Fusion protein containing Polh tag as well as preparation method and application of fusion protein

Abstract

The invention relates to the technical field of biology, and particularly discloses a fusion protein containing a Polh tag as well as a preparation method and application of the fusion protein. The fusion protein provided by the invention comprises an antibacterial peptide capable of inhibiting escherichia coli and staphylococcus aureus and a Polh tag, the amino acid sequence of the antibacterial peptide is as shown in SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 or SEQ ID No. 6. The fusion protein disclosed by the invention can be effectively and massively expressed in a prokaryotic expression system, and the antibacterial peptide obtained after splitting decomposition and purification still has ideal antibacterial activity. The invention provides a new scheme for large-scale preparation of the high-activity antibacterial peptide.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fusion protein containing a Polh tag and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides are a class of short peptides composed of amino acids with a broad antimicrobial spectrum. They are naturally occurring substances in organisms and usually act as part of the immune system to fight against the invasion of bacteria, viruses and other microorganisms. Due to their unique antimicrobial mechanisms, pathogens are not easily resistant to them, so they are expected to solve the problem of antibiotic resistance.

[0003] The preparation methods of antimicrobial peptides mainly include direct extraction, enzymatic hydrolysis, chemical synthesis and recombinant DNA technology. Among them, the recombinant DNA technology method is more suitable for large-scale production. However, in recombinant DNA technology, when the host cell expresses the antimicrobial peptide, it may be attacked by the antimicrobial peptide, thereby threatening its survival and normal physiological functions. Therefore, the expression of the antimicrobial peptide gene may be reduced and the degradation of the antimicrobial peptide may be increased. These will result in the inability to express the antimicrobial peptide, or the expression yield is limited and cannot meet the production requirements.

[0004] In the prior art, there are solutions to solve the above problems by co-expressing antimicrobial peptides with fusion tags, but not all fusion tags and antimicrobial peptides can achieve the desired effect. If the two are not properly matched, the fusion protein cannot be effectively expressed or the antimicrobial peptide cannot show its original antibacterial activity after being separated. Therefore, how to efficiently obtain antimicrobial peptides with ideal functions still needs further research. Summary of the invention

[0005] One of the purposes of the present invention is to provide a fusion protein that can be effectively expressed in large quantities through a prokaryotic expression system and can be cleaved to produce an antimicrobial peptide with high antibacterial activity.

[0006] The present invention provides a fusion protein, which comprises: an antimicrobial peptide capable of inhibiting Escherichia coli and Staphylococcus aureus and a Polh tag; the amino acid sequence of the antimicrobial peptide is shown in SEQ ID No.8, SEQ ID No.10, SEQ ID No.12 or SEQ ID No.6.

[0007] In genetic recombination engineering, the Polh (Polyhedrin) tag is a polyhedrin tag. Polyhedrin can promote the expression of foreign genes fused with it, making it easier for the target gene to synthesize the corresponding protein in large quantities in the expression system; and it can also form inclusion bodies that are soluble in alkaline buffers. However, not all antimicrobial peptides can be successfully expressed using this tag. If the fusion tag and the antimicrobial peptide do not work well together, there will be problems such as ineffective expression or inability to show the original antibacterial activity after the antimicrobial peptide is lysed and purified. However, there is no universal rule in the prior art for how to combine fusion tags and antimicrobial peptides to achieve the expected results.

[0008] After extensive research, the present invention finally provides a fusion protein that can effectively cooperate with an antimicrobial peptide and a Polh tag. The fusion protein can successfully achieve large-scale expression of inclusion body protein through Escherichia coli, and after lysis and purification, a highly active antimicrobial peptide can be obtained.

[0009] In the present invention, the inclusion body protein formed by the antimicrobial peptide and the Polh tag does not have the antimicrobial activity of the original antimicrobial peptide, so it will not kill the host bacteria, and after purification, hydroxylamine is added to lyse the target protein, which can release a separate antimicrobial peptide that still has a good antibacterial effect, that is, the fusion protein of the present invention can be used to prepare a large amount of antimicrobial peptides with ideal effects using a prokaryotic expression system.

[0010] In the fusion protein of the present invention, a cleavage site is further included between the Polh tag and the antimicrobial peptide, and the amino acid sequence of the cleavage site is NG.

[0011] In the fusion protein of the present invention, the amino acid sequence of the Polh tag is shown as SEQ ID No.1.

[0012] The antimicrobial peptides of the present invention can be co-expressed with the Polh fusion tag to form inclusion bodies.

[0013] The amino acid sequence of the fusion protein of the present invention is shown in SEQ ID No.16, SEQ ID No.18, SEQ ID No.20 or SEQ ID No.14.

