Preparation and application of bitter melon ribosome inactivating protein

Abstract

The present invention discloses the preparation and application of a bitter melon ribosome-inactivating protein, and belongs to the field of genetic engineering technology. The present invention successfully expresses a new bitter melon ribosome-inactivating protein, named bitter melon bryodin II‑like RIP protein, by constructing an in vitro prokaryotic expression system, and obtains a large amount of high-purity bitter melon ribosome-inactivating protein through purification by nickel column affinity chromatography. The target protein has good solubility, high expression level, biological activity, and a simple purification method. Using IPEC‑J2 as a cell model, the safe concentration range for the use of ribosome-inactivating protein was determined. It was then detected that under the action of protein concentrations of 50 μg / mL and 100 μg / mL, the ribosome-inactivating protein can inhibit the replication of pseudorabies virus, indicating that the protein has potential application prospects in anti-pseudorabies virus prevention and treatment drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to the preparation and application of a bitter melon ribosome inactivating protein. Background Art

[0002] Ribosome-inactivating proteins (RIPs) are a class of proteins with RNA N-glycosidase activity that are mainly found in plants. RIPs act on the 28S rRNA of the large subunit of the eukaryotic ribosome, destroying the ribosome structure and thus inhibiting protein synthesis.

[0003] Pseudorabies virus (PRV) is an enveloped, linear, double-stranded DNA virus morphologically similar to herpesviruses and classified as swine herpesvirus type 1. It can infect a variety of mammals, causing pseudorabies symptoms such as fever, severe itching (except in pigs), and encephalomyelitis. Mature PRV particles appear round or oval under an electron microscope, approximately 150-180 nm in diameter. They are composed of a double-stranded, linear genomic DNA, an icosahedral capsid, a protein envelope, and an envelope. The genome encodes 16 envelope proteins, 11 of which are glycosylated, such as gB, gC, gD, and gE.

[0004] Pigs are the natural reservoir for PRV, and symptoms of infection vary with age. Piglets primarily present with fever, neurological symptoms, and multi-organ failure, with a mortality rate of up to 100%. Adult pigs develop respiratory symptoms and can harbor the infection for life. Pregnant sows experience miscarriage and stillbirth. PRV can also cross species, infecting humans and causing endophthalmitis and encephalitis. It has become a significant threat to public health and animal husbandry. The development of anti-pseudorabies virus (PRV) drugs is crucial for PRV prevention and control. However, RIPs have not yet been used for PRV prevention and control. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation and application of a bitter melon ribosome inactivating protein to solve the problems existing in the above-mentioned prior art. The bitter melon ribosome inactivating protein can effectively inhibit the replication of pseudorabies virus.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of the bitter melon ribosome inactivating protein in any of the following:

[0008] (1) Application in the preparation of anti-pseudorabies drugs;

[0009] (2) Application in the preparation of drugs for inhibiting the replication of pseudorabies virus in cells;

[0010] The amino acid sequence of the bitter melon ribosome inactivating protein is shown in SEQ ID NO: 2.

[0011] The present invention also provides the use of a gene encoding a bitter melon ribosome inactivating protein in any of the following:

[0012] (1) Application in the preparation of anti-pseudorabies drugs;

[0013] (2) Application in the preparation of drugs for inhibiting the replication of pseudorabies virus in cells;

[0014] The nucleotide sequence of the coding gene is shown in SEQ ID NO: 1.

[0015] The present invention also provides the use of a recombinant vector containing a gene encoding a bitter melon ribosome inactivating protein in any of the following:

[0016] (1) Application in the preparation of anti-pseudorabies drugs;

[0017] (2) Application in the preparation of drugs for inhibiting the replication of pseudorabies virus in cells;

[0018] The amino acid sequence of the bitter melon ribosome inactivating protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1.

[0019] The present invention also provides the use of a recombinant bacterium containing a gene encoding a bitter melon ribosome inactivating protein in any of the following:

[0020] (1) Application in the preparation of anti-pseudorabies drugs;

[0021] (2) Application in the preparation of drugs for inhibiting the replication of pseudorabies virus in cells;

[0022] The coding gene is connected to an expression vector to construct a recombinant vector, so that the coding gene is integrated into the genome of the host bacteria through the recombinant vector; the amino acid sequence of the bitter melon ribosome inactivating protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the coding gene is shown in SEQ ID NO: 1.

