A prokaryotic expression system of SPRR2A with antibacterial activity and its construction method and application

Abstract

The invention discloses a SPRR2A prokaryotic expression system with antibacterial activity, a construction method and an application thereof, and belongs to the technical field of preparation of antimicrobial peptides. The invention discloses a SPRR2A prokaryotic expression system with antibacterial activity, using Escherichia coli Rosetta-gami (DE3) pLysS as an expression host, and comprising a pET32a-SPRR2A recombinant plasmid. The prokaryotic expression system provided by the invention is used to successfully obtain an active SPRR2A fusion protein with high expression efficiency for the first time using a prokaryotic expression system; the secondary structure of the prepared Trx-SPRR2A protein is mainly β-folding and random coiling, and contains five pairs of disulfide bonds possessed by the SPRR2A obtained by the eukaryotic expression system, and has excellent thermal stability and serum stability; it exhibits a significant inhibitory effect on the growth of Gram-positive bacteria, and as the concentration of the Trx-SPRR2A fusion protein increases, the antibacterial effect gradually increases; the prokaryotic expression system construction method is simple and easy to operate, has mild conditions, is green and environmentally friendly, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptide preparation, and in particular to a SPRR2A prokaryotic expression system with antimicrobial activity, a construction method and an application thereof. Background Art

[0002] Antibiotics play a vital role in fighting bacterial infectious diseases. By killing or inhibiting the growth of bacteria, they not only improve the treatment effect of infectious diseases, but also significantly improve the survival rate of patients. However, the widespread use and abuse of antibiotics has also brought about a problem that cannot be ignored - bacterial resistance. The increase in multidrug-resistant pathogens has made traditional treatments ineffective, posing an unprecedented challenge to public health. Faced with this severe challenge, scientists are actively engaged in the research and development of new candidate therapeutic drugs to provide new possibilities for future anti-infection treatments.

[0003] Among them, antimicrobial peptides have been widely used in many fields due to their remarkable advantages such as excellent antibacterial properties, low risk of drug resistance, broad biological activity, and ability to effectively fight a variety of pathogens (including bacteria, fungi, viruses, parasites and tumor cells).

[0004] When various pathogens in nature invade microorganisms, microorganisms will activate their self-defense mechanism, thereby producing antimicrobial peptides. Antimicrobial peptides are named Antibacterial peptides, ABPs (Chinese name for antimicrobial peptides) because of their outstanding antibacterial function. Subsequently, researchers found that antimicrobial peptides not only have a strong antibacterial effect on Gram-positive and Gram-negative bacteria, but some antimicrobial peptides also have a strong destructive power against some viruses, fungi, parasites and even cancer cells. Therefore, researchers prefer to call this type of active polypeptide "antimicrobial peptide", which means antimicrobial peptide.

[0005] Natural antimicrobial peptides have disadvantages such as difficulty in obtaining, low efficiency, high technology and cost required, and difficulty in large-scale production. Researchers have developed chemical synthesis, genetic engineering synthesis and other methods to synthesize antimicrobial peptides, thereby meeting people's needs for antimicrobial peptide production and cost savings, and can also improve the function of antimicrobial peptides to a certain extent.

[0006] Small proline-rich protein 2A (SPRR2A) is a proline-rich enteric bactericidal protein containing five pairs of intrachain disulfide bonds that was successfully expressed using the Sf9 insect cell expression system. During helminth infection, SPRR2A targets Gram-positive bacteria through membrane permeation, thereby protecting the intestinal barrier. However, obtaining active SPRR2A through prokaryotic expression strategies remains a huge challenge. Summary of the invention

[0007] The purpose of the present invention is to provide a SPRR2A prokaryotic expression system with antibacterial activity and a construction method and application thereof, so as to make up for the current lack of SPRR2A fusion protein with antibacterial activity expressed by a prokaryotic expression system.

[0008] To achieve the above object, the present invention provides a prokaryotic expression system of SPRR2A with antibacterial activity, wherein the prokaryotic expression system uses Escherichia coli Rosetta-gami (DE3) pLysS as an expression host and comprises a pET32a-SPRR2A recombinant plasmid.

[0009] Preferably, the amino acid sequence of the Trx-SPRR2A protein obtained by the prokaryotic expression system is as shown in SEQ ID NO.7.

[0010] A method for constructing a prokaryotic expression system of SPRR2A with antibacterial activity as described above comprises the following steps:

[0011] S1. Construction of pET32a-SPRR2A recombinant plasmid

[0012] The pUC57-SPRR2A vector synthesized by the company was used as a template, SPRR2A-F and SPRR2A-R were used as primers for PCR amplification, the PCR product was template digested, and the SPRR2A gene fragment was recovered and purified;

[0013] Using pET32a vector as template and pET32a-F and pET32a-R as primers, PCR amplification was performed, the PCR product was template digested, and the pET32a vector fragment was recovered and purified;

[0014] The SPRR2A gene fragment was connected with the pET32a vector fragment by seamless cloning to obtain the recombinant plasmid pET32a-SPRR2A;

[0015] S2. Transform the expression host to obtain an engineered bacterium capable of heterologously expressing the pET32a-SPRR2A recombinant plasmid

[0016] The volume ratio of expression host to recombinant plasmid was 5:1;

[0017] S3, heterologous expression pET32a-SPRR2A recombinant plasmid

[0018] The engineered bacteria obtained in step S2 were cultured and expanded. When the OD 600 When the value is close to 1.0, IPTG is added to induce expression;

[0019] S4. Collect the expression product and purify it to obtain the Trx-SPRR2A protein with antibacterial activity.

