Recombinant protein of Eriocheir sinensis serine protease inhibitor EsKPI-1 containing Kazal structural domain as well as preparation method and application of recombinant protein

Abstract

The invention belongs to the technical field of molecular biology, and relates to a recombinant protein of a serine protease inhibitor EsKPI-1 containing a Kazal structural domain of Eriocheir sinensis as well as a preparation method and application of the recombinant protein. The amino acid sequence of the recombinant protein rEsKPI-1 is shown as SEQ ID NO.1. The protein has the activity of combining aeromonas hydrophila, escherichia coli and staphylococcus aureus, and can effectively inhibit the growth of the microorganisms. The recombinant protein rEsKPI-1 shows an important application prospect in the fields of research and development of novel antibacterial immune preparations, aquatic feed additives and the like.

Description

Recombinant protein of Chinese mitten crab containing the Kazal domain serine protease inhibitor EsKPI-1, its preparation method and application Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to a method for preparing a recombinant protein of EsKPI-1, a serine protease inhibitor containing the Kazal domain, from the Chinese mitten crab and its application. Background Technology

[0002] Kazal-type serine proteinase inhibitors (KPIs) are a class of serine protease activity regulators widely found in plants and animals, participating in the regulation of many important life processes. KPIs participate in the fine-tuning of physiological processes such as growth, development, and immunity by modulating the activity of their target proteases. Currently, members of the KPI family are being used as potential diagnostic biomarkers for the early detection of various diseases.

[0003] KPIs can regulate the immune system by specifically modulating the activity of serine proteases, thus playing an important physiological role in the host's defense against pathogen invasion. KPIs can directly inhibit the activity of virulence-related proteases (including metalloproteinases and plasminogen activator) secreted by pathogens, blocking microbial colonization and spread. Existing studies have confirmed that KPIs from various biological sources have broad-spectrum antibacterial effects. For example, leech KPIs have strong in vitro bactericidal activity against Staphylococcus aureus. Penaeus monodon KPIs have antibacterial activity against the Gram-positive bacterium Bacillus subtilis. Currently, there are no reports on the application of EsKPI-1 protein from the Chinese mitten crab as an antibacterial immunomodulator. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant protein of EsKPI-1, a serine protease inhibitor containing the Kazal domain from the Chinese mitten crab.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: a recombinant protein of EsKPI-1, a serine protease inhibitor containing the Kazal domain of the Chinese mitten crab, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The second objective of this invention is to provide a method for preparing the recombinant protein of EsKPI-1 described above.

[0007] To achieve the above objectives, the technical solution adopted in this invention is: a method for preparing recombinant protein of Chinese mitten crab EsKPI-1, comprising the following steps: a. PCR amplification of the coding region fragment of the Chinese mitten crab EsKPI-1 gene using primer pair P1 and P2, wherein the DNA sequence of primer P1 is shown in SEQ ID NO.2 and the DNA sequence of primer P2 is shown in SEQ ID NO.3; b. PCR amplification product and pET30a vector are digested with BamHI and HindIII, ligated with T4 ligase, transformed, and sequenced to screen recombinants; c. The recombinants are transformed into Escherichia coli Transetta (DE3) expression strain for prokaryotic induction expression, and then purified and renatured to obtain the rEsKPI-1 recombinant protein with the amino acid sequence shown in SEQ ID NO.1.

[0008] Primer P1: CGCGGATCCCAGTGTCCCGGGGTGTGCACG; Primer P2: CCCAAGCTTATCACTGGGCACTGGCCGGGG.

[0009] A third objective of this invention is to provide applications of the aforementioned rEsKPI-1 recombinant protein.

