Norovirus GII.17 virus-like particles and their preparation method and application

Abstract

The present invention discloses a norovirus GII.17 virus-like particle and its preparation method and application, which relate to the field of biomedicine technology. The norovirus GII.17 virus-like particle of the present invention includes the VP1 protein of the mutant norovirus GII.17, and the amino acid sequence of the VP1 protein of the mutant norovirus GII.17 is shown as SEQ ID NO.2; or, the virus-like particle includes the VP1 protein of the norovirus GII.17, and the amino acid sequence of the VP1 protein of the norovirus GII.17 is shown as SEQ ID NO.1. The virus-like particle has the morphology of a typical norovirus particle, and the particle size can be observed to be uniform by electron microscopy, and it has good immunogenicity and can induce a good immune response. It can be used in combination with an adjuvant to prepare a norovirus vaccine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to a norovirus GII.17 virus-like particle and a preparation method and application thereof. Background Art

[0002] Human norovirus is one of the leading causes of acute gastroenteritis, primarily transmitted through the fecal-oral route. The main clinical symptoms of norovirus-associated acute gastroenteritis are vomiting and diarrhea, accompanied by abdominal pain, fever, and muscle aches. The disease is self-limiting, but it has a high incidence and can even lead to death in children, the elderly, and those with compromised immune systems. Norovirus types GI, GII, and GIV can cause human infection, with GII.4 being the predominant type, followed by other GII types, including GII.3 and GII.17. In China, outbreaks of norovirus-associated acute gastroenteritis have been increasing since 2006. The predominant types include GII.4, GII.17, GII.3, and GII.1. GII.17 was the predominant type from 2014 to 2015, and its prevalence has decreased since 2019, although strains other than GII.4 continue to emerge. Due to genetic diversity and antigenic diversity, there is currently no targeted treatment for norovirus infection, and vaccine development has become another effective measure.

[0003] Virus-like particles (VLPs) are considered an ideal vaccine format due to their morphology, which resembles natural virus particles and lacks a viral genome. They offer excellent safety and immunogenicity, making them an ideal vaccine. The production of VLPs relies on recombinant protein expression systems. Currently, mainstream recombinant protein expression systems include yeast, prokaryotic expression systems such as Escherichia coli, mammalian cell expression systems, and plant cell expression systems. These systems vary significantly in their effectiveness. Recombinant protein expression is a key technology in the development of VLP vaccines. Developing coding sequences capable of efficiently expressing exogenous proteins is crucial for VLP vaccine development.

[0004] Therefore, providing virus-like particles that can efficiently express foreign proteins and have good immunogenicity for application in the development of norovirus vaccines is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a norovirus GII.17 virus-like particle and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A polynucleotide comprising a nucleotide sequence encoding a VP1 protein of a mutant norovirus GII.17, wherein the nucleotide sequence encoding the VP1 protein of the mutant norovirus GII.17 is as shown in SEQ ID NO.4;

[0008] Alternatively, the polynucleotide includes a nucleotide sequence encoding the VP1 protein of norovirus GII.17 type, and the nucleotide sequence encoding the VP1 protein of norovirus GII.17 type is shown as SEQ ID NO.3.

[0009] Another object of the present invention is to provide a VP1 protein, the amino acid sequence of the VP1 protein is shown as SEQ ID NO.2 or as SEQ ID NO.1.

[0010] Another object of the present invention is to provide a biological material, which comprises the above-mentioned polynucleotide.

[0011] Preferably, the expression vector of the biological material is a yeast expression vector.

[0012] Another object of the present invention is to provide a cell line, wherein the above-mentioned polynucleotide is integrated into the genome of the cell line, or the cell line comprises the above-mentioned biological material.

[0013] Another object of the present invention is to provide a norovirus GII.17 virus-like particle, wherein the virus-like particle comprises a mutant norovirus GII.17 VP1 protein, and the amino acid sequence of the mutant norovirus GII.17 VP1 protein is shown in SEQ ID NO.2;

[0014] Alternatively, the virus-like particle comprises the VP1 protein of the norovirus GII.17 type, and the amino acid sequence of the VP1 protein of the norovirus GII.17 type is shown in SEQ ID NO.1.