[0020] Preferably, in step S1, the sequence of SPRR2A-F is shown as SEQ ID NO.1, the sequence of SPRR2A-R is shown as SEQ ID NO.2, the sequence of pET32a-F is shown as SEQ ID NO.3, and the sequence of pET32a-R is shown as SEQ ID NO.4.

[0021] Preferably, in step S2, the transformation of the expression host is carried out by heat shock transformation.

[0022] Preferably, in step S3, the method for culturing and expanding the engineered bacteria obtained in step S3 is: culturing with LB liquid culture medium containing Amp resistance at 37° C., and the working concentration of IPTG is 0.5 mM.

[0023] Preferably, in step S4, the protein is purified using a nickel ion column, washed with a wash buffer containing 50 mM imidazole, and the target protein is eluted with a wash buffer containing 200 mM imidazole.

[0024] An application of the prokaryotic expression system as described above in the preparation of SPRR2A antimicrobial peptides with antimicrobial activity.

[0025] An application of the prokaryotic expression system construction method as described above in the preparation of SPRR2A antimicrobial peptide with antimicrobial activity.

[0026] Therefore, the present invention provides a prokaryotic expression system of SPRR2A with antibacterial activity, a construction method and an application thereof, and the specific technical effects thereof are as follows:

[0027] (1) Using the prokaryotic expression system provided by the present invention, an active and highly efficient SPRR2A fusion protein, Trx-SPRR2A protein, was successfully obtained for the first time using the prokaryotic expression system. The yield of Trx-SPRR2A protein reached 7.18 mg / L, which contributed to significantly reducing the preparation cost of SPRR2A fusion protein and improving the yield of SPRR2A fusion protein;

[0028] (2) The secondary structure of the Trx-SPRR2A protein prepared by the prokaryotic expression system provided by the present invention is mainly β-folding and random coiling, including five pairs of disulfide bonds formed by eukaryotic expression of SPRR2A, and can maintain good thermal stability in the range of 25 to 80°C, and has excellent serum stability at pH values ​​of 6.5 and 7.4;

[0029] (3) The Trx-SPRR2A protein prepared by the prokaryotic expression system provided by the present invention can destroy the bacterial cell membrane, thereby showing a significant inhibitory effect on the growth of Gram-positive bacteria, and the antibacterial effect gradually increases with the increase of the concentration of the SPRR2A fusion protein;

[0030] (4) The prokaryotic expression system construction method provided by the present invention is simple and easy to operate, has mild conditions, is green and environmentally friendly, and is suitable for industrial production.

[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0033] Figure 1 This is an electrophoretic diagram of the linearized vector pET32a and the amplified SPRR2A gene in Example 1 of the present invention, wherein M is a marker, lane 1 is the linearized vector pET32a, and lane 2 is the SPRR2A gene;

[0034] Figure 2 is an electrophoresis diagram of PCR products using universal primers (a) and specific primers (b) after amplification of a single clone selected in Example 1 of the present invention, and c is a comparison diagram of sequencing results of the recombinant plasmid pET32a-SPRR2A, wherein M is a marker, lanes 1-4 in a are PCR identification results using universal primers, and lanes 1-3 are PCR identification results using specific primers;

[0035] Figure 3 It is the SDS-PAGE test result in Example 3 of the present invention, wherein M represents a protein marker with a molecular weight range of 10 to 150 kDa; lane 1 is the supernatant produced by lysing Rosetta-gami (DE3) pLysS bacteria containing empty plasmid pET32a after IPTG induction; lane 2 presents the supernatant after lysing Rosetta-gami (DE3) pLysS bacteria containing recombinant plasmid pET32a-SPRR2A without IPTG induction; lanes 3, 4, and 5 are the Rosetta-gami (DE3) pLysS bacteria lysate, supernatant, and precipitate containing recombinant plasmid pET32a-SPRR2A after IPTG induction, respectively;

[0036] Figure 4The SDS-PAGE electrophoresis results (a) of different elution conditions in Example 4 of the present invention and the SDS-PAGE results (b) after the purification of the Trx-SPRR2A protein in Experimental Test 1, wherein lane M represents a 14-120 kDa Marker, lanes 1-8 of a are target proteins eluted with 8 mL of Wash buffer containing 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, 200 mM, and 300 mM, respectively; lane 1 in b is the purified Trx-SPRR2A;

[0037] Figure 5 is the mass spectrometry analysis result in Experimental Test 2 of the present invention;

[0038] Figure 6 are CD spectra of Trx-SPRR2A dissolved in PB buffer at pH 7.4 (a) and MES buffer at pH 5.5 (b) at 25°C in Experimental Test 3 of the present invention;

[0039] Figure 7 This is the temperature change curve of Trx-SPRR2A in the third experimental test of the present invention, a is Trx-SPRR2A dissolved in 5mM PB buffer at pH 7.4; b is Trx-SPRR2A dissolved in 10mM MES buffer at pH 5.5;

[0040] Figure 8 It is the mass spectrometry analysis result in Experiment Test 4 of the present invention;

[0041] Fig. 9 It is the result of the serum stability test of Trx-SPRR2A protein in Experiment Test 5 of the present invention, wherein a is the SDS-PAGE test result after 20 μM Trx-SPRR2A and a cell culture medium with a pH value of 7.4 and 10% fetal bovine serum were incubated at 37°C for different times (0, 2, 6, 12, 24, 48h), c is the SDS-PAGE test result after 20 μM Trx-SPRR2A and a cell culture medium with a pH value of 7.4 and 10% fetal bovine serum were incubated at 37°C for different times (0, 2, 6, 12, 24, 48h), lane M is a protein marker with a molecular weight in the range of 20 to 200 kDa, the red arrow in the figure indicates the position of Trx-SPRR2A, b and d are the quantitative analysis results of the target bands in a and c, respectively;

