JNK recombinant protein as well as preparation method and application thereof
JNK recombinant protein was prepared by PCR amplification and homologous recombination technology, and purified by Ni column affinity chromatography. This solved the problem of micro/nanoplastics and heavy metal pollution in the Poyang Lake pleated crown mussel, revealed the protective role of JNK protein in cellular resistance to toxins, and promoted in-depth research on bioactive substances in shellfish.
Patent Information
- Application Number
- CN202511828287.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-01-23
AI Technical Summary
There is a lack of effective methods for monitoring pollutants and for studying the toxic effects of compound pollutants on aquatic organisms, especially for micro/nanoplastics and heavy metal pollution in the pleated crown mussel in Poyang Lake, and there is also a lack of effective methods for preparing and purifying recombinant proteins.
Recombinant plasmids were constructed using PCR amplification and homologous recombination techniques, and JNK recombinant protein was prepared by Ni column affinity chromatography. A 293T cell model treated with PSNPs and As was established to investigate the role of JNK protein in the cell's response to external stimuli.
An efficient preparation system for JNK protein was optimized, revealing its protective role against toxins, promoting the recycling and reuse of shellfish processing waste, and providing experimental reference for the in-depth research and development of shellfish bioactive substances.
Smart Images
Figure CN121380137A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of recombinant protein preparation and purification technology, specifically to a JNK recombinant protein, its preparation method, and its applications. Background Technology
[0002] As benthic organisms long exposed to extreme ecological environments such as high salinity and high pressure, shellfish have evolved unique metabolic regulatory networks and molecular defense systems. This adaptation is not only reflected in their specialized physiological structures (such as their thick calcareous shells) but also in their secondary metabolites. Studies have shown that shellfish contain abundant bioactive metabolites, including antimicrobial peptides (such as MGD-1 in mussels), lysozymes (such as CpLYZ3 in the pleated crown mussel), and polysaccharides. These molecules have been proven to possess antibacterial, anti-inflammatory, and antitumor activities and participate in various biological processes such as signal regulation and free radical scavenging. Research has found that quinoline pigments in invertebrates not only have strong antioxidant activity but are also inexpensive and readily available, making them an important source of natural antioxidants. Glycoproteins isolated from oysters have a highly efficient ability to scavenge hydroxyl radicals, and their efficacy is significantly superior to vitamin C. Modern biotransformation technology has enabled the efficient extraction of high-value-added products such as chitin and chitin from shellfish processing waste, promoting the deep application of shellfish in the pharmaceutical field.
[0003] The pleated crown mussel, an important freshwater molluskaloid in the Poyang Lake basin, is not only a raw material for processing folk art but also a major source of pearls. Its edible meat and medicinal uses make it a significant player in related industries. In recent years, the abundance of microplastics, primarily polystyrene polymers, in Poyang Lake has been increasing, and multiple heavy metals such as Mn, As, and Pb have been detected in the silt and shallow groundwater of some areas. Although Poyang Lake faces the dual threat of combined micro / nanoplastics and heavy metal pollution, effective pollutant monitoring methods are currently lacking, and research on the toxic effects of these combined pollutants on aquatic organisms is scarce.
[0004] Based on the above research, focusing on the pleated crown mussel, a freshwater mollusk that is unique to my country and widely distributed, how to prepare a recombinant protein, how to purify the recombinant protein, and how to analyze the pharmacological potential of the recombinant protein are urgent technical problems that need to be solved. Summary of the Invention
[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a JNK recombinant protein, its preparation method and application.
[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing JNK recombinant protein, comprising the following steps: S1. Using cDNA from the pleated crown mussel as a template, PCR amplification was performed using specific primers to obtain the amplification product; S2. Using homologous recombination technology, the amplification product is ligated to a vector to construct a recombinant plasmid. S3. The recombinant plasmid was purified after induction of expression to obtain the JNK recombinant protein.
[0007] Optionally, in S1, the specific primers are: qpJNK-F: CTGAAGCCCTCTAACATT, qpJNK-R: TGGTCACTACCTGGAAACA.