[0023] The present invention also provides an in vitro non-therapeutic method for inhibiting pseudorabies virus, comprising the step of mixing bitter melon ribosome inactivating protein with a sample containing pseudorabies virus to inhibit the replication of pseudorabies virus, wherein the bitter melon ribosome inactivating protein is ≤100 μg / mL.

[0024] The present invention also provides a method for preparing bitter melon ribosome inactivating protein, comprising the following steps:

[0025] The bitter melon ribosome inactivating protein encoding gene is connected to an expression vector to construct a recombinant vector, and the recombinant vector is then transformed into Escherichia coli to construct a recombinant bacterium; the bitter melon ribosome inactivating protein amino acid sequence is shown in SEQ ID NO: 2;

[0026] The recombinant bacteria were cultured using IPTG induction, and the cells were collected; the cells were ultrasonically disrupted and centrifuged, and the supernatant was collected;

[0027] The supernatant is filtered and the protein is eluted using a nickel column. The eluate is collected and concentrated using an ultrafiltration concentration tube with a molecular weight cut-off of 10 KDa to obtain the bitter melon ribosome inactivating protein.

[0028] Optionally, the conditions for IPTG-induced culture of the recombinant bacteria are: 0.8 mM IPTG, inducing expression at 28° C. for 8 hours.

[0029] Optionally, before eluting the protein through the nickel column, the nickel column is first equilibrated with a binding buffer, wherein the binding buffer is a solution containing 20 mM sodium phosphate, 0.5 M NaCl, pH 7.41.

[0030] Optionally, the elution buffer is: 20 mM sodium phosphate, 0.5 M NaCl, 200 mM imidazole; 20 mM sodium phosphate, 0.5 M NaCl, 400 mM imidazole; 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole; the pH of the buffer is 7.49.

[0031] The present invention also provides an anti-pseudorabies drug, comprising the bitter melon ribosome inactivating protein prepared by the preparation method, wherein the bitter melon ribosome inactivating protein has a molecular weight of 39 KDa.

[0032] The present invention discloses the following technical effects:

[0033] The present invention achieves soluble expression of the target protein (bitter melon ribosome-inactivating protein) by constructing an in vitro prokaryotic expression vector and utilizing an Escherichia coli expression system. By optimizing the process conditions for preparing the bitter melon ribosome-inactivating protein, not only is the protein yield increased, but the biological activity of the protein is also ensured, providing a sufficient material basis for subsequent experimental research and application development.

[0034] The bitter melon ribosome-inactivating protein prepared in this study was tested using IPEC-J2 cells as a cell model. Cytotoxicity experiments determined the safe concentration range for its use, providing a scientific basis for its safe application. The results showed that at concentrations of ≤100 μg / mL, the protein had no significant effect on cell survival and exhibited activity in inhibiting pseudorabies virus infection.

[0035] The bitter melon ribosome-inactivating protein prepared in this invention has broad application prospects in the fight against pseudorabies virus. RT-qPCR confirmed the protein's inhibitory effect on pseudorabies virus. The discovery and application of this protein is expected to provide a more effective and safer means of preventing and treating pseudorabies virus, with significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 This is a diagram showing the results of high-fidelity enzyme (KOD) amplification of the bitter melon gene in Example 1 of the present invention; M: DL2000 DNA Marker; 1-2: are the target genes of the bitter melon;

[0038] Figure 2 This is a diagram showing the double enzyme digestion results of the bitter melon gene and the empty pET-28a(+) in Example 1 of the present invention; M: DL5000 DNA Marker; 1: enzyme digestion identification of the bitter melon target gene; 2: enzyme digestion identification of the empty pET-28a(+);

[0039] Figure 3 Graph showing the effect of different inducer (IPTG) concentrations on target protein expression in Example 2 of the present invention; 1: empty pET-28a(+) control; M: Protein Marker; 2: whole cells before induction; 3-6: whole cells induced with IPTG at final concentrations of 0.1 mM, 0.5 mM, 0.8 mM, and 1 mM, respectively;