[0042] Fig.10 is a graph showing the antibacterial effect of Trx-SPRR2A on four bacteria detected by the plate colony counting method in the sixth experimental test of the present invention, and be is the quantitative analysis result of a;

[0043] Fig.11These are six SEM images of the experimental test of the present invention;

[0044] Fig.12 This is the observation result of the fluorescent inverted microscope in the sixth experimental test of the present invention, wherein a is Enterococcus faecalis, b is Listeria monocytogenes, c is Staphylococcus aureus, and d is Citrobacter rodentium;

[0045] Fig.13 It is the statistical result of PI intake in the sixth experimental test of the present invention. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0047] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0048] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0049] The BHI culture medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0050] Embodiment 1

[0051] The pET32a-SPRR2A recombinant plasmid was constructed. The specific steps are as follows:

[0052] S1.1. Use the pUC57-SPRR2A vector synthesized by the company as a template, SPRR2A-F (sequence as shown in SEQ ID NO.1) and SPRR2A-R (sequence as shown in SEQ ID NO.2) as primers, the reaction system shown in Table 1, and the amplification program shown in Table 2 for PCR amplification.

[0053] SEQ ID NO.1CTCGACGCTAACCTGGCCATGTCTTACCAGCAGCAG

[0054] SEQ ID NO.2GTGGTGGTGGGTGCTCGAGTTTAGATTTCGGCGGGTA

[0055] Table 1

[0056]

[0057]

[0058] Table 2

[0059]

[0060] S1.2. Add 1 μL Dpn1 and 5.1 μL 10× buffer to the PCR product tube, then react at 37°C for 2 h to digest the template. Then, perform 1% agarose gel electrophoresis on the amplified product. The results are as follows: Figure 1 As shown in lane 2, where M is a marker.

[0061] S1.3, using pET32a vector as template, pET32a-F (sequence as shown in SEQ ID NO.3) and pET32a-R (sequence as shown in SEQ ID NO.4) as primers, the reaction system shown in Table 1, and the amplification program shown in Table 2 (except that ③ is 60°C, 20s, ④ is 72°C, 1min), the pET32a vector was linearly amplified, the template was digested and purified in the same way as step S1.2, and then the amplified product was subjected to 1% agarose gel electrophoresis. The results are shown in FIG. Figure 1 As shown in lane 1, where M is a marker.

[0062] SEQ ID NO.3CTCGAGCACCACCACCACCACCACTGAG

[0063] SEQ ID NO.4GGCCAGGTTAGCGTCGAGGAACTCTTTC

[0064] Depend on Figure 1 It can be seen that obvious electrophoresis bands appeared between 5000bp-8000bp and 100bp-250bp in lane 1 and lane 2, respectively, which is consistent with the theoretical values ​​of the linearized vector pET32a (5698bp) and the target gene SPRR2A (216bp). The electrophoresis bands were cut and purified using a recovery kit according to the attached instructions to obtain the SPRR2A gene fragment and the pET32a vector fragment.

[0065] S1.4. Construct the pET32a-SPRR2A recombinant plasmid using the seamless cloning method. Add the reaction system shown in Table 3 into a sterile 1.5 mL centrifuge tube. Mix well and place in a 50°C water bath for 20 min. After cooling, immediately place the centrifuge tube on ice to form a closed loop through pairing of homologous sequences to obtain the recombinant plasmid pET32a-SPRR2A.

[0066] Table 3

[0067] Reagents volume 2×Seamless cloning Master Mix 10.0μL <![CDATA[Sterlized ddH2O]]> 8.9μL pET32a vector fragment 0.9μL SPRR2A 0.2μL Total volume 20.0μL

[0068] S1.5. Transform the pET32a-SPRR2A recombinant plasmid constructed in step S1.4 into E. coli DH5α competent cells to achieve plasmid cloning:

[0069] (1) Take out the E. coli DH5α competent cells that were previously stored at -80°C and place them on ice to slowly thaw;

[0070] (2) Gently mix 50 μL of competent cells with 10 μL of pre-cooled pET32a-SPRR2A recombinant plasmid, and then place the mixture on ice for 30 min;

[0071] (3) Slowly and carefully transfer the mixture to a constant temperature water bath pre-set to 42°C. After 45 seconds, quickly transfer to ice and let stand for 2 minutes.

[0072] (4) In a clean bench, take 600 μL of LB liquid medium without added resistance and transfer it to the mixture on ice in (3), then shake in a 37°C constant temperature shaker for 1 h to revive, with the speed set to 210 rpm;

[0073] (5) The revived bacterial solution was centrifuged at 4000 rpm for 2 min, 400 μL of supernatant was discarded, and the remaining supernatant was mixed with the bacterial pellet and evenly spread on the surface of LB solid culture medium containing 100 ng / μL Amp. It was then placed in a constant temperature incubator and cultured overnight at 37°C.

[0074] S1.6. Randomly pick 10 monoclonal colonies, add them to 20 μL LB medium and mix them thoroughly. Then, take out 1 μL from each mixed solution and use universal primers T7 (sequence as shown in SEQ ID NO.5) and T7-TER (sequence as shown in SEQ ID NO.6) as primers, the reaction system shown in Table 1, and the amplification program shown in Table 2 (except that ③ is 50°C, 20s) to perform PCR. The PCR products were subjected to agarose gel electrophoresis, and the bacterial solution corresponding to the brightest band of 721 bp was selected and inoculated into a test tube containing 10 mL LB medium and cultured at 37°C overnight.