[0010] Specifically, this involves the application of rEsKPI-1 recombinant protein in the preparation of antibacterial immunomodulators; the application of rEsKPI-1 recombinant protein in the preparation of immunomodulators that inhibit Gram-negative bacteria; the application of rEsKPI-1 recombinant protein in the preparation of immunomodulators that inhibit Gram-positive bacteria; the application of rEsKPI-1 recombinant protein in the preparation of immunomodulators that inhibit both Gram-negative and Gram-positive bacteria; and the application of rEsKPI-1 recombinant protein in the preparation of feed additives.

[0011] The advantages of this invention are as follows: This invention obtains a recombinant protein, *Eriocheir sinensis* containing a Kazal domain serine protease inhibitor, EsKPI-1, through prokaryotic recombinant expression technology. Experiments show that the rEsKPI-1 recombinant protein can bind to the fungus *Pichia pastoris*, the Gram-positive bacterium *Staphylococcus aureus*, and the Gram-negative bacteria *Escherichia coli* and *Aeromonas hydrophila*. The rEsKPI-1 recombinant protein significantly inhibits the growth of *A. hydrophila*, *E. coli*, and *S. aureus*. Furthermore, the rEsKPI-1 recombinant protein reduces the bacterial protease activity of *A. hydrophila*, *E. coli*, and *S. aureus*. The *Eriocheir sinensis* recombinant protein *EsKPI-1* containing a Kazal domain serine protease inhibitor obtained by this invention has application value in the preparation of antibacterial drugs, novel immunomodulators, and feed additives. Attached Figure Description

[0012] Figure 1 is a purification diagram of the recombinant protein EsKPI-1, a serine protease inhibitor containing the Kazal domain from the Chinese mitten crab, provided in an embodiment of the present invention; Lane M: protein molecular marker (kDa); Lane 1: uninduced; Lane 2: IPTG induced; Lane 3: after purification.

[0013] Figure 2 is a graph showing the binding activity of the recombinant protein EsKPI-1, a serine protease inhibitor containing the Kazal domain from the Chinese mitten crab, with microorganisms, provided in this embodiment of the invention.

[0014] Figures 3-5 show the detection effect of the recombinant protein EsKPI-1, a serine protease inhibitor containing the Kazal domain of the Chinese mitten crab, on the growth of A. hydrophila, E. coli, and S. aureus, provided in the embodiments of the present invention.

[0015] Figure 6 shows the detection effect of the recombinant protein EsKPI-1, a serine protease inhibitor containing the Kazal domain from the Chinese mitten crab, on the inhibition of bacterial protease activity of A. hydrophila, E. coli, and S. aureus, provided in the embodiments of the present invention. Detailed Implementation

[0016] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0017] The rEsKPI-1 recombinant protein obtained by prokaryotic recombinant expression technology in this invention has binding activity with the fungus P. pastoris, the Gram-positive bacterium S. aureus, and the Gram-negative bacteria E. coli and A. hydrophila, and also has inhibitory activity on the growth of A. hydrophila, E. coli, and S. aureus. It can be used for the development of antibacterial feed additives and novel immune enhancers.

[0018] The culture media involved in the following examples are: LB liquid medium: catalog number A507002-0250, Shanghai Sangon Biotech Co., Ltd.; 2216E medium: catalog number HB0132-1, Haibo Biotechnology Co., Ltd.; YPD medium: catalog number YP7840, Beijing Huayueyang Biotechnology Co., Ltd.

[0019] Example 11. Construction of the recombinant vector: The recombinant vector used was Novagen's pET-30a(+) prokaryotic expression vector. Using Chinese mitten crab cDNA as a template, primer pairs P1 / P2, with BamHI and HindIII restriction sites introduced at the 5' ends, were used to amplify the EsKPI-1 coding region of Chinese mitten crab by PCR. The sequence of primer P1 is shown in SEQ ID NO.2, and the sequence of primer P2 is shown in SEQ ID NO.3.

[0020] Primer P1: CGCGGATCCCAGTGTCCCGGGGTGTGCACG; Primer P2: CCCAAGCTTATCACTGGGCACTGGCCGGGG.

