A cmv virus-like particle for producing a vlp recombinant vaccine and a method for preparing the same

Abstract

The application discloses a CMV virus-like particle for producing a VLP recombinant vaccine and a preparation method thereof, the CMV virus-like particle is obtained by cloning a C-terminal G4SLPETG modified plant virus cucumber mosaic virus CMV capsid gene into a prokaryotic expression vector to obtain a recombinant expression vector, the recombinant expression vector is transfected into E. coli BL21 (DE3), and expression is obtained through the recombinant E. coli BL21 (DE3); and the amino acid sequence of the C-terminal G4SLPETG modified CMV is SEQ ID NO. 1. Test proves that the recombinant strain constructed by the application is stable to expression of an exogenous protein. The recombinant protein expressed by the application is used for preparing a virus-like particle for antigen coupling, the coupling efficiency is high, the antigen purity is high, the safety is good, the virus-like particle is not pathogenic to mice and other animals, and the safety evaluation is easy.

Description

[0001] This application is a divisional application of the patent application filed on December 15, 2020, with application number 202011476397.3 and invention title "CMV virus-like particles for producing VLP recombinant vaccines and a method for preparing the same". Technical Field

[0002] This invention relates to the fields of molecular biology, virology, immunology, and medicine. The invention comprises a CMV virus-like particle for producing a VLP recombinant vaccine, a VLP recombinant vaccine, and a method for preparing the CMV virus-like particle for producing the VLP recombinant vaccine. Background Technology

[0003] Plant viruses and their derived virus-like particles (VLPs) have attracted much attention, primarily due to the unique post-translational modification capabilities and cost-effectiveness of plants, making them an economical and rapid alternative platform for producing VLP vaccines with high safety, production speed, and scalability.

[0004] While progress has been made in VLP-based vaccine development, further unique VLP systems are still needed. In particular, non-response can occur in cases where elderly individuals generally have poorer antibody responses, and where vaccines induce variable antibody responses in immunized subjects and individuals, often spanning a range of more than 100-fold variation. Non-response is associated with certain class II MHC molecules; that is, the failure to induce a favorable T helper (Th) cell response is the cause of poor antibody responses in these individuals. Therefore, vaccines that induce a favorable Th cell response in virtually all subjects and individuals are an important goal in vaccine development.

[0005] Recently, a CMV VLP-based vaccine platform has been able to present different antigens on its surface using chemical linker conjugation technology. CMV VLPs are derived from CMV-modified capsid proteins with inserted T-cell-stimulating epitopes. However, the chemical linker modification efficiency remains low and uneven, failing to achieve highly efficient site-specific modification. This approach utilizes sortease A enzyme for site-specific condensation of the target antigen with CMV, exhibiting high catalytic efficiency and a simple process. Site-specific modification of CMV can be engineered. Currently, recombinant CMV vaccines serve as delivery systems, expressing hepatitis C virus (HCV)-derived epitopes on their outer surface. Serum samples from 60 patients with chronic hepatitis C showed significant immunoreactivity to crude plant extracts infected with chimeric CMV. Furthermore, CMV VLP-based expression systems have been described as potential vaccines against Alzheimer's disease and porcine circovirus-specific vaccines.