[0075] SEQ ID NO.5TAATACGACTCACTATAGG

[0076] SEQ ID NO.6TGCTAGTTATTGCTCAGCGG

[0077] The plasmid was extracted using a plasmid extraction kit, and pET32a-SPRR2A was used as a template. PCR amplification was performed using universal primers T7 (sequence shown in SEQ ID NO.5) and T7-TER (sequence shown in SEQ ID NO.6), the system shown in Table 1, and the amplification program shown in Table 2 (except that step ③ was 50°C and 20s). The results are shown in Figure 2 As shown in a, lanes 1-4 are 4 repeats, and all show clear bands in the range of 500bp-750bp. The lengths of these bands are consistent with the theoretical value of the fragment length between the universal primers in the pET32a-SPRR2A recombinant plasmid (721bp).

[0078] Using pET32a-SPRR2A as a template, PCR amplification was performed using specific primers SPRR2A-F (sequence shown in SEQ ID NO.1) and SPRR2A-R (sequence shown in SEQ ID NO.2), the system shown in Table 1, and the amplification program shown in Table 2, and the PCR product was subjected to agarose gel electrophoresis. The results are shown in Figure 2 As shown in b, lanes 1-3 all present clear bands in the range of 100 bp-250 bp, and the lengths of these bands are consistent with the theoretical value (216 bp) of the length of the fragment SPRR2A between the specific primers in the pET32a-SPRR2A recombinant plasmid.

[0079] Take 10 μL of the extracted pET32a-SPRR2A plasmid and send it to the company for sequencing. The results are as follows Figure 2 As shown in c, the sequencing results are consistent with the theoretical sequence of the expected target gene SPRR2A, thereby confirming that the recombinant plasmid pET32a-SPRR2A was successfully constructed.

[0080] Embodiment 2

[0081] The pET32a-SPRR2A recombinant plasmid was transformed into the expression host, Rosetta-gami (DE3) pLysS, as follows:

[0082] (1) Take out the Rosetta-gami (DE3) pLysS competent cells that were previously stored at -80°C and place them on ice to slowly thaw;

[0083] (2) Gently mix 50 μL of competent cells with 10 μL of pre-cooled pET32a-SPRR2A recombinant plasmid, and then place the mixture on ice for 30 min;

[0084] (3) Slowly and carefully transfer the mixture to a constant temperature water bath pre-set to 42°C. After 45 seconds, quickly transfer to ice and let stand for 2 minutes.

[0085] (4) In a clean bench, take 600 μL of LB liquid medium without added resistance and transfer it to the mixture on ice in (3), then shake in a 37°C constant temperature shaker for 1 h to revive, with the speed set to 210 rpm;

[0086] (5) The revived bacterial solution was centrifuged at 4000 rpm for 2 min, 400 μL of supernatant was discarded, and the remaining supernatant was mixed with the bacterial pellet and evenly spread on the surface of LB solid culture medium containing 100 ng / μL Amp. It was then placed in a constant temperature incubator and cultured overnight at 37°C.

[0087] Embodiment 3

[0088] Heterologous expression of pET32a-SPRR2A recombinant plasmid, the expressed target protein is called Trx-SPRR2A protein, the specific steps are as follows:

[0089] (1) Pick a single colony from the LB solid medium in which Rosetta-gami (DE3) pLysS was cultured overnight in Example 2 and place it in two 5 mL LB medium test tubes (containing 100 ng / μL Amp and CAP), numbered ①-②; take another test tube containing 5 mL LB liquid culture medium (containing 100 ng / μL Amp), inoculate Rosetta-gami (DE3) pLysS containing plasmid pET32a, numbered ③, and place the three test tubes in a constant temperature table shaker for 8 h, the temperature is set to 37°C, and the rotation speed is 210 rpm;

[0090] (2) The cultured bacterial liquid was inoculated into three bottles of 0.4 L LB liquid culture medium for expansion culture, numbered ④-⑥, with the temperature set to 37°C and the rotation speed set to 220 rpm;

[0091] (3) When OD 600 When the pH value was about 1.0, 200 μL of IPTG (final concentration 0.5 mM) was added to each of ④ and ⑥, and then the bacterial solutions of the three bottles ④-⑥ were cultured at 16°C and 180 rpm for 12 h;

[0092] (4) The cultured bacterial solution was centrifuged at 8000 rpm for 15 min in a large centrifuge, and then the supernatant was removed and the bacterial precipitate was collected and weighed;

[0093] (5) The weighed bacterial precipitates were mixed with 20 mM Tris buffer at a ratio of 1:12 (W / V) to prepare a uniform suspension. Subsequently, the bacterial cells were crushed using ultrasonic disruption technology at a power of 40% until the cells became transparent. During this process, the operation was carried out in a mode of 4s on and 6s off;

[0094] (6) After ultrasonic disruption, 40 μL of the bacterial solution was taken as a sample. The remaining portion was centrifuged at 12,000 rpm at 4°C for 40 min, and 40 μL of the supernatant was taken as a sample. The precipitate was resuspended in 20 mM Tris buffer at a ratio of 1:12 (W / V) to prepare the sample.

[0095] (7) The supernatant containing plasmid pET32a and induced, the supernatant containing the recombinant plasmid without the addition of IPTG inducer, the whole bacteria containing the recombinant plasmid with the addition of IPTG inducer, the supernatant and the precipitate samples were detected by SDS-PAGE.