[0040] Figure 4 Graph showing the effect of different induction times on target protein expression in Example 2 of the present invention; 1: empty pET-28a(+) control; M: Protein Marker; 2: whole cells before induction; 3-6: whole cells after induction at 28°C with 0.8 mM IPTG for 2, 4, 6, and 8 hours, respectively;

[0041] Figure 5 This is an SDS-PAGE electrophoresis diagram of large-scale induced expression and solubility analysis in Example 2 of the present invention; M: Protein Marker; 1: whole cells before induction; 2: whole cells after 28°C, 0.8 mM IPTG induction for 8 hours; 3: supernatant after ultrasonic disruption; 4: precipitate after ultrasonic disruption;

[0042] Figure 6This is an SDS-PAGE electrophoresis diagram of the purified protein eluted with gradient imidazole concentrations in Example 2 of the present invention; M: Protein Marker; 1: Protein flow-through; 2-7: Gradient imidazole elution of protein, 20 mM sodium phosphate, 0.5 M NaCl, containing 20 mM imidazole, 50 mM imidazole, 100 mM imidazole, 200 mM imidazole, 400 mM imidazole, and 500 mM imidazole, respectively;

[0043] Figure 7 This is a graph showing the results of a cytotoxicity test using CCK-8 to detect different concentrations of bitter melon bryodin II-like RIP protein in Example 3 of the present invention;

[0044] Figure 8 Figures 1 and 2 show the results of fluorescent quantitative PCR validation of the effect of bryodin II-like RIP protein in Momordica charantia on PRV replication in Example 3 of the present invention; A: Effect of bryodin II-like RIP protein in Momordica charantia on PRV replication at a concentration of 50 μg / mL; B: Effect of bryodin II-like RIP protein in Momordica charantia on PRV replication at a concentration of 100 μg / mL;

[0045] Figure 9 This is a graph showing the results of protein purification using the buffer used in the early stage of the present invention; M: Protein Marker; 1: Protein flow-through; 2-6: Gradient imidazole elution of protein, 20mM Tris, 50mM NaCl, containing 20mM imidazole, 50mM imidazole, 100mM imidazole, 200mM imidazole, and 400mM imidazole, respectively. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] This study used bioinformatics methods to screen the RIP gene family from the bitter melon (Momordica charantia L) database, identifying 25 bitter melon RIPs. A bitter melon RIP gene was selected, encoding a bryodinII-like RIP protein (SEQ ID NO: 2), a novel bitter melon ribosome-inactivating protein. A BLAST comparison of this protein with the known bitter melon MAP30 revealed 51.02% homology. The protein was expressed in an Escherichia coli expression system and purified by nickel affinity chromatography to obtain a soluble target protein. Its activity in inhibiting pseudorabies virus replication was tested.

[0052] Example 1

[0053] 1. Design and synthesis of primers for the bitter melon RIP gene

[0054] The present invention uses bioinformatics methods to screen the RIP gene family in the bitter melon (Momordica charantia L) database, obtaining 25 bitter melon RIPs. One bitter melon gene was selected and searched in the CugenDB database, obtaining the complete CDS sequence of the gene in the bitter melon (OHB3-1) v2 genome (see SEQ ID NO: 1). Based on the vector map of the prokaryotic expression vector pET-28a(+) and the CDS sequence of the target bitter melon gene, primer design software (PrimerPremier 5.0) was used to design full-length primers targeting the CDS of the target gene. Restriction enzymes that target the gene sequence were screened, and the enzymes used in the final design were Sac I and Sal I (underlined in Table 1). The upstream and downstream primers were synthesized by Sangon Biotech Co., Ltd.

[0055] Table 1 Primer sequences

[0056]

[0057] 2. High-fidelity enzyme (KOD) amplification of target gene

[0058] High-fidelity enzymes are also a type of DNA polymerase. Their unique feature is their proofreading function, enabling self-correction and significantly reducing the mismatch rate. The present invention uses KOD high-fidelity enzymes to perform PCR amplification of target genes. The reaction system and reaction procedure are shown in Tables 2 and 3 below.