[0014] The present invention also provides a DNA molecule encoding the above fusion protein.

[0015] The DNA molecule of the present invention has a nucleotide sequence as shown in SEQ ID No.17, SEQ ID No.19, SEQ ID No.21 or SEQ ID No.15.

[0016] The present invention also provides a biological material containing the above DNA molecule, wherein the biological material is an expression cassette, a vector or a host cell.

[0017] The present invention also provides the use of the above fusion protein, or DNA molecule, or biological material in the preparation of antimicrobial peptides.

[0018] The present invention also provides a method for preparing an antimicrobial peptide, comprising: (1) expressing the fusion protein in Escherichia coli; (2) cleaving the fusion protein using a hydroxylamine cleavage method to collect free antimicrobial peptides; (3) Purifying the antimicrobial peptide.

[0019] In step (3) of the method of the present invention, the antimicrobial peptide is purified by dialysis, and preferably, the dialysis fluid used in the dialysis is distilled water.

[0020] The purification method of the present invention is beneficial to ensuring the antibacterial activity of the renatured antimicrobial peptide.

[0021] The beneficial effects of the present invention are at least: The fusion protein of the present invention can be effectively expressed in large quantities in a prokaryotic expression system, and the antimicrobial peptide obtained after cleavage and purification still has ideal antimicrobial activity. The present invention provides a new solution for the large-scale preparation of highly active antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the map of Polh+E1~E4 in the pET-28a expression vector.

[0023] Figure 2 These are the gel electrophoresis results of antimicrobial peptide fusion proteins Polh+NG+E1, Polh+NG+E2, Polh+NG+E3, and Polh+NG+E4.

[0024] Figure 3 These are the results of the antibacterial test after the E1, E2, E3, and E4 antimicrobial peptides were dialyzed into acetonitrile.

[0025] Figure 4 These are the antibacterial test results of E1, E2, E3, and E4 antimicrobial peptides dialyzed into distilled water.

[0026] Figure 5 The results of plate inhibition test of E1, E2, E3, and E4 antimicrobial peptides dialyzed into distilled water.

[0027] Figure 6 The figure is the gel electrophoresis result of the antimicrobial peptide fusion protein of the comparative example expressing the soluble fusion protein. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0029] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0030] Example 1 This embodiment provides a method for preparing antimicrobial peptides E1, E2, E3, and E4, wherein the amino acid sequences of E1, E2, E3, and E4 are shown in SEQ ID No.6 (E1), SEQ ID No.8 (E2), SEQ ID No.10 (E3), and SEQ ID No.12 (E4); the corresponding base sequences are shown in SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, and SEQ ID No.13.

[0031] The specific preparation method is as follows: 1. Preparation of fusion proteins Polh+NG+E1, Polh+NG+E2, Polh+NG+E3, and Polh+NG+E4 containing Polh tags and cleavage sites: The amino acid sequence of the Polh fusion tag is shown in SEQ ID No. 1, and the codon-optimized base sequence thereof for E. coli expression is shown in SEQ ID No. 2.

[0032] The amino acid sequences of the E1, E2, E3, and E4 antimicrobial peptide fusion proteins Polh-NG-E1, Polh-NG-E2, Polh-NG-E3, and Polh-NG-E4 prepared in this example are shown in SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, and SEQ ID No. 20, respectively; the corresponding base sequences are shown in SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, and SEQ ID No. 21, respectively.

[0033] In this example, the Polh-NG-E1, Polh-NG-E2, Polh-NG-E3, and Polh-NG-E4 base sequences were commissioned to Shanghai Huajin Biotechnology Co., Ltd. to synthesize the gene fragments, and the synthesized gene fragments were inserted into the PET-28a expression vector through the restriction sites of BamH I (NEB Company, Catalog No.: R0136L) and Xho I (NEB Company, Catalog No.: R0146L), respectively. The schematic diagram is shown in Figure 1 .

[0034] In this example, the four expression plasmids constructed successfully above were transformed into Escherichia coli competent cells BL21 (DE3) (Merck). The specific process is as follows: (1) Place 1 µl of the above plasmid in 100 µl of competent E. coli cells BL21(DE3) and place on ice for 30 min.

[0035] (2) The mixture was heat-shocked in a 42°C water bath for 90 seconds and then quickly placed on ice for 2 minutes.

[0036] (3) Add 600 µl of resistance-free LB to the mixture and incubate at 37°C, 220 rpm for 1 h.

[0037] (4) Take 200 µl of the bacterial solution and spread it evenly on an LB plate containing kanamycin sulfate resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 µg / ml kanamycin).