[0096] The results are as follows Figure 3 As shown, lane 3 presents a unique target band in the region of about 20 kDa, which is very close to the expected molecular weight of Trx-SPRR2A protein. Compared with lanes 1 and 2, the band shown in lane 3 indicates that Trx-SPRR2A protein was successfully induced and expressed. The brightness of the target protein band in lane 4 is significantly higher than that in lane 5, indicating that the Trx-SPRR2A target protein is mainly expressed in a soluble form rather than in the form of inclusion bodies.

[0097] Embodiment 4

[0098] The purification conditions of the Trx-SPRR2A protein obtained in Example 3 were explored, and the specific steps were as follows:

[0099] (1) Nickel ions were subjected to three repeated column loading operations using 30 mL of 100 mM NiSO4;

[0100] (2) Equilibrate the nickel column with 40 mL of 20 mM Tris buffer;

[0101] (3) Repeat the column hanging operation twice on the protein supernatant after ultrasonic disruption and centrifugation;

[0102] (4) Pass the column with 2 times column volume of 20 mM Tris buffer to remove impurities in the sample;

[0103] (5) Explore the washing and elution concentrations of the target protein: Use different concentrations of imidazole solution to collect the protein. Specifically, use 20, 40, 60, 80, 100, 150, 200 and 300 mM imidazole solution, and use 8 mL of each concentration to ensure that the target protein is effectively separated and collected from each stage;

[0104] (6) The collected protein solution was mixed with 5× Loading buffer at a ratio of 1:4, oscillated evenly on an oscillator and boiled in a 100°C metal bath for 6 min to prepare the sample. Finally, the washing effect was observed by SDS-PAGE.

[0105] The results are as follows Figure 4 As shown in a, it was finally determined that wash buffer containing 50 mM imidazole was used for washing impurities, while wash buffer containing 200 mM imidazole was used for eluting the target protein.

[0106] (7) using 40 mL EDTA (pH 8.0) to react chemically with the nickel and protein on the nickel column, thereby removing the nickel and residual protein;

[0107] (8) Rinse the column material with 200 mL of deionized water;

[0108] (9) Nickel was plated again with 20 mL of 100 mM NiSO4 and the column was sealed.

[0109] Embodiment 5

[0110] The Trx-SPRR2A protein obtained in Example 3 was purified by the following method:

[0111] (1) Fill the chromatography column with nickel ions using 30 mL of 100 mM NiSO4;

[0112] (2) Equilibrate the nickel column with 40 mL of 20 mM Tris buffer;

[0113] (3) Repeat the column hanging operation twice on the protein supernatant after ultrasonic disruption and centrifugation;

[0114] (4) Pass the column with 2 times column volume of 20 mM Tris buffer to remove impurities in the sample;

[0115] (5) Pass 200 mL of 50 mM imidazole solution through the chromatography column to further remove impurities;

[0116] (6) eluting through the chromatography column with 40 mL of 200 mM imidazole solution to collect the target protein Trx-SPRR2A;

[0117] (7) using 40 mL of EDTA (pH 8.0) to react chemically with the nickel and protein on the nickel column, thereby achieving the purpose of removing nickel and residual protein;

[0118] (8) Rinse the column material with 200 mL of deionized water;

[0119] (9) Nickel-plating was performed again with 20 mL of 100 mM NiSO4 and the column was sealed;

[0120] (10) The eluted Trx-SPRR2A was desalted and concentrated by ultrafiltration centrifuge tube, dissolved in MES buffer, and then sterilized with a 0.22 μM filter to obtain the purified Trx-SPRR2A protein, the amino acid sequence of which is shown in SEQ ID NO.7, and the nucleotide sequence of which is shown in SEQ ID NO.8.

[0121] SEQ ID NO.7MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAMSYQQQQCKQPCQPPPVCPTPKCPEPCPPPKCPEPCPPPKCPQPCPPQQCQQKYPPVTPSPPCQSKYPPKSKLEHHHHHH

[0122] SEQ ID NO.8

[0123] ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCT GACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCAC TGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCATGTCTTACCAGCAGCAGCAGTGCAAACAGCCGTGCCAGCCGCCGCCGGTTTGCCCGACCCCGAAATGCCCGGAACCGTGCCCGCCGCCGAAATGCCC GGAACCGTGCCCGCCGCCGAAATGCCCGCAGCCGTGCCCGCCGCAGCAGTGCCAGCAGAAATACCCGCCGGTTACCCGTCTCCGCCGTGCCAGTCTAAATACCCGCCGAAATCTAAACTCGAGCACCACCACCACCACCAC

[0124] Experimental Test 1

[0125] The purified Trx-SPRR2A protein obtained in Example 5 was identified by SDS-PAGE in the following manner:

[0126] (1) The purified Trx-SPRR2A protein sample was mixed with 5× Loading buffer at a ratio of 1:4, shaken evenly on an oscillator, and the mixed sample was boiled in a metal bath at 100°C for 6 min;

[0127] (2) The protein marker and the boiled sample are loaded into the pre-prepared SDS-PAGE protein gel loading wells (15% separation gel and 5% stacking gel). When the sample is in the stacking gel, the electrophoresis instrument is operated at 60V. When the bromophenol blue indicator shows that the protein sample has reached the separation gel, the electrophoresis is continued at a constant 120V. The electrophoresis is stopped when the bromophenol blue migrates to a position 1 to 2 cm away from the front.

[0128] (3) After the electrophoresis is completed, the gel is placed in Coomassie Brilliant Blue staining solution for 2 hours. Then, the staining solution is removed; the surface of the gel is washed several times with distilled water, and then a destaining solution is added and replaced periodically until the protein bands are clearly visible;

[0129] (4) Observe and scan using a gel scanner. The results are as follows Figure 4 As shown in b, the purified Trx-SPRR2A showed a clear single band between 20-25 kDa, which was consistent with the theoretical molecular weight of Trx-SPRR2A of 21 kDa.

