuspended in 5 mL of sterile PBS solution. The precipitate was ultrasonically treated (ultrasonic power of 100 W, working for 5 s, interval of 5 s, 280 cycles), and the ultrasonically treated liquid was collected. The culture was centrifuged at 4°C and 11000 rpm for 30 min, and the precipitate and supernatant were collected respectively. The corresponding cell lysis liquid of LysSGF3 cell lysis protein without IPTG induction and the cell lysis supernatant were obtained as controls.

[0054] The expression of recombinant proteins in the above liquids was detected by SDS-PAGE electrophoresis. The results are shown in Figure 3 .Depend on Figure 3 It can be seen that the cell fragmentation liquid and cell fragmentation supernatant of the cleavage protein LysSGF3 without IPTG induction have a band at 14kDa, while the cell fragmentation liquid and cell fragmentation supernatant of the cleavage protein LysSGF3 induced by IPTG have a more obvious band at 14kDa, indicating that the cleavage protein LysSGF3 can produce a large amount of soluble expression in Escherichia coli BL21 (DE3) after IPTG induction.

[0055] (2) Protein purification

[0056] The cell disruption supernatant of the above-mentioned IPTG-induced cleavage protein LysSGF3 was taken and purified using a Ni-NTA protein purification kit (BBI, purchased from Sangon Biotech (Shanghai) Co., Ltd.). The specific process is as follows: the original buffer in the Ni-NTA protein purification column was discharged, and 2 volumes of Wash Buffer were added to the purification column to balance the purification column; the sample to be purified and Wash Buffer were mixed in a volume ratio of 1:1, added to the above-balanced purification column, and the liquid was drained by gravity, and the flow-through liquid was collected and passed through the column again once; 2 volumes of Wash Buffer were added to the column for washing, and the washing was repeated several times until the absorbance of the flow-through liquid at 280 nm was close to the baseline; after the washing was completed, a new collection tube was placed under the column to prepare for protein collection, and 2 volumes of Elution Buffer were added to the column to elute the 6×His-tagged recombinant protein on the column, eluted twice, and collected and stored separately in new collection tubes. This liquid is the purified cleavage protein LysSGF3.

[0057] The concentration of the purified cleavage protein LysSGF3 was determined by BCA protein concentration determination kit (about 0.6 mg / L), and the purification of the cleavage protein LysSGF3 was detected by SDS-PAGE electrophoresis. The results are shown in Figure 4 ,Depend on Figure 4 It can be seen that the purified cleavage protein LysSGF3 solution has an obvious band at 14 kDa, indicating that the purification effect is very good.

[0058] Based on Example 2, the sequence shown in SEQ ID NO.2 was replaced with the sequence shown in SEQ ID NO.1 (without codon optimization), and other condition parameters remained unchanged to construct a recombinant expression vector without codon optimization, and the recombinant strain was constructed and propagated according to the conditions of Example 3. The bacterial liquid was inoculated into LB liquid culture medium containing 50 μg / ml kanamycin at an inoculum of 1%, and cultured at 37°C and 180rpm for 3h. Various conditions were tried for IPTG induction experiments: IPTG was added to a final concentration of 0.5mmol / L, and cultured at 16°C for 16h / 25°C for 10h / 37°C for 4h at 180rpm, and SDS-PAGE analysis was performed. The results are shown in the table. Figure 5 AC, no obvious band appeared at 14kDa by SDS-PAGE electrophoresis, indicating that the recombinant plasmid without codon optimization was not expressed in Escherichia coli BL21 (DE3). It can be seen that the original gene sequence of the lytic protein LysSGF3 was successfully expressed in vitro only after codon optimization.

[0059] Example 4 Determination of properties of cleavage protein LysSGF3

[0060] 1. Analysis of bactericidal properties

[0061] (1) Detection of the inhibitory effect of the lytic protein LysSGF3 on the host bacterium Shigella flexneri CGMCC 1.10599

[0062] The bacterial liquid of Shigella flexneri CGMCC 1.10599 in the logarithmic growth phase was evenly spread on the LB solid culture medium, and after drying, four wells of equal volume were punched on the plate with the wide end of a sterile pipette tip and marked 1-4. The protein LysSGF3 purified in Example 3 was diluted to 0.6 mg / ml and 0.1 mg / ml and then added to wells 1 and 3 respectively. PBS buffer was added to wells 2 and 4 as a comparison. The plate was placed in a 37°C incubator for overnight culture, and the formation of the inhibition zone was observed the next day. The results are shown in FIG. Figure 6 .