[0006] Self-antigen proteins are generally difficult to induce antibody responses against their own antigens. One way to improve vaccination efficiency is to increase the reproducibility of the antigens used. Unlike isolated proteins, viruses can induce rapid and effective immune responses with and without the aid of T cells, without any adjuvants (Bachmann and Zinkernagel, Ann. Rev. Immunol: 15: 235-270 (1991)). They can trigger much stronger immune responses than their isolated components compared to a few proteins. For B cell responses, a key factor in viral immunogenicity is known to be the reproducibility and sequence of surface epitopes. Many viruses exhibit quasi-crystal surfaces with regularly arranged epitopes that can effectively crosslink epitope-specific immunoglobulins on B cells (Bachmann and Zinkernagel, Immunol. Today 17: 553-558 (1996)). This crosslinking of B cell surface immunoglobulins is a strong activating signal that directly induces cell cycle progression and antibody production of IgM. Furthermore, this triggered B cell activity can activate T helper cells, thereby inducing the conversion of IgM antibodies to IgG antibodies in B cells and the generation of long-lived B cell memory targets for any vaccination (Zinkernagel, Ann. Rev. Immunol. 15: 235-270 (1997)). Viral structure is even involved in the production of anti-antibodies in autoimmune diseases and is part of the natural response to pathogens (see Fehr, T., et al., J. Exp. Med. 185: 1785-1792 (1997)). Therefore, antigens presented by highly organized viral surfaces can induce strong antibody responses against antigens. Summary of the Invention

[0007] This invention addresses the shortcomings of existing chemical conjugation technologies by providing a CMV virus-like particle for the production of VLP recombinant vaccines and its preparation method. This method facilitates directional catalytic conjugation, is easily obtained through bacterial culture, has high expression yield, is suitable for industrial production, and enhances the immunogenicity of the target antigen and accelerates the immune response.

[0008] The first aspect of this invention provides a CMV virus-like particle for producing a VLP recombinant vaccine. The CMV virus-like particle is obtained by cloning the C4SLPETG-modified CMV capsid gene of a plant virus, cucumber mosaic virus (CVV), into a prokaryotic expression vector to obtain a recombinant expression vector. The recombinant expression vector is then transfected into Escherichia coli BL21(DE3), and the recombinant E. coli BL21(DE3) is expressed. The amino acid sequence of the C4SLPETG-modified CMV is SEQ ID NO.1.

[0009] The second aspect of the present invention provides a VLP recombinant vaccine, which is prepared by mixing CMV virus-like particles and target antigen in a molar ratio of 1:1 under the catalysis of Sortease A.

[0010] A third aspect of the present invention provides a method for preparing CMV virus-like particles for producing VLP recombinant vaccines, comprising the following steps:

[0011] (1) The his-CMV-G4SLPETG gene synthesis fragment was cloned into the pET28a prokaryotic expression vector to obtain the positive recombinant plasmid his-CMV-G4SLPETG-pET28a;

[0012] (2) Transform Escherichia coli BL21(DE3) with the recombinant plasmid that has been correctly sequenced in step (1) to obtain the recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21;

[0013] (3) The recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21 was cultured and IPTG was added to induce expression, and CMV virus-like particles were obtained.

[0014] Preferably, the positive recombinant plasmid his-CMV-G4SLPETG-pET28a is obtained by cloning the nucleotides shown in SEQ ID NO.2 into the NdeI and BamhI sites of the prokaryotic expression vector pET28a via homologous recombination.

[0015] Preferably, the recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21 contains the positive recombinant plasmid his-CMV-G4SLPETG-pET28a.

[0016] Preferably, when the recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21 reaches an OD600 of 0.6-0.8, IPTG at a final concentration of 1 mM is added to induce expression.

[0017] The present invention has the following beneficial effects:

[0018] First, CMV originates from the capsid of a cucumber mosaic virus (CMV). The capsid protein has the function of self-assembling into nanoparticles, is non-infectious, and has strong antigenic immunity. This is a unique technology of the applicant's VLP screening platform.

[0019] Second, the antigens coupled to CMV virus-like particles exhibit high antigenicity and can induce high levels of specific antibodies in mice, rats, cats, dogs, and horses, resulting in a rapid humoral immune response.

[0020] Third, the C-terminal G4SLPETG modified CMV virus-like particles in this invention can be expressed in large quantities by Escherichia coli, the production process is simple, and the CMV-G4SLPETG virus-like particles have high purity.