[0059] Table 2 PCR reaction system

[0060]

[0061] Table 3 PCR reaction procedure

[0062]

[0063] After the PCR reaction, 15 μL of 6× Loading Buffer was added to the system and 1% agarose gel electrophoresis was performed. The results are shown in Figure 1 The specific target band size was approximately 942 bp, which was consistent with expectations.

[0064] 3. Gel excision and recovery of PCR products of target gene

[0065] a) Equilibration of adsorption column: Place adsorption column CA2 in a collection tube, add 500 μL of equilibration solution BL, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and return the column to the collection tube.

[0066] b) Gel cutting and weighing: Use a gel imager to cut the single DNA band containing the target gene from the agarose gel, removing as much excess as possible, and then place it in a clean centrifuge tube and weigh it.

[0067] c) Dissolving the gel: Add an equal volume of solution PN to the gel and place in a 50°C water bath. Gently invert the tube upside down to ensure complete dissolution of the gel.

[0068] d) DNA Adsorption: Add the solution from the previous step to the adsorption column CA2 (place the adsorption column in the collection tube). Incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 30-60 seconds. Discard the waste liquid from the collection tube and return the adsorption column CA2 to the collection tube. If the sample volume is larger than 800 μL, add the solution in batches.

[0069] e) Rinsing the adsorption column: Add 600 μL of rinsing solution PW to the adsorption column CA2 (please check whether anhydrous ethanol has been added before use). Centrifuge at 12,000 rpm for 30-60 seconds. Discard the waste liquid in the collection tube and return the adsorption column CA2 to the collection tube.

[0070] f) Removal of residual rinse solution: Return the CA2 column to the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much of the rinse solution as possible. Allow the CA2 column to dry thoroughly at room temperature for several minutes to prevent residual rinse solution from interfering with subsequent experiments.

[0071] g) DNA Elution: Place the adsorption column CA2 in a clean centrifuge tube. Add an appropriate amount of Elution Buffer EB dropwise to the center of the adsorption membrane. Incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution. The DNA product should be stored at -20°C to prevent DNA degradation.

[0072] 4. Enzyme digestion of target gene and ligation with vector

[0073] The recovered PCR product and the empty vector pET-28a(+) were first digested with the restriction endonucleases Sac I and Sal I determined during primer design at 37°C for 3-4 hours. The digestion system is shown in Table 4 below.

[0074] Table 4 Enzyme digestion system

[0075]

[0076] After the reaction was completed, 15 μL of 6× Loading Bμffer was added to the reaction system. The PCR product was detected by 1% agarose gel electrophoresis. The results are shown in Figure 2 At this time, it is necessary to repeat the steps of cutting the gel and recovering the product by electrophoresis the target gene band and the vector band.

[0077] Vector ligation: The target gene product recovered from the gel was ligated with the vector product using T4 ligase at room temperature for 1 h. The ligation system is shown in Table 5 below.

[0078] Table 5 Connection system

[0079]

[0080] 5. Transformation of DH5α competent cells

[0081] Thaw the competent cells on ice. Add the target DNA to the competent cell suspension, gently mix the contents, and place on ice for 30 minutes. Heat shock the centrifuge tube in a 42°C water bath for 90 seconds, then quickly transfer to an ice bath and place for 2-3 minutes, taking care not to shake the centrifuge tube. Add 900 μL of sterile, antibiotic-free LB medium to the centrifuge tube and incubate at 37°C with shaking at 180 rpm for approximately 45 minutes. This is to allow expression of the relevant resistance marker gene on the plasmid and recovery of the bacteria. Spread 150 μL of the transformed competent cells evenly on an LB agar plate containing kanamycin (Kan, 50 mg / mL). Incubate the plate upside down at 37°C in a humidified incubator for 12-16 hours. Once colonies have grown, select positive single colonies based on their morphology and size for subsequent identification and culture.

[0082] 6. Single colony PCR and shaking

[0083] Remove the overnight cultured LB agar plate, select a single positive colony and mark it with a serial number. Prepare a new LB agar plate, mark the serial position, and add the selected positive single colony to the PCR system (see Table 6).

[0084] Table 6 Colony PCR system

[0085]

[0086] The PCR reaction program is shown in Table 7 below.