[0063] Depend on Figure 6 It can be seen that when the lytic protein LysSGF3 (concentration of 0.6 mg / ml) was added, a clear inhibition zone with obvious edge contours was produced; when the lytic protein LysSGF3 was added at a concentration of 0.1 mg / ml, the inhibition zone was smaller in area than when the concentration was 0.6 mg / ml, but the produced inhibition zone was still clear and had obvious contours, proving that when the concentration of the lytic protein LysSGF3 was 0.6 or 0.1 mg / ml, it had a better effect of lysing bacteria; no inhibition zone was produced when PBS solution was added.

[0064] (2) Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the lytic protein LysSGF3 against the host bacterium Shigella flexneri CGMCC 1.10599

[0065] The single colony of Shigella flexneri CGMCC 1.10599 was fully dispersed in MH liquid medium and prepared into 5×10 5 CFU / ml of the standard suspension, add 50 μL to each of the 1st to 12th wells of a sterile 96-well polyethylene plate. Use PBS buffer to dilute the purified cleavage protein LysSGF3 in Example 3 to 20 μg / ml, 50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml, 300 μg / ml, 350 μg / ml, 400 μg / ml, and 500 μg / ml, respectively, and add 50 μL to the 2nd to 11th wells in turn, add 50 μL PBS buffer to the 1st well, and only add 100 μL MH culture medium to the 12th well as a negative control. The 96-well polyethylene plate was incubated at 37°C for 16 to 20 hours, and the protein concentration was observed from low to high. The protein concentration value corresponding to the first clarified well is the MIC value. The results are shown in Table 2. Figure 7 .

[0066] Depend on Figure 7 As shown, there was obvious clarification starting from the third well, so the MIC value of the cleaved protein LysSGF3 was 50 μg / mL.

[0067] Pipette the bacterial solution from each well onto the MH plate and observe the protein concentration from low to high. The first one without colony growth is the MBC value. Figure 8 As shown, no colonies grew on the MH plate starting from the 8th figure. Therefore, the MBC value of the cleavage protein LysSGF3 was 300 μg / mL.

[0068] (3) Effect of the concentration of lytic protein LysSGF3 on the bactericidal effect

[0069] The Shigella CGMCC 1.10599 culture solution that was cultured overnight to the logarithmic growth phase was centrifuged at 8000g for 10 minutes, washed twice with 10mmol / L PBS (8.0), adjusted to OD600 = 0.8 with PBS, and added to a 96-well polyethylene plate. Different amounts of cleavage protein LysSGF3 were added to the culture solution to make the final concentration of cleavage protein LysSGF3 20, 50, 100, 200, 300μg / mL, and the total amount was 200μL. PBS was added as a blank control, and the absorbance OD was measured at different times. 600 Each condition was repeated 3 times. Fig. 9 shown.

[0070] Depend on Fig. 9 It can be seen that with the increase of time, the treatment effect of the lysis protein LysSGF3 in the first 40 minutes is very significant, and the higher the concentration of the lysis protein LysSGF3, the better the lysis effect; after 40 minutes, the treatment effect of the lysis protein LysSGF3 tends to be flat, and after treatment with 300μg / mL lysis protein, the OD600 of the bacterial suspension decreased by about 0.55 (68.75%). Comparing the lysis effects of different concentrations, considering the economic benefits, it is more appropriate to choose a lysis protein LysSGF3 concentration of 100μg / mL.

[0071] 2. Analysis of the antibacterial spectrum of the lytic protein LysSGF3

[0072] The selected bacteria are Shigella flexneri 1.10599, Shigella dysenteriae 1.1869, Shigella flexneri 1.1868), Salmonella enterica subsp. Enterica 1.10754, Salmonella typhimurium 1.1174, Escherichia coli ST100, Escherichia coli ST110, Escherichia coli ST120, Escherichia coli ST130, Escherichia coli sakai, Edwardsiella tarda 15947, Edwardsiella hoshinae 33379, Edwardsiella tarda 1K00243, Staphylococcus aureus aureus 1.2456, Staphylococcus aureus aureus 1.8721), Bacillus cereus Frankland 14579, Bacillus thuringiensis WY-197 were cultured to the logarithmic growth phase, and then the strains in the logarithmic growth phase were evenly spread on the solid LB medium with a coating rod. After drying, three wells of the same volume were punched on the plate with the wide end of a sterile gun tip, and PBS buffer and protein LysSGF3 with concentrations of 0.6 mg / ml and 0.1 mg / ml were dropped into the wells, respectively. The plate was placed upright in a 37°C incubator for overnight culture, and the formation of inhibition zones was observed, and whether inhibition zones were generated was counted. The results are shown in Table 1.