[0021] Fourth, this invention is suitable for industrialization. The selected recombinant Escherichia coli expression strain has the characteristics of fast growth, easy cultivation, simple genetic operation, fast reproduction, low requirements for culture conditions, inexpensive culture medium, ability to carry out high-density culture, and tolerance to high hydrostatic pressure, which facilitates industrial production. Attached Figure Description

[0022] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the recombinant plasmid after the C-terminal G4SLPETG modified CMV gene is ligated to the pET28a plasmid in an embodiment of the present invention.

[0024] Figure 2 This is a schematic diagram of SDS-PAGE running of CMV-G4SLPETG virus-like particle protein provided in an embodiment of the present invention. Detailed Implementation

[0025] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] The molecular biology experimental methods used in the embodiments of this invention, such as enzyme digestion and homologous recombination ligation, can be found in the second edition of *Molecular Cloning*. The basic materials for preparing the CMV-G4SLPETG virus-like particles of this invention include: the CMV-G4SLPETG protein nucleotide sequence pET28a plasmid, BL21(DE3) *E. coli* strain, Tiangen plasmid small-scale preparation kit, LB medium, IPTG, kanamycin, nickel column, and imidazole. The nucleotide sequence was sent to BGI Genomics to synthesize the his-TEV-CMV-G4SLPETG-pET28a recombinant plasmid.

[0028] Example 1

[0029] Example 1 of this invention provides a CMV virus-like particle for producing a VLP recombinant vaccine. The CMV virus-like particle is obtained by cloning the C4SLPETG-modified CMV capsid gene of the plant virus cucumber mosaic virus into a prokaryotic expression vector to obtain a recombinant expression vector. The recombinant expression vector is transfected into Escherichia coli BL21(DE3), and the recombinant Escherichia coli BL21(DE3) is expressed. The amino acid sequence of the C4SLPETG-modified CMV is SEQ ID NO.1.

[0030] Example 2

[0031] See Figure 1 and Figure 2 Embodiment 2 of the present invention provides a method for preparing CMV virus-like particles for producing VLP recombinant vaccines, comprising the following steps:

[0032] (1) Construction of recombinant plasmid: The CMV-G4SLPETG gene was directly synthesized by BGI Genomics and inserted into the NdeI and XhoI restriction sites of the pET28a vector to obtain the recombinant plasmid his-TEV-CMV-G4SLPETG-pET28a.

[0033] (2) Transformation of recombinant plasmid into expression strain: The recombinant plasmid his-TEV-CMV-G4SLPETG-pET28a was transformed into the Escherichia coli BL21(DE3) expression strain to obtain the recombinant expression strain his-TEV-CMV-G4SLPETG-pET28a-BL21;

[0034] (3) Bacterial culture and purification of his-TEV-CMV-G4SLPETG virus-like particles: The recombinant expression strain his-TEV-CMV-G4SLPETG-pET28a-BL21 was cultured and his-TEV-CMV-G4SLPETG virus-like particles were obtained by IPTG induction.

[0035] (4) Preparation of CMV-G4SLPETG virus-like particles: The his-TEV-CMV-G4SLPETG virus-like particles obtained in (3) were digested with his-TEV enzyme to separate the his-TEV tag and CMV-G4SLPETG. The digestion solution was purified by nickel column purification, and the permeate was pure CMV-G4SLPETG virus-like particles.

[0036] The specific steps for CMV-G4SLPETG virus-like particle expression and purification are as follows:

[0037] (a1) Preparation of his-TEV-CMV-G4SLPETG-pET28a recombinant plasmid

[0038] 5 μL of the glycerol-containing bacteria containing the plasmid was pipetted into 5 mL of 2YT medium (containing 50 μg / mL kanamycin) and incubated at 37°C with shaking for 14–16 hours. After incubation, 1 mL of the bacterial culture was sent for sequencing. The remaining bacterial culture was used to extract the plasmid using a plasmid mini-extraction kit, and the nucleic acid concentration was detected using a protein nucleic acid detector.