[0008] Optionally, S3 includes the following specific methods: (1) IPTG was used to induce expression. After induction, the bacterial cells were collected, washed with PBS, and the precipitate was collected. Binding / Wash Buffer was added to the precipitate to resuspend the bacterial cells and obtain the resuspended bacterial solution. (2) Sonicate the resuspended bacterial solution in an ice bath and collect the total protein in the supernatant; add the total protein in the supernatant to the buffer solution, mix the protein in the metal bath, and collect the supernatant; (3) The supernatant was purified by resin to obtain JNK recombinant protein.
[0009] Optionally, the method of step (1) includes: Take 50 μL of the correctly sequenced bacterial culture and inoculate it into 100 mL of LB medium containing the corresponding antibiotic. Incubate at 37°C until the OD600 value is 0.6-0.8. Add IPTG at a concentration of 0.5 mM-2 mM and induce at 20°C-37°C for 4-12 h. After induction, centrifuge at room temperature to collect the bacterial cells. Wash with 1×PBS and collect the precipitate. Add 2-5 mL of Binding / WashBuffer to the precipitate to resuspend the bacterial cells.
[0010] Optionally, in step (1), the IPTG concentration is 1 mM, and induction is performed at 25°C for 8 h.
[0011] Optionally, the method of step (2) includes: Transfer the bacterial culture to a clean EP tube, sonicate in an ice bath, rinse the probe with deionized water, and then place the sample tube in a beaker containing an ice-water mixture, with the ice-water interface slightly higher than the bacterial culture surface, and immerse the probe in the bacterial culture. Sonicate at 60% power for 2 seconds, with a 2-second interval, for 1-5 minutes, repeating until the bacterial culture becomes clear or no longer viscous. Centrifuge at 8000 r / min and 4℃ for 10 minutes, and collect the supernatant total protein. Pipette 100 uL of the supernatant into a centrifuge tube, add 25 uL of 5×protein loading buffer, and mix well. Denature the protein mixture in a 95℃ metal bath for 10 minutes, centrifuge at 12000 r / min and 4℃ for 5 minutes, and collect the supernatant, which is the protein sample.
[0012] Optionally, the method of step (3) includes: The storage buffer in the Ni-NTA agarose purification resin was effluxed by gravity, and the column was equilibrated with 10 mL of Binding / Wash Buffer. The sample solution was added to the column, and the flow-through was collected into a centrifuge tube and allowed to pass through twice. The column was washed with 10 mL of Binding / Wash Buffer. The His-tagged protein in the column was eluted with 10 mL of Elution Buffer, and the elution was repeated twice until the absorbance of the eluent at 280 nm was close to the baseline. The column material was eluted with 25 mL of Elution Buffer, and then the column was equilibrated with 25 mL of Binding / Wash Buffer. The column packing material was washed with 25 mL of ddH2O, and the column was protected with 20% ethanol and stored at 4°C.
[0013] Secondly, the present invention provides a JNK recombinant protein, which is obtained by the preparation method described above.
[0014] Thirdly, the present invention provides the application of the aforementioned JNK recombinant protein in the preparation of antioxidant products.
[0015] Fourthly, the present invention provides the application of the aforementioned JNK recombinant protein in the preparation of antitoxic products.
[0016] This invention has at least one of the following beneficial effects: This invention constructs a recombinant plasmid using PCR amplification and homologous recombination techniques. After induced expression and purification, the recombinant plasmid yields a JNK recombinant protein. This invention optimizes the efficient preparation system conditions for JNK protein, capturing the target protein using nickel (Ni) column affinity chromatography. To reveal the protective role of JNK protein in cellular resistance to toxins, a 293T cell model treated with PSNPs and As was established. JNK was transfected into 293T cells to investigate the role of JNK protein in cellular responses to external stimuli. Furthermore, the resistance of JNK protein to H2O2 was assessed using the H2O2 disk diffusion method, revealing the universality of JNK protein in cross-species cells. This provides crucial data support for subsequent pharmacodynamic studies. Moreover, research on high-value-added products such as JNK protein promotes the recycling and reuse of shellfish processing waste, providing experimental reference for the in-depth research and development of bioactive substances from shellfish. Attached Figure Description
[0017] Figure 1 Optimization of purification conditions for JNK recombinant protein; where A: protein standard curve; B: IPTG concentration screening; C: induction time screening; D: induction temperature screening.