[0130] The purity of Trx-SPRR2A protein was analyzed using Image J image processing software, and the results showed that the purity of Trx-SPRR2A was 94.5%, indicating that the target protein can be effectively separated from a complex protein mixture by nickel column affinity chromatography. After measuring the protein concentration using the BCA method, the yield of Trx-SPRR2A was calculated to be 7.18 mg / L, indicating that the expression and purification of the protein has reached a relatively high efficiency.

[0131] Experimental Test 2

[0132] The purified Trx-SPRR2A protein obtained in Example 5 was identified by mass spectrometry in the following manner:

[0133] The purified Trx-SPRR2A band stained with Coomassie Brilliant Blue was cut off and placed in an EP tube and sent to the company for mass spectrometry analysis using a high-quality, high-sensitivity, accurate and high-resolution matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometer (MALDI-TOF / TOF).

[0134] The results are shown in Table 4 and Figure 5 As shown, the sequence coverage of Trx-SPRR2A was 89% and the molecular weight was 21547 Da, which was consistent with the theoretical value and the experimental data of SDS-PAGE, indicating that the Trx-SPRR2A protein was successfully expressed.

[0135] Table 4

[0136]

[0137] Experimental Test 3

[0138] The purified Trx-SPRR2A protein obtained in Example 5 was subjected to circular dichroism analysis as follows:

[0139] (I) Secondary structure detection of Trx-SPRR2A

[0140] (1) The purified Trx-SPRR2A was desalted and concentrated using an ultrafiltration centrifuge tube, and Trx-SPRR2A was dissolved in 5 mM PB buffer at pH 7.4 and 10 mM MES buffer at pH 5.5, respectively, to a final concentration of 15 μM.

[0141] (2) Analyze Trx-SPRR2A using circular dichroism (CD), select a cuvette with a pathlength of 0.05 cm, and scan in the wavelength range of 195 to 260 nm at 25°C and a speed of 1 nm / s;

[0142] (3) Scan the CD spectrum of Trx-SPRR2A with 5 mM PB buffer (pH 7.4) and 10 mM MES buffer (pH 5.5) as the baseline;

[0143] (4) Based on the CD spectral data, the secondary structure type and content of Trx-SPRR2A were analyzed using CDNN software.

[0144] Figure 6The CD spectra of Trx-SPRR2A at 25℃ provide preliminary information on the secondary structure of Trx-SPRR2A. In order to gain a deeper understanding of the secondary structure of Trx-SPRR2A, the CDNN software was used to process the data to obtain the proportions of various secondary structures of Trx-SPRR2A in two buffers. The results showed that when Trx-SPRR2A was present in 5mMPB buffer at pH 7.4, its secondary structure included 11.3% α-helix, 35.9% β-sheet (parallel and antiparallel), 19.5% β-turn and 34.2% random coil. When Trx-SPRR2A was present in 10mM MES buffer at pH 5.5, its secondary structure included 12.9% α-helix, 31.8% β-sheet (parallel and antiparallel), 20.1% β-turn and 34.8% random coil. It can be seen that the secondary structure of Trx-SPRR2A is mainly β-fold and random coil.

[0145] (II) Thermal stability analysis of Trx-SPRR2A

[0146] (1) The purified Trx-SPRR2A was desalted and concentrated using a 10 kD ultrafiltration centrifuge tube, and then replaced with PB buffer (pH 7.4) and MES buffer (pH 5.5), respectively, with the final protein concentration of 15 μM;

[0147] (2) collecting the CD spectrum of Trx-SPRR2A in the range of 25 to 95°C by circular dichroism at a frequency of scanning once every 5°C rise, and the heating rate of the circular dichroism spectrometer was 2°C / min;

[0148] (3) Based on the CD spectrum data, the temperature change curve of Trx-SPRR2A was drawn. The results are as follows Figure 7 As shown in the figure, the CD spectra of Trx-SPRR2A at 25-80°C showed a high degree of similarity, indicating that Trx-SPRR2A can maintain good thermal stability in this temperature range; however, when the temperature exceeds 85°C, it can be observed that the CD spectra of Trx-SPRR2A in the two buffers show obvious changes. This phenomenon shows that under high temperature conditions, the secondary structure of Trx-SPRR2A will undergo changes, indicating that Trx-SPRR2A has good thermal stability.

[0149] Experimental Test 4

[0150] The disulfide bonds of the purified Trx-SPRR2A protein obtained in Example 5 were identified as follows:

[0151] The purified, desalted, concentrated Trx-SPRR2A protein obtained in Example 5 and dissolved in MES buffer was sent to the company for disulfide bond analysis of the Trx-SPRR2A protein using high-resolution mass spectrometry - Q-Exactive (Thermo Fisher) mass spectrometry. The protein was enzymatically digested under non-reducing conditions, and the disulfide bond-containing peptides were identified using high-resolution mass spectrometry to confirm the disulfide bond connection mode.

[0152] The results are as follows Figure 8 As shown in Table 5, Trx-SPRR2A has a total of 7 pairs of disulfide bonds, whose sites are C132-C136 (or C141-C145), C172 / C172 (interchain disulfide bond), C121-C127, C132, C136, C141, C145, C150, C154, C159 / C172, C117 / C172, and C33-C36, including five pairs of disulfide bonds formed by eukaryotic expression of SPRR2A.