[0087] Table 7 PCR reaction procedure

[0088]

[0089] After the reaction, prepare a 1% agarose gel for electrophoresis. Load 7 μL of sample and 5 μL of DL2000 marker. Maintain a constant voltage of 110 V for 25 minutes and image the gel under a UV gel imager. Based on the electrophoresis pattern, identify the correct band (i.e., a positive single colony) on the streaked plate. Use a pipette tip to streak the colony and inject it into the prepared LB liquid medium. Incubate the plate in a shaker at 37°C and 180 rpm overnight.

[0090] 7. Plasmid extraction and testing

[0091] Use the Gold Plasmid Miniprep Kit according to the manufacturer's instructions: Centrifuge the overnight bacterial culture at 10,000 g for 1 minute and discard the supernatant. Add colorless RB solution containing RNase A and vortex to resuspend the bacterial pellet; no small bacterial clumps should remain. Add blue LB solution and gently invert the mixture 4-6 times to fully lyse the cells, forming a clear blue solution. Then add yellow NB solution and gently mix 5-6 times until a firm yellow clumping mass forms. Let it stand at room temperature for 2 minutes. Centrifuge the solution at 12,000 g for 1 minute and discard the flow-through. Finally, add 650 μL of WB solution and centrifuge at 12,000 g for 1 minute. Discard the flow-through and centrifuge again at 12,000 g for 1-2 minutes to completely remove any residual WB. Place the spin column in a clean microcentrifuge tube and add 31 μL of LB to the center of the column. Let it stand at room temperature for 1 minute, then centrifuge at 10,000 g for 1 minute. Elute the DNA and store at -20°C.

[0092] Example 2

[0093] 1. Transformation of recombinant plasmid

[0094] Add the constructed plasmid to a 1.5mL EP tube containing 100μL of BL21(DE3) competent cells. Place the tube on ice for 30 minutes, heat shock the tube in a 42°C water bath for 90 seconds, and place it on ice again for 3 minutes. Add 900μL of LB liquid medium (without antibiotics) to the tube, mix thoroughly, and incubate the tube with shaking at 37°C for 1 hour to allow the bacteria to resume normal growth and express the antibiotic resistance gene encoded by the plasmid. Remove the EP tube and centrifuge at 2500 rpm for 5 minutes. Remove the supernatant and gently pipette the suspended bacteria. Spread the suspension onto a selection plate containing kanamycin. Once the suspension has been completely absorbed by the medium, invert the plate and incubate overnight at 37°C.

[0095] 2. Optimization of expression conditions for bitter melon bryodin II-like RIP protein induced by IPTG in a small amount

[0096] Different induction conditions were set to test the expression of bitter melon bryodin II-like RIP protein. In order to determine the optimal induction concentration and time, a small amount of expression was induced at a uniform temperature of 28°C. To explore the effect of different inducer (IPTG) concentrations on the expression of the target protein, glycerol bacteria transformed into BL21 (DE3) and pET-28a (+) were taken out and shaken overnight. The next day, the bacterial liquid was inoculated into a new LB medium containing antibiotics and shaken at 37°C at 200 rpm until the OD 600At around 0.6-0.8, 1 mL of bacterial culture was removed for lysis as the pre-induction sample. IPTG was then added to final concentrations of 0.1 mM, 0.5 mM, 0.8 mM, and 1 mM, respectively. The cells were shaken at 28°C, 160 rpm, and induced for 8 h. Again, 1 mL of bacterial culture was removed for lysis as the post-induction sample. Analysis was performed on 12% separating gel by SDS-PAGE.

[0097] See the results Figure 3 A new protein band appeared at approximately 39KDa, which was basically consistent with the theoretical prediction value. After BL21 was transformed with empty pET-28a(+), no protein band appeared at the same position. The results showed that 0.8mM can induce the expression of the target protein. With the increase of concentration, the expression level of the target protein did not change much. Therefore, 0.8mM was selected as the induction concentration in subsequent experiments.