[0038] (5) Place the plate upside down in a 37°C incubator for about 20 hours until clearly visible colonies appear.

[0039] In this embodiment, a monoclonal colony was picked from the transformed LB plate and cultured in 10 ml LB (containing 100 μg / ml kanamycin) medium for 12 h-16 h, and then transferred to 2×YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) at a ratio of 1:100 for expansion culture, and cultured at 37°C and 220 rpm until the bacterial solution OD600 was 0.4-0.6, and then added with a final concentration of 0.5 mM IPTG (Sigma, catalog number: I5502-1G) for induction expression, and the induction conditions were 18°C ​​and 180 rpm for 20 h. Finally, the bacterial cells were collected by centrifugation and stored at -20°C or immediately entered the next step of purification.

[0040] In this example, about 50 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride) was used to resuspend (1 L) the bacterial pellet, and the bacteria were lysed using a high-pressure lysis instrument (Xinzhi Biotechnology) and then centrifuged at 13,000 rpm for 30 min to fully separate the soluble protein from the inclusion bodies.

[0041] In this example, the inclusion bodies were washed twice with 30 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride), centrifuged at 13000 rpm for 30 min, and the washed inclusion bodies were collected. The inclusion bodies were fully dissolved with 20 ml of denaturing buffer (6 M guanidine hydrochloride, 40 mM NaH2PO3, 500 mM NaCl, pH 7.8). The fusion protein was collected in the supernatant by centrifugation at 13000 rpm for 30 min. The obtained product can be analyzed for concentration and purity by SDS-PAGE electrophoresis. The specific process is as follows: take 40µl of protein solution, add 10µl of 5× protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100℃ boiling water for 10min, then add 10µl per well to SDS-PAGE protein gel, run at 80V for 2h, use Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization. The gel electrophoresis results of the fusion protein are shown in Figure 2 , from which it can be seen that each fusion protein is expressed correctly.

[0042] 2. Preparation of E1, E2, E3, E4 antimicrobial peptides: In this embodiment, the fusion protein is cleaved by hydroxylamine cleavage to release free E1, E2, E3, and E4 antimicrobial peptides. The fusion protein solution of Polh-NG-E1, Polh-NG-E2, Polh-NG-E3, and Polh-NG-E4 is mixed with hydroxylamine lysis solution (4M hydroxylamine, pH 9.0) in a volume ratio of 1:1, adjusted to pH 9.0, and closed in a 45-degree water bath environment. The reaction can be completed in about 4 to 6 hours. Hydroxylamine solution can specifically cut the Asn-Gly site inside the protein in a weakly alkaline environment, which can just release the E1, E2, E3, and E4 antimicrobial peptides in the fusion protein. The progress of the enzyme cleavage reaction was detected by SDS-PAGE electrophoresis.

[0043] The E1, E2, E3, and E4 antimicrobial peptides released in this embodiment can be renatured and purified by dialysis, and then subjected to activity detection or freeze-dried for standby use. The specific process is: transfer the E1, E2, E3, and E4 antimicrobial peptides to a dialysis bag with a molecular weight cutoff of 1 kDa, and place them in 100 times the volume of the dialysate in distilled water and acetonitrile, respectively, and dialyze for 24 hours in a 4°C environment with continuous stirring. The distilled water and acetonitrile can be replaced every 4 hours. Then collect the solution in the dialysis bag, which is the purified E1, E2, E3, and E4 antimicrobial peptides.

[0044] Example 2 In this example, the antibacterial activity of the antimicrobial peptides E1, E2, E3, and E4 dialyzed into acetonitrile and distilled water in Example 1 was tested to verify their antibacterial activity. The specific method is as follows: (1) Thaw the frozen Escherichia coli and Staphylococcus aureus strains separately and inoculate them into 100 μl solid culture medium. Pick a single clone at 37 degrees overnight. Select a single clone colony and inoculate it into a 20 ml liquid culture medium (LB) flask. Place it on a shaker at 180 rpm and incubate it at 37 degrees for 16 hours as a bacterial stock solution.

[0045] (2) Blank control group: Take 1 ml of bacterial stock solution, add 5 ml of liquid culture medium LB, mix well, culture at 37 degrees for 4 hours on a shaker, and measure the OD value at 600 nm.

[0046] (3) Antimicrobial peptide experimental group: 1 ml of bacterial stock solution was taken, and the corresponding antimicrobial peptide was added at a final concentration of 50 ug / ml. The mixture was mixed evenly, and cultured on a shaker at 37°C for 4 h. The OD value at 600 nm was measured.

