Griffithsin-IL18 fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a Griffithsin-IL18 fusion protein and a preparation method and application thereof.The Griffithsin-IL18 fusion protein comprises a Griffithsin protein fragment, an IL18 protein fragment and a flexible connecting peptide connecting the Griffithsin protein fragment and the IL18 protein fragment, the Griffithsin protein fragment is located at the N end of the fusion protein, and the IL18 protein fragment is located at the C end of the fusion protein. According to the present invention, the dual functions of virus infection blocking and immune activation are achieved, the Griffithsin fragment is specifically combined with host cell surface integrin to induce internalization and block virus endocytosis, and the IL-18 fragment activates NK cells so as to improve the overall antiviral efficiency and achieve the precise targeting intervention on the infected cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Griffithsin-IL18 fusion protein, its preparation method, and its application. Background Technology

[0002] Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections worldwide, and persistent infection with high-risk types has become a major cause of malignant tumors such as cervical cancer, anal cancer, and oropharyngeal cancer. This virus is transmitted through mucosal contact, has a long incubation period, and is difficult to detect, resulting in millions of new infections globally each year and causing numerous cancer-related deaths. Although preventative HPV vaccines, such as virus-like particle-based vaccines, have been developed and have shown excellent results in reducing the risk of new infections, their effectiveness in treating patients already infected is limited. Current treatment options mainly rely on surgical interventions, topical medications, or immunosuppressants, but these methods often fail to completely eliminate the virus, leading to frequent relapses, and lack precise interventions targeting the viral lifecycle. Particularly in developing countries, limited medical resources further exacerbate the public health burden of HPV-related diseases.

[0003] Griffithsin is a lectin protein isolated from red algae with broad-spectrum antiviral activity. Its main mechanism involves high-affinity binding to glycoproteins on the viral envelope, such as the L1 major capsid protein of HPV, thereby blocking the interaction between the virus and host cell receptors and preventing viral adsorption and entry into cells. Multiple studies have confirmed that Griffithsin exhibits significant inhibitory effects against various HPV types, including high-risk types such as HPV16 and HPV18. The broad-spectrum nature and low toxicity of this protein make it a potential local antiviral agent. However, Griffithsin has limitations: it primarily acts in the early stages of viral transmission and lacks the ability to directly activate the host immune system, failing to effectively clear infected cells, potentially leading to viral residues and chronic infection.

[0004] Interleukin-18 (IL-18), a member of the IL-1 cytokine family, is an important pro-inflammatory cytokine that plays a central role in immune regulation. NK cells (natural killer cells) highly express IL-18 receptors on their surface. IL-18 can strongly activate NK cell function by binding to these receptors, including promoting the production of interferon-γ (IFN-γ) and enhancing cytotoxic activity. This activation helps the host immune system recognize and eliminate virus-infected cells, thereby enhancing the overall antiviral response. Currently, various antiviral strategies, such as antiviral drugs and small molecule inhibitors, have been explored clinically. However, these methods often have limited functionality and cannot simultaneously achieve both viral blockade and immune enhancement. While there are reports of using cytokines such as IL-18 to enhance immunotherapy, these strategies are susceptible to neutralizing antibodies and lack specific targeting mechanisms, leading to unstable efficacy. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Griffithsin-IL18 fusion protein.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned Griffithsin-IL18 fusion protein.

[0007] Another object of the present invention is to provide the application of the above-mentioned Griffithsin-IL18 fusion protein.

[0008] The technical solution of the present invention is as follows:

[0009] A Griffithsin-IL18 fusion protein comprises a Griffithsin protein fragment, an IL18 protein fragment, and a flexible linker peptide connecting the Griffithsin and IL18 protein fragments, wherein the Griffithsin protein fragment is located at the N-terminus of the fusion protein and the IL18 protein fragment is located at the C-terminus of the fusion protein.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of the Griffithsin protein fragment is shown in SEQ ID NO.01, and the amino acid sequence of the IL18 protein fragment is shown in SEQ ID NO.03.

[0011] In a preferred embodiment of the present invention, the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO. 05.