[0153] Table 5

[0154]

[0155]

[0156] Experimental Test 5

[0157] The purified Trx-SPRR2A protein obtained in Example 5 was tested for serum stability in the following manner:

[0158] (1) The purified Trx-SPRR2A was desalted and concentrated using an ultrafiltration centrifuge tube (10 kD). Subsequently, it was replaced with 20 mM Tris buffer;

[0159] (2) Trx-SPRR2A was added to cell culture medium at pH 6.5 and 7.4 at a final concentration of 20 μM, both of which contained 10% fetal bovine serum;

[0160] (3) Place it in a CO2 cell culture incubator, strictly control the incubation temperature to 37°C, and take it out at time points of 0, 2, 6, 12, 24, and 48 h, respectively, use 5× Loading Buffer to prepare SDS-PAGE protein samples, and perform SDS-PAGE.

[0161] The results are as follows Fig. 9 As shown, after Trx-SPRR2A was incubated in a CO2 cell culture incubator for 48 hours, clear target protein bands were successfully detected at the expected position, indicating that Trx-SPRR2A has high stability under two different pH conditions.

[0162] In order to more accurately evaluate the stability of Trx-SPRR2A, the grayscale values ​​of these protein bands were analyzed using Image J software. The results showed that after 48 hours of incubation with complete culture medium with different pH values, Trx-SPRR2A did not show significant degradation.

[0163] Based on the obtained gray value data, the relative intensity was further calculated, which was the ratio of the gray value of the protein band at different incubation time points to the gray value of the band at 0 h, indicating that the band intensity of the target protein was always maintained above 90%.

[0164] These results fully demonstrate that Trx-SPRR2A exhibits excellent serum stability both in neutral and acidic environments.

[0165] Experimental Test 6

[0166] The antibacterial activity of the purified Trx-SPRR2A protein obtained in Example 5 was detected as follows:

[0167] 1. Cell culture

[0168] Three Gram-positive bacteria, Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus, and one Gram-negative bacterium, Citrobacter rodentium, were selected as experimental subjects.

[0169] Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus were cultured in BHI medium at 37°C; Citrobacter rodentium was cultured in LB medium at 37°C, and the aseptic operation process was strictly followed to avoid exogenous microbial contamination.

[0170] (II) Plate colony counting method

[0171] (1) Inoculate the four bacteria into 10 mL of culture medium respectively and wait for them to grow to the logarithmic growth phase;

[0172] (2) The bacterial solution was centrifuged at 8000 rpm for 5 min, and then washed and resuspended with 10 mL of MES buffer (10 mM MES and 25 mM NaCl, pH 5.5);

[0173] (3) Different bacterial solutions were mixed evenly with different concentrations of Trx-SPRR2A at various dilutions and incubated at 37°C for 2 h;

[0174] (4) The incubated mixtures were evenly spread on solid culture medium and cultured at 37° C. overnight;

[0175] (5) Count the surviving colonies and calculate the percentage relative to the remaining colonies in the control sample.

[0176] The results are as follows Fig.10 As shown, with the increase of Trx-SPRR2A concentration, the colony numbers of Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus gradually decreased, indicating that Trx-SPRR2A has an inhibitory effect on the growth of these three Gram-positive bacteria.

[0177] Further observations revealed that Enterococcus faecalis was most sensitive to Trx-SPRR2A, and the number of remaining colonies after treatment with 20μM and 40μM Trx-SPRR2A dropped to 44.5% and 0%, respectively, indicating that Trx-SPRR2A had the most significant antibacterial effect on Enterococcus faecalis. In contrast, Trx-SPRR2A had very weak activity against Citrobacter rodentium, with a remaining colony percentage of 70% at 40μM. This indicates that Trx-SPRR2A can selectively kill Gram-positive bacteria, which is consistent with the results of existing literature.

[0178] (III) Scanning electron microscopy observation of cell morphology

[0179] (1) Inoculate the four bacteria into 10 mL of culture medium respectively and wait for them to grow to the logarithmic growth phase;

[0180] (2) The bacterial solution was centrifuged at 8000 rpm for 5 min, and then washed and resuspended with 10 mL of MES buffer (10 mM MES and 25 mM NaCl, pH 5.5);

[0181] (3) Take 1 mL of bacterial solution and centrifuge at 8000 rpm for 5 min, mix evenly with different concentrations of Trx-SPRR2A, and incubate at 37°C for 2 h;

[0182] (4) The incubated mixture was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the cells were collected;

[0183] (5) Suspend the cells in the fixative solution by pipetting, fix at room temperature in the dark for 30 min, and then transfer to 4°C for overnight storage;

[0184] (6) Conduct SEM inspection.

[0185] The results are as follows Fig.11As shown, the untreated bacterial cells have intact morphology, while the integrity of the bacterial cell membranes of the three Gram-positive bacteria treated with Trx-SPRR2A is damaged, with morphological changes such as depression, breakage, and leakage of contents, indicating that Trx-SPRR2A kills bacteria by binding to the bacterial cell membrane, destroying the integrity of its cell wall and cell membrane, leading to cytoplasm leakage. The cell morphology of Citrobacter rodentium did not change significantly. This shows that Trx-SPRR2A selectively kills Gram-positive bacteria. Consistent with the results of (ii), this may be related to the differences in the composition and structure of the cell wall and cell membrane of Gram-positive bacteria and Gram-negative bacteria.

[0186] (IV) N01 / PI live-dead staining method: use the kit according to the instructions attached to the kit.

[0187] The results are as follows Fig.12 As shown, Gram-positive bacteria treated with Trx-SPRR2A showed stronger red fluorescence and weaker green fluorescence ( Fig.12 ac). This indicates that Trx-SPRR2A treatment leads to damage of bacterial cell membrane, which in turn allows PI dye to enter the cells, making the bacteria show red fluorescence.