[0047] (4) Antibiotic control group: 1 ml of bacterial stock solution was taken, and antibiotics were added at a final concentration of 50 μg / ml. The mixture was mixed well, and cultured on a shaker at 37°C for 4 h. The OD value at 600 nm was measured.

[0048] The results of the antibacterial test of E1-E4 antimicrobial peptides dialyzed into acetonitrile are shown in Figure 3 The antibacterial test results of E1, E2, E3, and E4 antimicrobial peptides dialyzed into distilled water are shown in Figure 4 From the test results, it can be seen that after the E1, E2, E3, and E4 antimicrobial peptides were dialyzed into acetonitrile, only E3 had good antibacterial activity, while E1, E2, and E4 had poor antibacterial activity. However, using the distilled water purification process, the antibacterial activities of E1, E2, E3, and E4 can all be activated.

[0049] The inhibition rate of E1, E2, E3, and E4 antimicrobial peptides dialyzed into distilled water was further calculated based on the test results. Inhibition rate = (OD value of bacterial solution in the blank group - OD value of bacterial solution in the experimental group) / OD value of bacterial solution in the blank group × 100%.

[0050] The inhibitory effects of the four antimicrobial peptides dialyzed into distilled water are as follows: at a concentration of 50ug / ml, the inhibition rate of antimicrobial peptide E1 against Escherichia coli was 100%, the inhibition rate of antimicrobial peptide E2 was 100%, the inhibition rate of antimicrobial peptide E3 was 97.80%, and the inhibition rate of antimicrobial peptide E4 was 100%; at a concentration of 50ug / ml, the inhibition rate of antimicrobial peptide E1 against Staphylococcus aureus was 95.23%, the inhibition rate of antimicrobial peptide E2 was 97.21%, the inhibition rate of antimicrobial peptide E3 was 100%, and the inhibition rate of antimicrobial peptide E4 was 95.33%.

[0051] Example 3 In this example, the plate antibacterial activity test was performed on the E1, E2, E3, and E4 antimicrobial peptides dialyzed into distilled water in Example 1, as follows: According to the E. coli culture, several plates were laid out and lines were drawn to distinguish the sample positions; water was dropped as a blank group, antimicrobial peptide was dropped as an experimental group, and kanamycin was dropped as a control group, and cultured overnight. Figure 5 It can be seen that, for E. coli, E1-E4 antimicrobial peptides all showed plaque phenomenon, which is similar to the effect of kanamycin.

[0052] Comparative Example In this comparative example, the Polh tag was fused with I20, R21, and G22 antimicrobial peptides according to the method described in Example 1, wherein the amino acid sequences of I20, R21, and G22 are shown in SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, respectively; however, the expression yield of these fusion proteins is low, and the specific test results are shown in Figure 6 Therefore, it can be seen that not all antimicrobial peptides can be expressed using the Polh tag.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A fusion protein, characterized in that include: An antimicrobial peptide and Polh tag capable of inhibiting Escherichia coli and Staphylococcus aureus; the amino acid sequence of the antimicrobial peptide is shown in SEQ ID No.8, SEQ ID No.10, SEQ ID No.12 or SEQ ID No.

6.

2. The fusion protein according to claim 1, characterized in that A cleavage site is also included between the Polh tag and the antimicrobial peptide, and the amino acid sequence of the cleavage site is NG.

3. The fusion protein according to claim 1 or 2, characterized in that The amino acid sequence of the Polh tag is shown in SEQ ID No.

1.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID No.16, SEQ ID No.18, SEQ ID No.20 or SEQ ID No.

14.

5. A DNA molecule encoding the fusion protein according to any one of claims 1 to 4.

6. The DNA molecule according to claim 5, characterized in that It has a nucleotide sequence as shown in SEQ ID No.17, SEQ ID No.19, SEQ ID No.21 or SEQ ID No.

15.

7. A biological material containing the DNA molecule according to claim 5 or 6, wherein the biological material is an expression cassette, a vector or a host cell.

8. Use of the fusion protein according to any one of claims 1 to 4, or the DNA molecule according to claim 5 or 6, or the biomaterial according to claim 7 in the preparation of antimicrobial peptides.

9. A method for preparing an antimicrobial peptide, characterized in that: include: (1) causing Escherichia coli to express the fusion protein according to any one of claims 1 to 4; (2) cleaving the fusion protein using a hydroxylamine cleavage method to collect free antimicrobial peptides; (3) Purifying the antimicrobial peptide.

10. The method according to claim 9, characterized in that In step (3), the antimicrobial peptide is purified by dialysis, and preferably, the dialysis fluid used in the dialysis is distilled water.

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