[0073] Table 1 Antibacterial spectrum analysis of lytic protein LysSGF3

[0074]

[0075]

[0076] CGMCC is the China General Microbiological Culture Collection Center, CDC is the China Center for Disease Control and Prevention, MCCC is the Marine Microbiological Culture Collection Center, ATCC is the American Cell Culture Collection Center, (+) has an inhibition zone, (-) has no inhibition zone

[0077] As shown in Table 1, the lytic protein LysSGF3 has a good lysis effect on Gram-negative bacteria such as Shigella, Escherichia coli, and Edwardsiella, but has no obvious lysis effect on Salmonella; it also has a significant lysis effect on Gram-positive bacteria such as Staphylococcus aureus and Bacillus, indicating that the host spectrum of the lytic protein LysSGF3 is relatively broad.

[0078] Example 5 Structural analysis of cleavage protein LysSGF3

[0079] (1) Based on the amino acid sequence of the cleavage protein LysSGF3 shown in SEQ ID NO.3, the amino acid composition was analyzed as shown in Table 2.

[0080] Table 2 Amino acid composition of cleavage protein LysSGF3

[0081]

[0082]

[0083] As shown in Table 2, the total number of positively charged (Arg+Lys) and negatively charged (Asp+Glu) amino acid residues of the cleavage protein LysSGF3 is 19 and 0, respectively.

[0084] Using Expasy-ProtParam (http: / / web.expasy.org / protparam / ) software, based on the amino acid sequence of the cleavage protein LysSGF3 shown in SEQ ID NO.3, the atomic composition of the protein was predicted. The results are shown in Table 3.

[0085] Table 3 Atomic composition of cleavage protein LysSGF3

[0086]

[0087] As shown in Table 3, the atomic formula of the cleavage protein LysSGF3 is: 611 H 985 N 187 O 167 S5, total number of atoms: 1955.

[0088] (2) SOPMA (Self-Optimized Prediction Method with Alignment, website: https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html) was used to predict the secondary structure model of the protein. The results are shown in Fig.10The results showed that the proportions of α-helix, extended chain, β-turn and random coil in the lytic protein LysSGF3 were 32.80%, 24.00%, 7.20% and 36.00%, respectively.

[0089] SWISS-MODEL (https: / / swissmodel.expasy.org / ) was used to predict the tertiary structure model of the protein. The results are shown in Fig.11 The tertiary structure of the protein is mainly composed of α-helix, β-turn, extended chain and random coil, and its prediction results are consistent with those of the secondary structure.

[0090] The TMHMM Server V.2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ) software was used to predict and analyze the transmembrane region of the protein. The results are shown in Fig.12 , the results showed that the cleavage protein LysSGF3 had no transmembrane domain.

[0091] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0092] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of the fragment at positions 4033-4410 of the whole gene of Shigella flexneri microphage SGF3 in encoding a lytic protein for lysing Shigella.

2. A lytic protein gene Lys for lysing Shigella, characterized in that: The fragment of positions 4033-4410 of the full gene of the Shigella flexneri microphage SGF3 described in claim 1 is obtained after codon optimization, and the specific nucleotide sequence is shown in SEQ ID NO.

2.

3. The lytic protein gene Lys for lysing Shigella according to claim 2, characterized in that: Codon optimization was performed using the GeMS software based on the codon usage frequency in Escherichia coli.

4. A lytic protein LysSGF3, characterized in that The nucleotide sequence as shown in SEQ ID NO.2 of claim 2 is used for encoding, and the encoded amino acid sequence is shown in SEQ ID NO.

3.

5. A method for preparing the cleavage protein LysSGF3 according to claim 4, characterized in that: The following steps are involved: (1) constructing a recombinant expression vector pET30a-Lys using the sequence shown in SEQ ID NO.2, and then constructing a recombinant strain using the recombinant expression vector pET30a-Lys for cultivation and propagation; (2) inducing the recombinant strain to express protein; (3) Isolate and purify the cleavage protein LysSGF3.

6. The method for preparing the lysate protein LysSGF3 according to claim 5, characterized in that: The method for constructing the recombinant expression vector in step (1): The pET30a plasmid and the Lys gene shown in SEQ ID NO.2 were double-digested with NdeI and XhoI, respectively. The double-digested products were connected and transformed into Escherichia coli. Positive clones were screened, and plasmids were extracted to obtain the recombinant expression vector pET30a-Lys.

7. The method for preparing the lysate protein LysSGF3 according to claim 5, characterized in that: The recombinant strain construction method of step (1) is as follows: the recombinant expression vector pET30a-Lys is transformed into the competent state of Escherichia coli to obtain the recombinant strain.

8. Use of the fragment at positions 4033-4410 of the whole gene of Shigella flexneri microphage SGF3 according to claim 1 or the lytic protein gene Lys for lysing Shigella according to claim 2 or the lytic protein LysSGF3 according to claim 4 in the preparation of drugs for treating bacterial malaria.

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