[0021] The PCR reaction program was as follows: first, pre-denaturation at 94℃ for 5 min, followed by the following cycles: denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 90 s, for a total of 25 cycles, and a final extension at 72℃ for 10 min; after agarose gel electrophoresis, the PCR product was excised and recovered, and ligated into the pMD19-T vector; positive clones were screened after transformation, plasmids were extracted, and double-digested with BamHI and HindIII restriction enzymes; the target fragment was recovered and ligated into the expression vector pET-30a(+) which had been double-digested with BamHI and HindIII to complete the construction of the recombinant expression plasmid.

[0022] 2. Expression of the EsKPI-1 recombinant protein: The successfully constructed recombinant expression plasmid was transformed into Escherichia coli Transetta (DE3) competent cells. After screening for positive clones, single colonies were inoculated into 400 mL LB medium and cultured at 220 × g and 37°C until OD500. 600= 0.4; Add IPTG (final concentration 1 mmol / L), continue culturing at 37℃ for 12 h, then centrifuge at 12,000 ×g for 30 min at 4℃ to collect the bacterial cells, and freeze at -80℃ for later use; at the same time, take 1 mL of bacterial culture, centrifuge, discard the supernatant, add 80 μL of water and 20 μL of 5 × SDS-PAGE protein loading buffer (containing 5% β-mercaptoethanol), boil at 99℃ for 10 min, and detect the expression product by SDS-PAGE.

[0023] 3. Purification and refolding of EsKPI-1 recombinant protein The recombinant protein was purified using a nickel agarose gel FF column, and the obtained denatured recombinant protein was refolded by dialyzing with dialysis buffer. The specific operation steps are as follows: (1) Pack a nickel agarose gel FF column, 1.6 × 20 cm, with a column bed volume of 10 mL; (2) Equilibrate 2 to 5 column bed volumes with buffer I (50 mmol / L Tris-HCl buffer, pH 9.0, 50 mmol / L NaCl, 8 mol / L urea) at a flow rate of 2 mL / min; (3) Resuspend the IPTG-induced cells with buffer I, sonicate at 150 W for 40 min, centrifuge at 12,000 × g, 4℃ for 30 min, filter the supernatant through a 0.45 μm filter membrane, and cycle the sample through the column for 4 h at a flow rate of 1 mL / min; (4) Wash 2 to 5 column bed volumes with buffer I at a flow rate of 2 mL / min; (5) Use a solution containing 50 mmol / L Tris-HCl buffer, pH 9.0, 50 mmol / L NaCl, 8 mol / L urea. (6) Wash with 2-5 column volumes of mmol / L imidazole buffer I at a flow rate of 2 mL / min; (7) Elute the target protein with 400 mmol / L imidazole buffer I at a flow rate of 2 mL / min and collect the purified protein; (8) Detect the expression of the fusion protein by SDS-PAGE; (9) Place the purified deformed protein in refolding buffer to remove urea by dialysis so that the protein can refold correctly and restore the correct conformation. The denatured and purified protein product was dialyzed in a refolding buffer (2 mM reduced glutathione, 0.4 mM oxidized glutathione, 1 mM EDTA, 50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 1% glycine, and a gradient of decreasing urea). The urea concentration was initially 6 M, gradually changed to 4 M, 3 M, 2 M, 1 M, and 0 M, and finally dialyzed into a urea-free refolding buffer (without glycerol). Dialysis was performed at 4°C for 12 h each time to obtain the fully refolded EsKPI-1 recombinant protein containing the Kazal domain from the Chinese mitten crab. The amino acid sequence is shown in SEQ ID NO.1, and the results are shown in Figure 1.