[0015] Another object of the present invention is to provide a method for preparing the above-mentioned norovirus GII.17 virus-like particles, comprising the following steps:

[0016] S1: artificially synthesized norovirus GII.17 type codon-optimized VP1 gene, the nucleotide sequence of which is shown in SEQ ID NO.4 or SEQ ID NO.3;

[0017] S2: Ligate the codon-optimized VP1 gene obtained in step S1 with the Pink-HC vector to obtain the corresponding expression vector;

[0018] S3: Transform the expression vector obtained in step S2 into Pichia pastoris competent cells; induce with methanol; continue culturing for 24 hours and then collect the bacterial solution;

[0019] S4: The bacterial cells of the bacterial liquid obtained in step S3 were broken and purified, the VP1 protein was identified by Western Blot, and the particle morphology was confirmed by electron microscopy to confirm that norovirus GII.17 virus-like particles were obtained.

[0020] Another object of the present invention is to provide an immunogen, which is the above-mentioned VP1 protein or the above-mentioned norovirus GII.17 virus-like particles.

[0021] Another object of the present invention is to provide the use of the above-mentioned VP1 protein or the above-mentioned norovirus GII.17 virus-like particles in the preparation of products for preventing norovirus infection.

[0022] Another object of the present invention is to provide a pharmaceutical composition for preventing norovirus infection, which comprises the above-mentioned VP1 protein or the above-mentioned norovirus GII.17 virus-like particles, and a pharmaceutically acceptable carrier.

[0023] Beneficial effects:

[0024] (1) The GII.17 norovirus virus-like particles prepared by the present invention are suitable for preparing vaccines after being combined with an adjuvant: the virus-like particles have the morphology of typical norovirus particles, and the particles can be observed to be uniform in size through electron microscopy, and have strong immunogenicity and can induce a good immune response.

[0025] (2) Easy to produce on an industrial scale: The expression level of exogenous proteins is high; the yeast expression system has the characteristics of simple culture, rapid growth, suitability for high-density fermentation and low cost.

[0026] (3) Easy to obtain efficient expression strains: The present invention optimizes the design of the VP1 protein gene sequence of GII.17 norovirus and constructs a recombinant expression vector, which can achieve efficient expression of virus-like particles in Pichia pastoris.

[0027] (4) Good stability: The recombinant protein expression vector can be integrated into the host yeast genome through homologous recombination, is not easily lost, and the product is not easily degraded. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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 or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0029] Figure 1 This is the map of the recombinant GII.17 norovirus VP1 expression vector; A: expression vector map; B: Sanger sequencing map of the 3' junction between the vector and the insert; C: Sanger sequencing map of the 5' junction between the vector and the insert; D: Sanger sequencing map of the Mut-type insert.

[0030] Figure 2 This is the peak diagram of norovirus GII.17 VLPs purified by chromatography column and the identification result of Coomassie Brilliant Blue staining; among them, G11.17 is wild-type GII.17 VLPs; GII.17M2 is mutant GII.17 VLPs.

[0031] Figure 3 Western Blot detection of norovirus GII.17 VP1 protein; Note: NC: blank control; WT: wild-type VLPs; MUT: mutant VLPs.

[0032] Figure 4 This is a transmission electron microscope observation of norovirus GⅡ.17 VLPs; the left picture shows wild-type norovirus GII.17 virus-like particles, and the right picture shows mutant norovirus GII.17 virus-like particles; the small and medium pictures are corresponding magnifications, the full scale is 200nm, and the particle size is about 10nm.

[0033] Figure 5 This is the serum antibody level induced by norovirus GⅡ.17 VLPs. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. The GII.17 type norovirus-like particles involved in the present invention can be prepared in a Pichia pastoris expression system and realize the effect as a vaccine, but it should not be understood as a limitation of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions, such as those described in "Molecular Cloning: A Laboratory Manual".

[0035] Example 1GII. Construction of Norovirus VP1 Expression Vector

[0036] 1. Construction of wild-type GII.17 norovirus VP1 expression vector

[0037] To optimize expression, the GII.17 norovirus VP1 amino acid sequence (SEQ ID NO. 1) was optimized and synthesized based on the codon preference of Pichia pastoris (nucleotide sequence shown in SEQ ID NO. 3). This sequence does not include the yeast secretion signal peptide or the transcription termination signal recognized by yeast. This sequence was cloned into the multiple cloning site of the Pink-HC vector using the EcoR I and Fse I restriction sites to generate the Pink-HC-GII.17-VP1 expression vector.