[0012] The preparation method of the above-mentioned Griffithsin-IL18 fusion protein includes the following steps:

[0013] (1) Construction of expression vector: Insert the nucleotide sequence of Griffithsin protein fragment as shown in SEQ ID NO.02, the nucleotide sequence of flexible linker peptide as shown in SEQ ID NO.06, and the nucleotide sequence of IL18 protein fragment as shown in SEQ ID NO.04 into the expression vector to form a fusion protein expression cassette;

[0014] (2) Transformation of host cells: The expression vector constructed in step (1) is transformed into host cells;

[0015] (3) Induction of expression: Under appropriate conditions, the host cells obtained in step (2) are induced to express the Griffithsin-IL18 fusion protein;

[0016] (4) Extraction and purification: The host cells obtained in step (3) were lysed, and the Griffithsin-IL18 fusion protein was extracted and purified.

[0017] In a preferred embodiment of the present invention, the expression vector is pET-28a(+).

[0018] In a preferred embodiment of the present invention, the host cell is Escherichia coli DH5α and / or BL21(DE3).

[0019] The application of the above-mentioned Griffithsin-IL18 fusion protein in the preparation of pharmaceutical compositions that enhance the antiviral function of NK cells.

[0020] The application of the above-mentioned Griffithsin-IL18 fusion protein in the preparation of pharmaceutical compositions for the prevention or treatment of HPV infection and related diseases.

[0021] A pharmaceutical composition comprising the above-mentioned Griffithsin-IL18 fusion protein as its active ingredient.

[0022] In a preferred embodiment of the present invention, the dosage form is a gel, spray, or injection.

[0023] The beneficial effects of this invention are:

[0024] 1. This invention achieves the dual functions of blocking viral infection and activating immunity. By specifically binding to host cell surface integrins through the Griffithsin fragment, it induces internalization and blocks viral endocytosis. At the same time, the IL-18 fragment activates NK cells, enhances overall antiviral efficacy, and achieves precise targeted intervention on infected cells.

[0025] 2. This invention enhances the cytotoxic activity of NK cells, promotes targeted killing of virus-infected cells, improves the host's immune system response, and reduces the risk of viral residue and recurrence in the body.

[0026] 3. This invention increases the level of IFN-γ secreted by NK cells, strengthens the immune regulation mediated by inflammatory factors, helps in virus clearance and infection control, and enhances the body's resistance to various viruses.

[0027] 4. This invention is applicable to the prevention and treatment of HPV infection and related diseases, such as cervical intraepithelial neoplasia, genital warts, anal cancer and oropharyngeal cancer, and has potential clinical translational value and broad application prospects. It can be used as a supplementary therapy to existing vaccines.

[0028] 5. The fusion protein of the present invention has a stable structure, and the flexible linker peptides ensure independent folding of functional domains and maintenance of activity, which facilitates optimized production in different expression systems and reduces the risk of protein degradation.

[0029] 6. The preparation method of the present invention is highly efficient, including plasmid construction, host transformation, expression induction and purification steps, and is easy to implement on a large scale and control quality, making it suitable for industrial-grade protein production processes;

[0030] 7. The present invention can be formulated into a variety of pharmaceutical compositions, such as gels, sprays or injections, to improve the convenience and stability of local administration, expand its applicability in the biomedical field, and improve patient compliance.

[0031] 8. This invention exhibits low toxicity and biocompatibility, reducing potential side effects such as excessive immunogenicity or cytotoxicity, which is beneficial for long-term use and clinical safety assessment.

[0032] 9. This invention provides a novel multifunctional biological agent design paradigm that can be extended to treatment strategies for other viral infections, promoting innovation and development in the biomedical field. Attached Figure Description

[0033] Figure 1 This displays a map of the fusion protein expression plasmid in Example 1 of the present invention.

[0034] Figure 2 The image shows the purified fusion protein analyzed by SDS-PAGE in Example 1 of this invention.

[0035] Figure 3 This demonstrates that in Example 2 of the present invention, the fusion protein activates NK cells to secrete INF-γ.

[0036] Figure 4 This demonstrates the antiviral infection effect of the Griffithsin-IL18 fusion protein in Example 3 of the present invention.