[0024] SEQ ID NO.1: QCPGVCTFEYNPVCGSDGRTYGNLCQLEAERRCNNALLIAVARGECGTQHQDDCPGVCTLEYNPVCGSDGKTYGNLCQLEAEKRCVNPRLSVASQGECQNQHQSDCPGICTLEYNPVCGSDGNTYGNLCQLEAERRCVNAKLTLVSKGECHKYECPGVCTKEYRPVCGSDGQTYGNLCL FSTQKRCVNPLLTLISQGECKVRQCPDVCTRELRPVCGSDGVTYNNLCLFEAQRCRNPQLILVSKGRCASLPNKGQQCDFVCTLQLDPVCGSDGLTYGNLCMLTAQRKCSNPDLSFARRGECDTGLKEKKCSGVCTLEYNPVCGSDGVTYGNLCQLRHTSRCKDPSITLAYKGECGIIPGQCPV.

[0025] Length: 363 Type: Amino acid chain type: Single chain Characteristics: Molecular weight is 39.45 kDa, isoelectric point is 7.52, and it has a Kazal domain.

[0026] Source: Chinese mitten crab. Experimental results: The results showed that a distinct band of about 45 kDa was observed in the SDS-PAGE results, which is consistent with the predicted molecular weight of rEsKPI-1 (45.45 kDa) (Figure 1).

[0027] Example 2: Assay of the binding activity of recombinant EsKPI-1 protein to microorganisms. The binding activity of recombinant protein rEsKPI-1 to Gram-negative bacteria (Aeromonas hydrophila, Vibrio anguillarum, and Escherichia coli), Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, and Bacillus subtilis), and fungi (Pichia pastoris) was determined using Western blotting. The bacterial strains used were sourced as follows: Aeromonas hydrophila and Vibrio anguillarum were purchased from the Beijing Culture Collection Center for Microbial Culture, Escherichia coli was purchased from TransGen Biotech, Staphylococcus aureus was purchased from the Beijing Culture Collection Center for Microbial Culture, Micrococcus luteus and Bacillus subtilis were purchased from the Beijing Culture Collection Center for Microbial Culture, and Pichia pastoris GS115 was purchased from Invitrogen.

[0028] The specific operations are as follows: (1) The above 7 microorganisms were cultured overnight. The culture method was as follows: Bacillus subtilis, Micrococcus luteus and Escherichia coli were cultured in LB medium at 37°C for 20 h, Vibrio anguillarum was cultured in 2216E medium at 28°C for 20 h, Aeromonas hydrophila and Staphylococcus aureus were cultured in LB medium at 28°C for 20 h, and Pichia pastoris was cultured in YPD medium at 28°C for 20 h; (2) The bacterial cells were collected by centrifugation, washed three times with TBS buffer and resuspended, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL; (3) Take 100 μL of microbial suspension and mix it with an equal volume of the recombinant protein rEsKPI-1 obtained in the above example, and incubate at room temperature for 30 min by rotation; (4) Centrifuge at 10,000×g for 2 min to collect the bacterial cells, and wash the bacterial cells four times with TBS buffer; (5) After washing, collect the bacterial cells and resuspend them with 80 μL of sterile water; (6) Add 20 μL of 5× SDS-PAGE protein loading buffer, heat at 99℃ for 10 min, and separate the protein samples by SDS-PAGE electrophoresis; (7) After electrophoresis, remove the gel and cut out NC membranes and filter paper of the same size and immerse them together in electroporation buffer and let them stand for 10 min; (8) Place the filter paper, NC membrane, gel and filter paper into the electroporator in order from top to bottom, set the corresponding current according to the size of the gel block area, and the transfer time is 45 min; (9) Take out the NC membrane and wash it three times with TBST buffer, 5 μL each time. (10) Immerse the NC membrane in 5% skim milk powder (dissolved in TBST) and block at room temperature for 2 h; (12) Take out the NC membrane and wash it three times with TBST buffer for 5 min each time; (13) Immerse the NC membrane in the incubation solution (5% skim milk powder, TBST buffer) of mouse anti-His tag monoclonal antibody (purchased from Shanghai Sangon Biotech) diluted at a ratio of 1:1000 and incubate at room temperature for 3 h; (14) Take out the NC membrane and wash it three times with TBST buffer for 5 min each time.