[0038] The core elements of the Pink-HC-GII.17-VP1 expression vector include: promoter, terminator, replicon, resistance gene and GII.17-VP1 insert-6xHis tag (see Appendix Figure 1 -A). After insertion, the expression vector was sequenced by Sanger to verify that the fragments were correctly connected (see Appendix Figure 1 -B and 1-C).

[0039] 2. Construction of mutant GII.17 norovirus VP1 expression vector

[0040] The mutant GII.17 norovirus VP1 sequence was modified based on the norovirus VP1 structure in a public dataset (PDB ID: 7MRY), and the G190C and G192C mutations were made on this sequence (shown in SEQ ID NO. 2). The amino acid sequence of the mutant GII.17 norovirus VP1 was optimized and synthesized based on the codon preference of Pichia pastoris (the nucleotide sequence is shown in SEQ ID NO. 4). This sequence does not include the sequence of the yeast secretion signal peptide or the transcription termination signal recognized by yeast. This sequence was cloned into the multiple cloning site of the Pink-HC vector using the EcoR I and Fse I restriction sites to obtain the Pink-HC-Mut-GII.17-VP1 expression vector.

[0041] The core elements of the vector include: a promoter, a terminator, a replicon, a resistance gene and a mutant GII.17-VP1 insertion fragment-6xHis tag.

[0042] SEQ ID NO.1:

[0043] MKMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ;

[0044] SEQ ID NO.2:

[0045] MKMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNCSCDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ;

[0046] SEQ ID NO.3:

[0047]

[0048] SEQ ID NO.4:

[0049]

[0050] Example 2 Induced expression of yeast strains and purification and identification of GⅡ.17 type norovirus VLPs

[0051] The Pink-HC-GII.17-VP1 expression vector (linked to the norovirus GII.17 VP1 protein sequence), the Pink-HC-Mut-GII.17-VP1 expression vector (linked to the mutant norovirus GII.17 VP1 protein sequence), and the Pink-HC empty plasmid were digested with Spe I overnight at 37°C. To the linearized DNA solution, 1 / 10 volume of 3M sodium acetate solution (pH 5.2) and 2.5 volumes of 95%-100% anhydrous ethanol were added. The mixture was then placed in a -20°C refrigerator for at least 1 hour to promote DNA precipitation. The DNA was precipitated by centrifugation at 12,000-15,000 rpm at 4°C for 15-30 minutes and then redissolved in 10 μL of deionized water for later use. Mix 80 μL of Pichia competent cells with 10 μL (5-10 μg) of linearized plasmid DNA, transfer to a pre-cooled 0.2 cm electroporation cup, and incubate on ice for 5 minutes. Electroporate for 5 ms at 2000 V, 25 μF capacitance, and 200 Ω resistance. Immediately after electroporating the cells, add 1 mL of YPDS medium to the electroporation cup. Incubate the cells at 28.8°C for at least 2 hours. Then, take 300 μL of the cell mixture and evenly spread it on the PAD selection plate. Incubate at 28.8°C for 3-10 days until obvious colonies are formed. Select the positive clones whose target fragments are integrated into the yeast genome and inoculate them into BMGY medium and continue to culture for 5-10 days.

[0052] A well-growing single colony was inoculated into BMGY medium. Methanol was added to the original medium to a final concentration of 0.5% and cultured for another 24 hours before the bacterial suspension was collected. After washing three times with 0.01M PBS, the yeast was disrupted using a high-pressure homogenizer and centrifuged at 4°C. The supernatant was concentrated 3-5 times before purification. A Monomix Core 1000 composite chromatography column was connected in the forward direction to a protein purification system. The preservative solution in the column and system was replaced with 20% ethanol, rinsed with water, and then equilibrated with 0.01M PBS. After equilibration, the protein sample was loaded according to the standard protocol of the purification instrument and equilibrated again with PBS. Finally, the column was eluted with NaCl, and the flow-through and eluate were collected and characterized by SDS-PAGE. After initial purification, a Q column was connected in the forward direction to the purification system and the above steps of replacement, rinse, equilibration, sample loading, and equilibration were repeated. Elution was then performed with 1M NaCl, and the flow-through and eluate were collected and characterized. The eluent is 0.5M imidazole. When the eluent concentration is about 30% to 40%, the protein begins to elute. The stock solution is the unpurified sample. The flow-through is the sample that does not bind to the nickel column when the sample passes through the nickel column. The elution is a gradient elution using the eluent. The eluent is 1mol / L NaCl. The concentrated stock solution is the sample after the unpurified protein stock solution is concentrated. The flow-through is the sample that does not bind to the purification column when the sample passes through the purification column. The elution is a 100% direct elution using the eluent (see Appendix). Figure 2 ).