[0037] Figure 5 This demonstrates that the Griffithsin-IL18 fusion protein in Example 4 of the present invention promotes NK cell killing activity. Detailed Implementation

[0038] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0039] Example 1: Construction and preparation of Griffithsin-IL18 fusion protein

[0040] (1) Plasmid construction

[0041] The amino acid sequence of Griffithsin is shown in SEQ ID NO.01:

[0042] SLTHRKFGGSGGSPFSGLSSIAVRSGSYLDAIIIDGVHHGGSGGNLSPTFTFGSGEYISNMTIRSGDYIDNISFETNMGRRFGPYGGSGGSANTLSNVKVIQINGSAGDYLDSLDIYYEQY,

[0043] Nucleotide sequence such as SEQ ID NO.02 shows: agtctgacccatcgtaaatttggcggcagtggtggtagtccgtttagcggtctgagtagcattgcagttcgcagcggtagctatctggatgcaattattattgatggcgttcatcatggtggcagtggcggcaatctgagcccgacctttacctttggcagtggtgaatatattagta atatgaccatccgtagtggtgactatattgataatattagtttcgagaccaacatgggccgtcgctttggtccgtatggtggtagtggcggcagcgccaataccctgagcaatgttaaagttattcagattaacggtagcgcaggtgactatctggatagtctggatatctattatgaacagtat;

[0044] The amino acid sequence of IL18 is shown in SEQ ID NO.03:

[0045] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED,

[0046] Nucleotide sequence such as SEQ ID NO.04 shows: tattttggtaaactggaaagtaaactgagtgtgattcgtaatctgaatgatcaggtgctgtttattgatcagggtaatcgcccgctgtttgaagatatgaccgatagtgatt gccgtgataatgccccgcgtaccatttttattattagcatgtataaggacagccagccgcgtggtatggccgtgaccattagtgtgaaatgcgaaaaaattagcaccctgagctgcgaaa ataagattattagctttaaggagatgaacccgccggataatattaaggataccaaaagtgatatcatcttctttcagcgtagcgttccgggccatgataataagatgcagtttgaaagta gcagttatgaaggctattttctggcctgcgaaaaagaacgtgatctgtttaaactgattctgaaaaaagaggatgagctgggtgaccgcagtattatgtttaccgttcagaatgaagat;

[0047] Using pET-28a(+) as the expression vector, Griffithsin and IL18 protein expression sequences were inserted into the multiple cloning site of the pET-28a vector. The two sequences were then linked into a fusion protein expression cassette using a linker (amino acid sequence as shown in SEQ ID NO.05: GGGGSGGGGSGGGS, nucleotide sequence as shown in SEQ ID NO.06: ggcggcggcggcagcggcggcggcggcggcggcggcggcggcggcggcggcggcggc). This fusion protein was synthesized by General Biotech and named pET28a-Griffithsin-IL18 plasmid. Figure 1 The image shown is a map of the fusion protein expression plasmid.

[0048] (2) Amplification of pET28a-Griffithsin-IL18 plasmid:

[0049] The synthesized pET28a-Griffithsin-IL18 plasmid was electroporated into Escherichia coli DH5α competent cells, and single colonies of the recombinant strain were obtained after kanamycin resistance screening.

[0050] Single colonies were inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 200 rpm; the culture was continued until OD600 = 0.6~0.8; the plasmid was extracted and purified using an endotoxin-free plasmid extraction kit.

[0051] (3) Production of fusion proteins:

[0052] The purified pET28a-Griffithsin-IL18 was electrotransformed into competent Escherichia coli BL21(DE3) cells. The culture was added to 500 μL of antibiotic-free LB medium and incubated at 37°C with shaking at 200 rpm for 1 h. Then, 5 mL of LB medium containing 50 μg / mL kanamycin was added, and the culture was incubated at 37°C with shaking at 200 rpm until the culture became turbid. A portion of the culture was centrifuged, and the culture was preserved with 15% glycerol. The remaining culture was transferred to 250 mL of LB medium and incubated overnight at 37°C with shaking at 200 rpm. Then, 250 mL of the culture was transferred to 1 L of LB medium containing 50 μg / mL kanamycin and incubated at 37°C with shaking at 200 rpm until the logarithmic growth phase (OD600 = 0.5). IPTG (final concentration 1 mM) was added, and the culture was incubated overnight at 37°C with shaking at 200 rpm.