[0029] (15) Immerse the NC membrane in the incubation solution (5% skim milk powder, TBST buffer) of HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (purchased from Shanghai Sangon Biotech) diluted at a ratio of 1:1000 and incubate at room temperature for 1 h; (16) Take out the NC membrane and wash it three times with TBST buffer for 10 min each time; (17) Perform the development reaction using ECL chemiluminescence method and record the Western blotting results using a chemiluminescence imager. The results are shown in Figure 2.

[0030] Experimental Results: The results showed that the recombinant EsKPI-1 protein containing the Kazal domain from the Chinese mitten crab in this embodiment exhibited varying degrees of binding activity against Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, and Pichia pastoris, with the strongest binding activity against Aeromonas hydrophila. No obvious band was observed in the rTrx negative control group (Figure 2). These results indicate that rEsKPI-1 possesses broad-spectrum binding activity.

[0031] Example 3: Detection of the inhibitory activity of recombinant EsKPI-1 protein on bacterial growth (growth curve method). Recombinant EsKPI-1 protein from the Chinese mitten crab was incubated with Gram-negative bacteria *Aeromonas hydrophila* and *Escherichia coli*, and Gram-positive bacteria *Staphylococcus aureus*. The OD values ​​of the bacteria were then detected using a microplate reader (Tecan Infinite M1000 PRO). 600 The activity of the recombinant EsKPI-1 protein from the Chinese mitten crab in inhibiting bacterial growth was analyzed using the growth curve method.

[0032] The strains used were sourced as follows: Aeromonas hydrophila was purchased from Beijing Microbial Culture Collection Center, Escherichia coli was purchased from TransGen Biotech, and Staphylococcus aureus was purchased from Beijing Microbial Culture Collection Center.

[0033] The specific operation is as follows: (1) Collect the bacteria cultured overnight by centrifugation, and adjust the bacterial concentration to 4×10⁻⁶ using TBS buffer. 4 CFU / mL; (2) Take 100 μL of rEsKPI-1 protein and mix it with 100 μL of the above bacterial solution, and incubate at room temperature for 2 h; (3) After incubation, centrifuge at 10,000 ×g for 2 min to collect the bacterial cells, and wash them 3 times with TBS buffer; (4) Take 20 μL of the above recombinant protein EsKPI-1 and bacterial solution mixture, add it to the wells of a 96-well plate, and add 200 μL of LB medium to the wells. At the same time, set up negative control wells (rTrx), with a final volume of 220 μL per well; (5) Place the plate in an ELISA reader and shake at an appropriate temperature for 18 h to reach the bacterial growth plateau phase. During this period, detect OD every 30 min. 600 (6) Plot the growth curves of each detected bacteria for comparison. The results are shown in Figure 3-5.

[0034] Experimental Results: The results showed that the recombinant protein EsKPI-1, a serine protease inhibitor containing the Kazal domain, from the Chinese mitten crab in this embodiment of the invention inhibited the growth of Aeromonas hydrophila (Figure 3), Staphylococcus aureus (Figure 4), and Escherichia coli (Figure 5). The results indicate that the recombinant protein rEsKPI-1 has a significant inhibitory effect on the growth of Aeromonas hydrophila, Staphylococcus aureus, and Escherichia coli.

[0035] Example 4: Detection of Bacterial Protease Activity Inhibited by Recombinant EsKPI-1 Protein from the Chinese Eriocheir sinensis. EsKPI-1, a serine protease inhibitor, can inhibit bacterial protease activity. The OD value of bacterial protease activity after incubation with rEsKPI-1 was measured using a Tecan Infinite M1000 PRO microplate reader. 440 The inhibitory effect of the recombinant EsKPI-1 protein from the Chinese mitten crab on bacterial protease activity was analyzed.