[0018] Figure 2 SDS-PAGE images of JNK recombinant proteins after combined treatment with PSNPs and As; where, channel 1: total protein band of blank group; channel 2: marker; channel 3: purified protein band of blank group; channel 4: total protein band of PA group; channel 5: purified protein band of PA group; channel 6: total protein band of IPTG group; channel 7: purified protein band of IPTG group; channel 8: total protein band of IPTG + PA group; channel 9: purified protein band of IPTG + PA group; PA: PSNPs 100 + As 400 μg / L.
[0019] Figure 3 The effect of combined treatment with PSNPs and As on JNK protein expression.
[0020] Figure 4 To investigate the effect of JNK overexpression on the resistance of 293T cells to PSNPs and As-induced toxicity.
[0021] Figure 5 The results of the paper disc diffusion experiment of Escherichia coli BL21 of JNK were used to express the results. Note: 0: ddH2O; 1: 50 mM H2O2; 2: 100 mM H2O2; 3: 200 mM H2O2; 4: 400 mM H2O2. Detailed Implementation
[0022] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
[0023] The prokaryotic expression vector pET32a required for the experiment was all stored in the laboratory; the E. coli competent cells BL21(DE3) required for the experiment were purchased from Novizan Biosciences Co., Ltd.
[0024] PSNPs refers to polystyrene nanoplastic particles, and As refers to arsenic.
[0025] Example 1 This embodiment provides a method for preparing JNK recombinant protein, including the following steps: 1. Construction of recombinant protein particles (1) Using cDNA from the pleated crown mussel as a template, PCR amplification was performed using specific primers to obtain the amplification product.
[0026] Specific primers were designed based on the JNK sequence. The specific primers are: qpJNK-F: CTGAAGCCCTCTAACATT, qpJNK-R: TGGTCACTACCTGGAAACA.
[0027] Prepare the PCR reaction system according to Table 1.
[0028] Table 1 PCR reaction system Gently pipette the prepared PCR reaction solution to mix well, and perform PCR amplification according to the system in Table 2.
[0029] Table 2 PCR Amplification Program PCR products were recovered using the Novizan FastPure Gel DNA Extraction Mini Kit (DC301-01) and stored at -20°C for later use.
[0030] (2) The amplification product is ligated to the desired vector using homologous recombination technology to construct a recombinant plasmid. The reaction system is shown in Table 3.
[0031] Table 3 Homologous recombination system Ligation was performed at 37℃ for 150 min; after transformation, single bacterial selection, and PCR of the ligation product, the corresponding bacterial culture was sent to the company for sequencing; the successfully sequenced strains were then amplified.
[0032] 2. Recombinant protein particle induction (1) Small amount of induction 50 μL of correctly sequenced bacterial culture was inoculated into 100 mL of LB medium containing the appropriate antibiotic and cultured at 37°C until the OD600 value reached 0.6-0.8. IPTG concentration gradients of 0.5 mM, 1 mM, and 2 mM were then added, and induction was performed at temperature gradients of 20°C, 25°C, 28°C, and 37°C for 4, 8, and 12 h, respectively. After induction, the cells were centrifuged at 5000 rpm for 10 min at room temperature, and the cells were collected. The cells were washed three times with 1 mL of 1×PBS, and the precipitate was collected. The cells were resuspended in 2-5 mL of Binding / Wash Buffer.
[0033] (2) Ultrasonic fragmentation Transfer the bacterial culture to a clean 10 mL EP tube, sonicate in an ice bath, rinse the probe with deionized water, and then place the sample tube in a beaker containing an ice-water mixture, with the ice-water interface slightly higher than the bacterial culture surface, and the probe immersed in the bacterial culture. Sonicate at 60% power for 2 seconds, with 2-second intervals, for 1-5 minutes, repeating until the bacterial culture becomes clear or no longer viscous (when the bacterial count is large). Centrifuge at 8000 r / min, 4℃ for 10 minutes, and collect the supernatant total protein. Transfer 100 μL of the supernatant to a centrifuge tube, add 25 μL of 5× protein loading buffer, and mix well. Denature the protein mixture in a 95℃ metal bath for 10 minutes, then centrifuge at 12000 r / min, 4℃ for 5 minutes. The supernatant is the protein sample. Prepare an SDS-PAGE gel, run the gel, stain the protein gel with Coomassie Brilliant Blue, and destain overnight in distilled water on a shaker. After destaining, take a photograph.