[0098] To investigate the effect of different induction times on the expression of the target protein, the glycerol bacteria transformed into BL21 (DE3) and the pET-28a (+) empty load were taken out and shaken overnight. The next day, the bacterial liquid was inoculated into new LB medium and shaken at 37°C and 200 rpm until the OD 600 At around 0.6-0.8, 1 mL of bacterial solution was taken out for lysis as the pre-induction sample, and then IPTG with a final concentration of 0.8 mM was added. The mixture was placed in a shaker at 28°C and 160 rpm. After 2 h, 4 h, 6 h, and 8 h of induction, 1 mL of bacterial solution was taken out for lysis as the post-induction sample and detected by SDS-PAGE using 12% separation gel.

[0099] See the results Figure 4 , indicating that with the extension of induction time, the expression level of the target protein gradually increased. After 8 hours of induction, the expression level at 39KDa was the highest, so the induction time was selected as 8 hours.

[0100] Based on the above experiments, we determined that the optimal induction conditions for the expression of the bitter melon bryodin II-like RIP protein were 0.8 mM IPTG and 28°C for 8 hours. This not only achieved efficient expression but also increased the expression level and solubility of the target protein. We then used these conditions to express the target protein in large quantities.

[0101] 3. Inducible expression of bryodin II-like RIP protein in bitter melon and purification of protein by affinity chromatography

[0102] At a 1:500 inoculation rate, add the glycerol bacteria to LB medium containing kanamycin and culture overnight at 200rpm and 37℃. The next day, inoculate all the bacteria into a 3L LB conical flask containing kanamycin resistance and culture at 200rpm and 37℃ until the OD 600The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.8 mM, and the cells were cultured in a shaker at 28°C and 160 rpm for 8 h. The cells were then centrifuged to collect the bacteria and extract the protein.

[0103] Harvest the cells (all steps below should be performed at low temperature): Centrifuge at 4500 rpm for 15 minutes, remove the supernatant, resuspend the pellet in PBS at a volume of 10 mL / 1 g, and collect in a small vial. Sonicate on ice (5 seconds, 5 seconds intervals, 80% power, for a total of 5 minutes, until the solution becomes transparent, which is sufficient to effectively disrupt cells without destroying the target protein). Centrifuge at 10000 rpm for 30 minutes, remove the pellet, and sterilize the supernatant by filtering through 0.45 μm and 0.22 μm filters. Place on ice until needed.

[0104] First, fill the silicone tube with water and fix it with a peristaltic pump to keep liquid at the connection to avoid introducing bubbles into the system. TM Connect the column to a 5mL HP nickel column. Remove the stopper from the bottom of the column and wash away the ethanol with 3 column volumes of deionized water. Equilibrate the column with at least 5 column volumes of 20mM sodium phosphate, 0.5M NaCl, pH 7.41 buffer at a flow rate of 2mL / min. Then, flow the treated supernatant into the nickel column via a peristaltic pump at a rate of 1mL / min. After the supernatant is loaded onto the column, elute with buffer containing various imidazole concentrations: 20mM, 50mM, 100mM, 200mM, 400mM, and 500mM, maintaining a flow rate of 2mL / min. Prior to this, the inventors used a binding buffer of 20mM Tris, 50mM NaCl, pH 7.5, and an elution buffer of 20mM Tris, 50mM NaCl, 400mM imidazole, pH 7.5 for their initial purification. The elution buffer was diluted to different imidazole concentrations, namely 20mM, 50mM, 100mM, 200mM, and 400mM. The lower ionic strength (only 50mM NaCl) resulted in a less tight binding of the target protein to the affinity medium, affecting the purification effect. Figure 9 shown.

[0105] After the improvement, the collected proteins eluted under different gradients were tested by 12% SDS-PAGE. The electrophoresis results are shown in Figure 5 and Figure 6 ,first Figure 5 This indicates that the expression was successfully induced after adding IPTG, and the protein appeared in the supernatant in a soluble form. Figure 6It was shown that the target protein could be eluted at imidazole concentrations of 200mM, 400mM, and 500mM. The collected fluids at these three concentrations were concentrated using an ultrafiltration tube with a 10kDa molecular cutoff. After concentration to a certain volume, an imidazole-free buffer was added. The salt concentration was diluted by changing the fluid, and the protein after removing the imidazole was packaged and added with glycerol to a final concentration of 10%. The solution was quick-frozen with liquid nitrogen and stored at -80°C.