[0053] (4) Protein extraction:

[0054] Pre-cool the centrifuge, centrifuge the bacterial culture obtained in step (3) at 4℃ and 5000 g for 10 min, collect the precipitate, resuspend it in PBS (pH 7.4) (bacterial precipitate volume: PBS volume = 1:5), add a mixture of protease phosphatase inhibitors (general type, 50×), freeze at -80℃ for 30 min, take it out and thaw it, and then place it on ice for ultrasonic disruption (on ice, power 200 W, working for 2 s, intermittent for 4 s, total duration 30 min, intensity 100%). Centrifuge the obtained lysate at 4℃ and 10000 g for 30 min, and collect the supernatant.

[0055] (5) Protein purification:

[0056] The supernatant obtained in step (4) was passed through a Ni-NTA agarose affinity chromatography column (pre-equilibrated). Impurities were washed with wash buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0), followed by elution with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 8.0). The eluted protein was desalted using an ultrafiltration tube (molecular weight cutoff 10 kDa) and stored at -20°C for later use. An appropriate amount of the purified protein was analyzed by SDS-PAGE. Figure 2 As shown, a single clear band is observed at approximately 33.5 kDa, with a purity >95%.

[0057] Example 2: NK cell activation experiment

[0058] NK cells (purity >90%) were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy volunteers using an NK cell isolation kit. The NK cells were then processed at a concentration of 1 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 1 / mL in 96-well plates, with 100 μL of KBM581 medium (containing 200 U / mL IL-2 and 80 U / mL gentamicin) per well. GRFT-IL18 fusion protein (final concentration 100 ng / mL) was added, while the control group received an equal volume of PBS or IL18 alone (100 ng / mL). Cells were co-cultured at 37°C and 5% CO2 for 24 h. After culture, the supernatant was collected, and the IFN-γ concentration in the supernatant was detected using a human IFN-γ ELISA kit, following the kit instructions. Absorbance was read at 450 nm using a microplate reader. Figure 3 As shown, the IFN-γ secretion level in the fusion protein treatment group was significantly higher than that in the IL18-only group and the control group. The IFN-γ secretion level in the 100 ng / mL fusion protein treatment group was 1550 pg / mL, which was significantly higher than that in the IL18-only group (580 pg / mL) and the control group (<50 pg / mL).

[0059] Example 3: Antiviral infection experiment using Griffithsin-IL18 fusion protein

[0060] 293FT cells were infected using fluorescently labeled HPV pseudovirus (GFP reporter gene). First, 293FT cells were seeded at a density of approximately 1.5 × 10⁴ cells per well in 96-well plates and pre-cultured at 37°C with 5% CO₂ for 4–6 h to allow adhesion. The experimental setup included: a blank control group with only cells cultured, a virus control group with only HPV pseudovirus, and an experimental group with both HPV pseudovirus and Griffithsin-IL18 fusion protein (fusion protein concentration 100 ng / mL). The HPV pseudovirus was diluted with DMEM medium to an MOI (multiple of infection) of 1, and the experimental groups were pre-incubated with the fusion protein at room temperature for 30 min. Then, the virus suspension (control group) or the virus-protein mixture (experimental group) was added to the corresponding wells, and the cells were cultured for another 60–72 h. After culturing, the culture medium was discarded, and the cells were washed three times with PBS. The number of cells expressing green fluorescence was then observed and counted using a fluorescence microscope. By comparing the number of fluorescent cells in the experimental group and the virus control group, the inhibition rate of the fusion protein against viral infection was calculated. Figure 4 As shown, the infection efficiency of HPV pseudoviruses was significantly reduced after the addition of the fusion protein (P < 0.0001), indicating that the Griffithsin-IL18 fusion protein has the effect of resisting viral infection.

[0061] Example 4: Experiment on the promotion of NK cell killing effect by Griffithsin-IL18 fusion protein

[0062] HPV16-positive 293FT cells (target cells) and NK cells (effective cells) isolated from healthy volunteers were co-cultured in 96-well plates at an effector-to-target ratio of 10:1, with each well containing 200 μL of KBM581 medium (containing IL-2 200 U / mL and gentamicin 80 U / mL). The experimental group received Griffithsin-IL18 fusion protein (100 ng / mL), while the control group received either equal concentrations of IL18 alone (100 ng / mL) or PBS. After 4 h of co-culture, the killing efficiency was assessed using the Calcein-AM release assay: target cells were pre-labeled with Calcein-AM (5 μM) for 30 min, washed, and then co-cultured with effector cells. After culture, the supernatant was collected, and fluorescence values ​​were read using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The release rate was calculated as [(experimental group fluorescence - spontaneous fluorescence) / (maximum fluorescence release - spontaneous fluorescence)] × 100%. Figure 5 As shown, the fusion protein group had a kill rate of 76.5%, which was significantly higher than that of the PBS control group and the IL18-only group.