[0034] (3) Purification According to the instructions for BBI Ni-NTA agarose purification resin (C600793), the storage buffer in the column was drained by gravity, and the column was equilibrated with 10 mL Binding / Wash Buffer. The sample solution was added to the column, and the flow-through was collected into a centrifuge tube and allowed to pass through twice. The column was washed with 10 mL Binding / Wash Buffer. The His-tagged protein in the column was eluted with 10 mL Elution Buffer, and the elution was repeated twice until the absorbance of the eluent at 280 nm was close to the baseline. The column material was eluted with 25 mL Elution Buffer, and the column material was equilibrated with 25 mL Binding / Wash Buffer. The column packing was washed with 25 mL ddH2O, and the column was protected with 20% ethanol and stored at 4°C.
[0035] (4) Determination of protein concentration by BCA method According to the instructions of the BCA protein quantification kit (G2026-200T) provided by Saivei, a protein standard curve was plotted using the enzyme-linked immunosorbent assay (ELISA) method, and the protein content of the sample was detected.
[0036] (5) Massive induction Expand 200 ml of bacterial culture; set up groups: blank group (PA group, PSNPs: 50 μg / L, As 2.5 μM), empty vector group (pET32a), inducer group (IPTG group) and experimental group (pET32a-JNK + PA group, PA: PSNPs 50 μg / L, As 2.5 μM); add 1 mM IPTG to each group of bacterial culture, induce at 28℃ for 8 h, after induction, centrifuge at 5000 rpm at room temperature for 10 min to collect bacterial cells, resuspend the bacterial cells in 5-10 mL Binding / Wash Buffer for purification.
[0037] The measurement results are as follows: (1) Optimization of JNK recombinant protein purification process Using *Cyprinus pleuropsus* cDNA as a template, a target band of approximately 1207 bp was amplified and ligated into the pET32a(+) plasmid via a source genome approach to form the recombinant plasmid pET32a-JNK. pET32a-JNK protein was induced using time- and IPTG concentration gradients; the detected OD values were substituted into a pre-plotted protein standard curve. Figure 1 The protein content obtained in A) was highest at an IPTG concentration of 1 mM, reaching 259.7 μg / mL. Figure 1 B), the highest protein level was 708.63 μg / mL after 8 h of IPTG induction (B). Figure 1 C), with an induction temperature of 25℃, the highest concentration was 720.65 μg / mL ( Figure 1 D).
[0038] (2) Effects of PSNPs and As stress on JNK protein expression The pET32a-JNK protein in each group was induced using the optimal induction conditions selected above; SDS-PAGE electrophoresis analysis was performed. Figure 2 Results: No purified band of pET32a-JNK protein was found in the blank group and the PA-treated group. A clear band appeared at 55 kDa in both the IPTG group and the IPTG + PA group, consistent with the predicted protein molecular weight. Compared with the IPTG group, the amount of pET32a-JNK protein was significantly reduced after treatment with PSNPs and As. Figure 3These results indicate that PSNPs and As can reduce the expression level of JNK protein in *Ceratophyllum demersum*.
[0039] (3) Antitoxic effects of JNK protein To investigate the role of JNK protein in the resistance of 293T cells to PSNPs and As-induced cytotoxicity, the pcDNA3.1-JNK plasmid was transfected into 293T cells; the experimental results are as follows. Figure 4 As shown, compared with the control group, PSNPs and As combined treatment for 48 h produced a certain cytotoxic effect on 293T cells; at the same time, the survival rate of 293T cells in the JNK overexpression group increased by 6.34%.
[0040] (4) Antioxidant effect of JNK protein like Figure 5 As shown, the paper disc diffusion method was used to investigate whether the JNK protein of the pleated crown mussel has antioxidant activity. Compared with the pET32a group, the inhibition zone of pET32a-JNK was reduced by 0.75, 0.71 and 0.76 times at H2O2 concentrations of 50 mM, 100 mM and 200 mM, respectively, but there was no significant change in the size of the inhibition zone at H2O2 concentration of 400 mM.
[0041] In summary, this invention optimized the induction and purification conditions of the key protein, JNK protein, from the pleated clam shell, through recombinant protein particle construction. Experimental studies revealed that the expression level of JNK protein reached its maximum of 720.65 μg / mL when the IPTG concentration was 1 mM, the induction temperature was 25℃, and the induction time was 8 h. Further investigation into the functional characteristics of JNK protein showed that it resists the combined toxicity of PSNPs and As, effectively promoting the survival of 293T cells. Simultaneously, JNK protein exhibited significant resistance to oxidative stress generated by H2O2, demonstrating clear antioxidant activity.