[0036] The strains used were sourced as follows: Aeromonas hydrophila was purchased from Beijing Microbial Culture Collection Center, Escherichia coli was purchased from TransGen Biotech, and Staphylococcus aureus was purchased from Beijing Microbial Culture Collection Center.

[0037] The specific operation is as follows: (1) The above three bacteria were cultured overnight. The culture method was as follows: Escherichia coli was cultured on LB medium at 37°C for 20 h, Aeromonas hydrophila and Staphylococcus aureus were cultured on LB medium at 28°C for 20 h, and then the bacteria cultured overnight were collected by centrifugation and prepared with TBS solution to a concentration of about 10. 7 (2) Take 500 μL of bacterial suspension and centrifuge at 4℃, 1,000 ×g for 5 min, and resuspend in 500 μL of lysis buffer.

[0038] (3) After placing the sample in an ice bath for 15 min, centrifuge at 16,000 ×g for 15 min at 4℃, and transfer 500 μL of the supernatant, which is the bacterial protease extract for later experimental detection; (4) Mix 500 μL of rEsKPI-1 recombinant protein with 500 μL of each bacterial protease extract separately, and set up a negative control (rTrx). Incubate at room temperature for 2 h, then mix 100 μL of the sample with 400 μL of the reaction solution and incubate at 37℃ for 30 min; (5) Add 500 μL of stop solution, vortex for 5 s, incubate in an ice bath for 15 min, and centrifuge at 3,000 ×g for 15 min at 4℃; (6) Transfer 500 μL of the supernatant to a new centrifuge tube, add 500 μL of neutralization solution, mix well, and then use an enzyme-linked immunosorbent assay (ELISA) reader to detect OD. 440 Value; (7) The bacterial protease activity is calculated using the following formula: △OD 440 = (OD 440(测试) - OD 440(空白) ) / (0.1 × incubation time (min)), where △OD 440 This indicates the change in absorbance, with an incubation time of 30 minutes.

[0039] Experimental Results: The results showed that after incubation with the recombinant EsKPI-1 protein containing the Kazal domain from the Chinese mitten crab, the bacterial protease activities of Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus were reduced in the embodiments of the present invention (Figure 6). The results indicate that the rEsKPI-1 recombinant protein can inhibit the bacterial protease activities of Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus.

Claims

1. A recombinant protein from the Chinese mitten crab containing the Kazal domain serine protease inhibitor EsKPI-1, characterized in that: The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. A method for preparing the recombinant protein of *Eriocheir sinensis* containing the Kazal domain serine protease inhibitor EsKPI-1 as described in claim 1, characterized in that... Includes the following steps: a. The coding region of the EsKPI-1 gene of the Chinese mitten crab was amplified by PCR using primer pairs P1 and P2, wherein the sequence of primer P1 is shown in SEQ ID NO.2 and the sequence of primer P2 is shown in SEQ ID NO.3; b. The PCR amplification product and pET30a vector were digested with BamHI and HindIII, respectively, and then ligated with T4 DNA ligase to obtain the recombinant expression vector; c. The recombinant vector was transformed into Transetta (DE3) competent cells, and after expression induced by IPTG, the recombinant protein rEsKPI-1 was purified and renatured.

3. The application of the recombinant protein rEsKPI-1 according to claim 1 in the preparation of antibacterial immunomodulators.

4. The use of the recombinant protein rEsKPI-1 of claim 1 in the preparation of an immunomodulatory agent for inhibiting Gram-negative bacteria.

5. The use of the recombinant protein rEsKPI-1 according to claim 1 in the preparation of an immunomodulatory agent for inhibiting Gram-positive bacteria.

6. The use of the recombinant protein rEsKPI-1 of claim 1 in the preparation of an immunomodulatory agent that inhibits Gram-negative and Gram-positive bacteria.

7. The use of the recombinant protein rEsKPI-1 according to claim 1 in the preparation of feed additives.

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