[0188] Further analysis showed that the red fluorescence intensity of bacteria gradually increased with the increase of Trx-SPRR2A concentration. This indicates that Trx-SPRR2A has a more obvious damaging effect on bacterial cell membrane at higher concentrations, making it easier for PI dye to enter the cell, thereby enhancing red fluorescence. In contrast, strong green fluorescence and weak red fluorescence were observed in the Gram-negative bacterium Citrobacter.

[0189] (V) PI uptake determination

[0190] (1) Enterococcus faecalis was inoculated into 10 mL of culture medium and allowed to grow to the logarithmic growth phase;

[0191] (2) The bacterial solution was centrifuged at 8000 rpm for 5 min, and then washed and resuspended with 10 mL of MES buffer (10 mM MES and 25 mM NaCl, pH 5.5);

[0192] (3) Dilute PI with MES to a final concentration of 11 μg / mL. Add 200 μL of 10 μg / mL diluted PI to 180 μL of the diluted PI. 6 times of bacterial solution to mix it evenly;

[0193] (4) (3) Divide the mixture into 6 equal parts, and add equal amounts of 0.05% SDS and different concentrations of Trx-SPRR2A to make the final concentrations 0, 5, 10, 20, and 40 μM, respectively;

[0194] (5) Add (4) to a 96-well plate, 100 μL per well, and use a microplate reader to measure the fluorescence intensity every 5 minutes (excitation wavelength: 535 nm; emission wavelength: 617 nm) for 1 hour. The 0.05% SDS treatment group was used as a positive control, and the PI uptake rate of each treatment group was calculated.

[0195] The results are as follows Fig.13 As shown, as the concentration of Trx-SPRR2A increases, the bacterial uptake of PI also increases, thereby enhancing the bactericidal effect. This indicates that Trx-SPRR2A exerts its antibacterial activity by destroying the permeability of bacterial cell membranes. This further proves that Trx-SPRR2A has the ability to destroy bacterial cell membranes, which in turn causes the bacteria to lose protection and eventually die.

[0196] Therefore, by applying the prokaryotic expression system provided by the present invention, an active SPRR2A fusion protein with high expression efficiency was successfully obtained for the first time; the secondary structure of the prepared Trx-SPRR2A protein was mainly β-folding and random coiling, containing five pairs of disulfide bonds formed by eukaryotic expression of SPRR2A, and had excellent thermal stability and serum stability; it showed a significant inhibitory effect on the growth of Gram-positive bacteria, and with the increase of the concentration of SPRR2A fusion protein, the antibacterial effect gradually increased; the method for constructing the prokaryotic expression system is simple and easy to operate, the conditions are mild, it is green and environmentally friendly, and it is suitable for industrial production.

[0197] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A prokaryotic expression system of SPRR2A with antibacterial activity, characterized in that: The prokaryotic expression system uses Escherichia coli Rosetta-gami (DE3) pLysS as the expression host, including pET32a- SPRR2A The amino acid sequence of the Trx-SPRR2A protein obtained by the recombinant plasmid and prokaryotic expression system is shown in SEQ ID NO.

7.

2. A method for constructing a prokaryotic expression system of SPRR2A with antibacterial activity as claimed in claim 1, characterized in that: Here are the steps: S1. Construction of pET32a- SPRR2A Recombinant plasmid pUC57- SPRR2A The vector is used as a template. SPRR2A- F and SPRR2A- R is the primer for PCR amplification, the PCR product is digested by template, and recovered and purified to obtain SPRR2A Gene fragments; Using pET32a vector as template and pET32a-F and pET32a-R as primers, PCR amplification was performed, the PCR product was template digested, and the pET32a vector fragment was recovered and purified; Using seamless cloning method SPRR2A The gene fragment was connected with the pET32a vector fragment to obtain the recombinant plasmid pET32a - SPRR2A ; SPRR2A- The sequence of F is shown in SEQ ID NO.1, SPRR2A- The sequence of R is shown in SEQ ID NO.2, the sequence of pET32a-F is shown in SEQ ID NO.3, and the sequence of pET32a-R is shown in SEQ ID NO.4; S2, transform the expression host to obtain heterologous expression pET32a- SPRR2A Engineering bacteria with recombinant plasmid capability The volume ratio of expression host to recombinant plasmid was 5:1; S3, heterologous expression pET32a- SPRR2A Recombinant plasmid The engineered bacteria obtained in step S2 were cultured and expanded. When the OD 600 When the value is close to 1.0, IPTG is added to induce expression; S4. Collect the expression product and purify it to obtain the Trx-SPRR2A protein with antibacterial activity.

3. The method for constructing a prokaryotic expression system of SPRR2A with antibacterial activity according to claim 2, characterized in that: In step S2, the expression host is transformed by heat shock transformation.

4. The method for constructing a prokaryotic expression system of SPRR2A with antibacterial activity according to claim 2, characterized in that: In step S3, the method for culturing and expanding the engineered bacteria obtained in step S3 is: culturing with LB liquid culture medium containing Amp resistance at 37° C., and the working concentration of IPTG is 0.5 mM.

5. The method for constructing a prokaryotic expression system of SPRR2A with antibacterial activity according to claim 2, characterized in that: In step S4, the protein was purified using a nickel ion column, washed with a wash buffer containing 50 mM imidazole, and the target protein was eluted with a wash buffer containing 200 mM imidazole.

6. Use of the prokaryotic expression system as claimed in claim 1 in preparing SPRR2A antimicrobial peptide with antimicrobial activity.

7. Use of the prokaryotic expression system construction method according to any one of claims 2 to 5 in the preparation of SPRR2A antimicrobial peptides with antimicrobial activity.

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