[0106] These results demonstrate that the improved binding buffer, consisting of 20 mM sodium phosphate, 0.5 M NaCl, pH 7.41, utilizes a higher NaCl concentration (0.5 M) to facilitate binding of the target protein to the affinity matrix. The elution buffer, consisting of 20 mM sodium phosphate, 0.5 M NaCl, and 500 mM imidazole, pH 7.49, provides stronger competitive binding and more efficient elution of the target protein from the matrix. Furthermore, the sodium phosphate buffer system exhibits superior buffering capacity and chemical stability in the presence of high salt and imidazole concentrations, maintaining a stable pH during the elution process. By diluting the elution buffer to varying imidazole concentrations (20 mM, 50 mM, 100 mM, 200 mM, 400 mM, and 500 mM) for gradient elution, the target protein can be more precisely separated from contaminants based on the strength of protein-matter binding, significantly improving protein elution efficiency and purity.

[0107] Example 3

[0108] 1. CCK-8 assay to detect the effect of bitter melon bryodin II-like RIP protein on cytotoxicity

[0109] A healthy IPEC-J2 cell culture flask grown to 80% confluency was obtained. After digestion, the suspension was transferred to a centrifuge tube and pipetted evenly. A cell control group, a cell-free blank group, and an experimental group were set up. 10 μL of the suspension was counted, diluted as needed, and evenly plated onto a 96-well plate. 100 μL of cell suspension was added to each well of the experimental and cell control groups, and 100 μL of complete culture medium was added to each well of the cell-free blank group. After incubation at 37°C, 5% CO₂ to 80% confluence, a protein concentration gradient was established (from high to low, 200, 100, 50, 25, 12.5, and 6.25 μg / mL). The protein was diluted with culture medium, with six replicate wells set for each concentration. The culture medium was aspirated and discarded. The experimental group was treated with culture medium containing protein, while the cell control and cell-free blank groups were treated with 100 μL of complete culture medium. The plates were incubated at 37°C, 5% CO₂ for 24 h. Take out the 96-well plate and add 10 μL of CCK-8 solution to each well. When adding the reagent, add it slowly to avoid bubbles. Place it in a 37°C, 5% CO2 incubator and culture for 2 hours until a distinct orange-yellow color appears. Place it in a multifunctional microplate reader to measure the absorbance at 450 nm.

[0110] According to the cell viability formula, the results are as follows Figure 7 As shown in the results, it was found that protein concentrations of 50, 25, 12.5, and 6.25 μg / mL had little effect on cell survival, while a protein concentration of 100 μg / mL had some effect, but the effect was not significant. The cell viability values ​​were all above 80% compared with the control group. A protein concentration of 200 μg / mL had a more significant effect on cell survival. Therefore, a protein concentration in the range of 6.25-100 μg / mL can be selected.

[0111] 2. Fluorescence quantitative PCR verification of the effect of bitter melon bryodin II-like RIP protein on PRV replication

[0112] IPEC-J2 cells were inoculated into 24-well cell culture plates and cultured in a 37°C, 5% CO2 incubator. When the cells in the 24-well plate grew to 80%-90% confluence, IPEC-J2 cell group, PRV group, and PRV + protein experimental group were set up, with 3 replicate wells set up for each group.

[0113] ① Add 2% FBS DMEM medium to the IPEC-J2 cell group and PRV group. Add 100 μg / mL and 50 μg / mL bitter melon RIP protein (diluted with 2% FBS DMEM medium) to the PRV+protein experimental group, respectively. Each well contains 500 μL and pre-treat in a 37°C, 5% CO2 incubator for 1 hour.

[0114] ② After treatment, the cells were discarded and washed three times with PBS. Then, serum-free PRV virus solution (MOI = 0.1) was added to the PRV group and PRV+protein experimental group, respectively. 100 μg / mL and 50 μg / mL of bitter melon RIP protein diluted in serum-free medium were also added to the virus solution, respectively. Serum-free DMEM was added to the IPEC-J2 cell group and incubated in a 37°C, 5% CO2 incubator for 1.5 h.