[0053] After purification, Western blot analysis was performed using a GII.17 norovirus VP1 polyclonal antibody (Thermo Fisher, PA5112040). It was observed that both the wild-type and mutant GII.17 virus-like particles after codon optimization had distinct bands, and under the same loading amount, the Mut type had a significantly higher expression level (see Appendix). Figure 3 ); and VLPs particles with typical viral structures can be observed under an electron microscope (see Appendix Figure 4 ), under the electron microscope, it can be seen that the particles are neat and uniform in size, with a typical virus-like particle structure, the particle size is about 10nm, and the particle density is high under the field of view, indicating that this purification route can obtain higher quality VLPs.

[0054] Example 3GII. Immunogenicity of Norovirus VLPs Type 17

[0055] Immunization experiments were conducted on 6-8 week old SPF-grade BALB / c female mice, with 5 mice per group. The control group received a 200 μL PBS vaccination; the experimental group received a single 10 μg immunization with 50 μg of AL(OH)3 as an adjuvant. Two weeks after the initial vaccination, a supplemental dose was administered intramuscularly. Tail vein blood was collected at weeks 0, 2, 4, 6, and 8. All blood samples were centrifuged after coagulation, and the supernatant was stored at -20°C until use.

[0056] The immune effect of VLP was evaluated by serum antibody titer assay (ELISA). Norovirus VLPs were coated on 96-well plates at a concentration of 10 μg / ml, and 100 μL was added to each well and incubated at 4°C overnight. The plates were washed three times with phosphate-buffered saline containing Tween (PBST). The wash solution remained in the wells for more than 1 minute and was shaken horizontally to ensure thorough washing. PBST with 5% skim milk powder was added for incubation for 1 hour for blocking, and the plates were washed three times again with PBST. Diluted mouse serum (1:1000 dilution, using 1% skim milk powder) was added. The plate was washed three times with PBST, and diluted goat anti-mouse IgG (HRP) antibody was added to the 96-well plate and incubated at 37°C for 1 hour. The plate was washed again with PBST five times. TMB color development solution (100 μL / well) was added and incubated at 37°C in the dark for 5 minutes. After the incubation, 100 μL / well of 1 M sulfuric acid (H2SO4) was immediately added to terminate the reaction. The optical density (OD) value of each well at a wavelength of 450 nm was measured and recorded using a microplate reader within 5 minutes to evaluate the antibody titer.

[0057] It can be seen that after codon optimization, both wild-type and mutant norovirus GII.17 VLPs can induce strong humoral immune responses in combination with vaccine adjuvants. And as time goes by, the performance of mutant norovirus GII.17 VLPs is more prominent (see Appendix Figure 5 ).

[0058] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0059] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A VP1 protein, characterized in that The amino acid sequence of the VP1 protein is shown in SEQ ID NO.

2.

2. A norovirus GII.17 virus-like particle, characterized in that The virus-like particle includes the VP1 protein of the mutant norovirus GII.17 type, and the amino acid sequence of the VP1 protein of the mutant norovirus GII.17 type is shown in SEQ ID NO.

2.

3. An immunogen, characterized in that The immunogen is the VP1 protein described in claim 1 or the norovirus GII.17 virus-like particle described in claim 2.

4. Use of the VP1 protein according to claim 1 or the norovirus GII.17 virus-like particles according to claim 2 in the preparation of a product for preventing norovirus infection.

5. A pharmaceutical composition for preventing norovirus infection, characterized in that: The pharmaceutical composition comprises the VP1 protein according to claim 1 or the norovirus GII.17 virus-like particles according to claim 2, and a pharmaceutically acceptable carrier.

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