[0042] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for preparing a JNK recombinant protein, characterized by, The method comprises the following steps: S1, using cDNA of Echinoconchae as template, specific primers are used for PCR amplification to obtain amplification product; S2, using homologous recombination technology, the amplification product is connected to the vector to construct recombinant plasmid; S3, after the recombinant plasmid is induced and expressed, the JNK recombinant protein is purified.
2. The production method according to claim 1, characterized by, In S1, the specific primers are qpJNK-F: CTGAAGCCCTCTAACATT and qpJNK-R: TGGTCACTACCTGGAAACA.
3. The production method according to claim 1, characterized by, S3 comprises the following specific method: (1) IPTG is used for induction expression, after the induction is completed, the bacterial body is collected, PBS is used for washing and collecting the precipitate, Binding / Wash Buffer is added to the precipitate to resuspend the bacterial body to obtain resuspended bacterial liquid; (2) the resuspended bacterial liquid is subjected to ice bath ultrasonic treatment, and the total protein in the supernatant is collected; the total protein in the supernatant is added into buffer solution, and the metal bath protein mixture is collected to obtain the supernatant; (3) the supernatant is purified by using resin to obtain the JNK recombinant protein.
4. The production method according to claim 3, characterized by, The method of step (1) comprises: 50 μL of the bacterial liquid with correct sequencing is inoculated into 100 mL of LB culture medium containing corresponding antibiotics, and the culture is carried out at 37℃ until the OD600 value is 0.6-0.8; then, 0.5 mM-2 mM of IPTG is added, and the induction is carried out at 20℃-37℃ for 4-12 h; after the induction is completed, the bacterial body is collected by centrifugation under room temperature condition, and the precipitate is collected after being washed by 1×PBS; 2-5 mL of Binding / Wash Buffer is added to the precipitate to resuspend the bacterial body.
5. The preparation method according to claim 4, characterized in that, In step (1), the concentration of IPTG is 1 mM, and the induction is carried out at 25℃ for 8 h.
6. The preparation method according to claim 3, characterized in that, The method of step (2) comprises: the bacterial liquid is transferred to a clean EP tube, and ice bath ultrasonic treatment is carried out; the probe is cleaned with deionized water, and then the sample tube is placed in a beaker with ice water mixture, and the ice water interface is slightly higher than the bacterial liquid surface; the probe is immersed in the bacterial liquid; the power is 60%, the ultrasonic time is 2 sec, the interval is 2 sec, the ultrasonic time is 1-5 min, and the repeated ultrasonic treatment is carried out until the bacterial liquid becomes transparent or the bacterial liquid is no longer viscous; centrifugation is carried out at 8000 r / min and 4℃ for 10 min to collect the total protein in the supernatant; 100 uL of the supernatant is taken into a centrifuge tube, 5×protein loading Buffer 25 uL is added, and mixing is carried out; denaturation is carried out at 95℃ for 10 min, centrifugation is carried out at 12000 r / min and 4℃ for 5 min, and the supernatant is collected, which is the protein sample.
7. The preparation method according to claim 3, characterized in that, The method of step (3) comprises: The storage buffer in the Ni-NTA agarose purification resin was drained by gravity, and the column was equilibrated with 10 mL Binding / Wash Buffer; the sample was added to the column, and the flow-through was collected in a centrifuge tube; the flow-through was recirculated 2 times; the bound column was washed with 10 mL Binding / Wash Buffer; the His-tagged protein was eluted from the column with 10 mL Elution Buffer, and the elution was repeated twice until the absorbance of the eluate at 280 nm was close to the baseline; the column was washed with 25 mL Elution Buffer, and then equilibrated with 25 mL Binding / Wash Buffer; the column was washed with 25 mL ddH2O, and then protected with 20% ethanol and stored at 4°C.
8. A JNK recombinant protein, characterized by, The JNK recombinant protein is prepared by the method of any one of claims 1-7.
9. Use of the JNK recombinant protein of claim 8 in the preparation of an antioxidant product.
10. Use of the JNK recombinant protein of claim 8 in the preparation of an anti-toxicity product.