[0115] ③ After challenge, cells were washed three times with PBS. The IPEC-J2 and PRV groups were replaced with virus maintenance medium (2% FBS-containing DMEM). The PRV and bitter melon protein groups were replaced with 2% FBS-containing DMEM containing 100 μg / mL and 50 μg / mL protein, respectively. Culture was continued for 48 hours. Microscopic observation of cells for CPE after 48 hours revealed a series of pathological changes associated with PRV infection, including cell rounding, detachment, and floating. The cells were stored in a -80°C freezer and subjected to three freeze-thaw cycles. The supernatant was collected by centrifugation at 12,000 rpm for 5 minutes. 200 μL of the viral stock solution was transferred to a 1.5 mL centrifuge tube. Viral DNA was extracted using the SteadyPμre Viral DNA / RNA Extraction Kit and subjected to SYBR Green qPCR. The primers are shown in Table 8.

[0116] Table 8 Primer sequences

[0117]

[0118] The results showed that IPEC-J2 cells were pretreated with 50 μg / mL and 100 μg / mL of bitter melon bryodin II-like RIP protein and then infected with PRV for 48 hours. The PRV viral copy number was detected by RT-qPCR. Figure 8 The results showed that 48 hours after infection with PRV, the infection amount of PRV was reduced to varying degrees after pretreatment with the two protein concentrations, and the amount of virus was reduced, indicating that the replication of PRV could be effectively inhibited.

[0119] SEQ ID NO: 1, which is the CDS sequence of the bitter melon bryodin II-like RIP protein:

[0120] 。

[0121] SEQ ID NO: 2 is the amino acid sequence of the protein ribosome-inactivating protein bryodin II-like [Momordica charantia] encoded by the gene shown in SEQ ID NO: 1:

[0122] MECVRSSEPFIQPLPFNDIGIHGVHANGAVNAPNSNVSFSLLGATGKTYQQFIQNLRNALTINSKIVYNIPVLAATALSSARFILVHLTNYKNETITVAIDVVNVYIVAYQAGNKAYFLKDASKEARDVLFKGIKQEILPYKGNYDGLETAAGK ISREKIDLGFSELGSAIGNMYHYNAGTSVPRAFIVMIQTISEAARFKYIETKVSQNVETKFKPDPGFLSLENRWSDLSEQVQIAQNRKGEFARPIEVRSVTNKPILVTNVKSKVVEGMALLLYSKPRGNGDLMELMEEFNKEWENGRGEFGIDF.

[0123] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of bitter melon ribosome inactivating protein in any of the following: (1) Application in the preparation of anti-pseudorabies drugs; (2) Application in the preparation of drugs for treating pseudorabies virus infection; The amino acid sequence of the bitter melon ribosome inactivating protein is shown in SEQ ID NO:

2.

2. Use of the gene encoding the bitter melon ribosome inactivating protein in any of the following: (1) Application in the preparation of anti-pseudorabies drugs; (2) Application in the preparation of drugs for treating pseudorabies virus infection; The nucleotide sequence of the coding gene is shown in SEQ ID NO:

1.

3. Use of a recombinant vector containing a gene encoding a bitter melon ribosome-inactivating protein in any of the following: (1) Application in the preparation of anti-pseudorabies drugs; (2) Application in the preparation of drugs for treating pseudorabies virus infection; The amino acid sequence of the bitter melon ribosome inactivating protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

1.

4. Use of a recombinant bacterium containing a gene encoding a bitter melon ribosome-inactivating protein in any of the following: (1) Application in the preparation of anti-pseudorabies drugs; (2) Application in the preparation of drugs for treating pseudorabies virus infection; The coding gene is connected to an expression vector to construct a recombinant vector, so that the coding gene is integrated into the genome of the host bacteria through the recombinant vector; the amino acid sequence of the bitter melon ribosome inactivating protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the coding gene is shown in SEQ ID NO:

1.

5. A method for inhibiting pseudorabies virus in vitro for non-therapeutic purposes, characterized in that: The method comprises the steps of mixing bitter melon ribosome inactivating protein with a sample containing pseudorabies virus to inhibit the replication of pseudorabies virus, wherein the concentration of the bitter melon ribosome inactivating protein is 50-100 μg / mL, and the amino acid sequence of the bitter melon ribosome inactivating protein is shown in SEQ ID NO: 2.

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