[0063] Example 5: Preparation of Pharmaceutical Composition

[0064] The purified Griffithsin-IL18 fusion protein (concentration 50-500 μg / mL) from Example 1 was formulated into a gel product with xanthan gum (0.5% w / v), hyaluronic acid (0.5% w / v), propylene glycol (1% v / v), p-hydroxyacetophenone (0.5% w / v), 1,2-hexanediol (0.5% v / v), and purified water. Specific steps: First, xanthan gum and hyaluronic acid were dissolved in a portion of purified water and stirred until homogeneous to form a matrix; propylene glycol, p-hydroxyacetophenone, and 1,2-hexanediol were added and mixed thoroughly; finally, the fusion protein solution was slowly added, stirring until homogeneous, avoiding the formation of bubbles. After sterilization by filtration through a 0.22 μm filter membrane, the product was aliquoted into sterile containers and stored at 2-8°C for 12 months. This gel product is suitable for topical application for the prevention or treatment of HPV-related infections.

[0065] The experimental results of the above embodiments show that the fusion protein Griffithsin-IL18 prepared in this invention can block viral entry into the host and inhibit viral replication and spread. It also activates NK cells, secretes IFN-γ, and kills virus-infected cells. It demonstrates good potential in antiviral treatment, has broad application prospects, and is easy to use. It can be used to treat HPV infection and related diseases, such as cervical intraepithelial neoplasia and genital warts.

[0066] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A Griffithsin-IL18 fusion protein, characterized in that: It contains a Griffithsin protein fragment, an IL18 protein fragment, and a flexible linker peptide connecting the Griffithsin and IL18 protein fragments, wherein the Griffithsin protein fragment is located at the N-terminus of the fusion protein and the IL18 protein fragment is located at the C-terminus of the fusion protein.

2. The Griffithsin-IL18 fusion protein as described in claim 1, characterized in that: The amino acid sequence of the Griffithsin protein fragment is shown in SEQ ID NO.01, and the amino acid sequence of the IL18 protein fragment is shown in SEQ ID NO.

03.

3. The Griffithsin-IL18 fusion protein as described in claim 1, characterized in that: The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.

05.

4. A method for preparing the Griffithsin-IL18 fusion protein according to any one of claims 1 to 3, characterized in that: Includes the following steps: (1) Construction of expression vector: Insert the nucleotide sequence of Griffithsin protein fragment as shown in SEQ ID NO.02, the nucleotide sequence of flexible linker peptide as shown in SEQ ID NO.06, and the nucleotide sequence of IL18 protein fragment as shown in SEQ ID NO.04 into the expression vector to form a fusion protein expression cassette; (2) Transformation of host cells: The expression vector constructed in step (1) is transformed into host cells; (3) Induction of expression: Under appropriate conditions, the host cells obtained in step (2) are induced to express the Griffithsin-IL18 fusion protein; (4) Extraction and purification: The host cells obtained in step (3) were lysed, and the Griffithsin-IL18 fusion protein was extracted and purified.

5. The preparation method according to claim 4, characterized in that: The expression vector is pET-28a(+).

6. The preparation method according to claim 4, characterized in that: The host cells are Escherichia coli DH5α and / or BL21 (DE3).

7. The use of the Griffithsin-IL18 fusion protein according to any one of claims 1 to 3 in the preparation of pharmaceutical compositions that enhance the antiviral function of NK cells.

8. The use of the Griffithsin-IL18 fusion protein according to any one of claims 1 to 3 in the preparation of pharmaceutical compositions for the prevention or treatment of HPV infection and related diseases.

9. A pharmaceutical composition, characterized in that: Its active ingredient includes the Griffithsin-IL18 fusion protein as described in any one of claims 1 to 3.

10. A pharmaceutical composition according to claim 9, characterized in that: Its dosage form is gel, spray or injection.

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