[0039] (a2) expression of his-TEV-CMV-G4SLPETG virus-like particles

[0040] The plasmid described in (a1) was transformed into the BL21(DE3) expression strain. After shaking, the culture was concentrated by centrifugation, plated on kanamycin-resistant plates, and incubated overnight at 37°C. Colonies were scraped off and inoculated into 10 mL of 2×YT medium containing ampicillin. The culture was shaken at 180 rpm at 37°C until the OD value of the bacterial culture reached approximately 0.8. The culture was then transferred to 500 mL of 2×YT medium containing kanamycin and shaken at 180 rpm at 37°C until the OD value of the bacterial culture reached approximately 0.8. A final concentration of 0.8 mM IPTG inducer was added, and the culture was incubated overnight at 30°C. On the third day, the bacterial cells were collected by centrifugation at 4500 rpm for 15 min. The bacterial cells were then sonicated for 45 min (3 s on, 4 s off) at 125 W. After sonication, the supernatant was collected at 9500 rpm for 20 min at 4°C.

[0041] (a3) Purification of his-TEV-CMV-G4SLPETG virus-like particles

[0042] Centrifuge the supernatant, purify it using a nickel column, and equilibrate the nickel column with 5 column volumes of PBS. Load the sample, wash with 5 column volumes of PBS, wash with 25 mM imidazole for 2 column volumes, and then wash with 25 mM imidazole for another 5 column volumes. Then elute continuously with 50, 100, 150, 200, and 250 mM imidazole for 5 column volumes each time. Collect the eluent from sample loading to 500 mM imidazole.

[0043] Take 10 μL from each collection solution and add it to 10 μL of 2× protein electrophoresis loading buffer, then heat at 100°C for 4 min. Perform protein electrophoresis, stain, and observe the concentration of the target band in the collection solution. Ultrafilter the target protein using a 3 kDa ultrafiltration tube, replacing the solvent with PBS.

[0044] (a4) CMV-G4SLPETG virus-like particle purification

[0045] The his-TEV-CMV-G4SLPETG virus-like particles obtained in (a3) ​​were dissolved in PBS solution (10 mM), and his-TEV recombinase was added for digestion. Digestion was performed at a ratio of 10 μg TEV enzyme to 1 mg of recombinant protein, with shaking at 30°C for 3 hours. The digestion buffer was then passed through a nickel column. The permeate was the CMV-G4SLPETG virus-like particles, while the his-TEV enzyme and incompletely digested recombinant protein remained on the nickel column. The permeate (CMV-G4SLPETG virus-like particles) was concentrated using a 3K concentration tube. The target protein was then ultrafiltered using a 3 kDa ultrafiltration tube, replacing the PBS solvent.

[0046] Example 3

[0047] Example 3 of the present invention provides a VLP recombinant vaccine, which is prepared by mixing the CMV virus-like particles from Example 1 with the target antigen at a molar ratio of 1:1 and then catalyzing it under the catalysis of Sortease A, thus realizing simplified vaccine production.

[0048] Specifically, the CMV-G4SLPETG virus-like particles and the target antigen modified with three glycine residues at the N-terminus were prepared into 1 mg / ml solutions using PBS (10 mM) and mixed at a 1:1 ratio. His-Sotase A5 enzyme was added, and the mixture was catalyzed at 37°C with shaking for 5 hours at a ratio of 100 μg catalyzing 2 mg of the mixed protein. The catalytic solution was then passed through a nickel column. The permeate was CMV-G4SLPETGGG-target antigen virus-like particles, while the his-Sotase A recombinant enzyme remained on the nickel column. The permeate, namely CMV-G4SLPETGGG-target antigen virus-like particles, was concentrated using a 3K concentrator to obtain the VLP recombinant vaccine modified with CMV virus-like particles.

[0049] sequence:

[0050] SEQ ID NO.1:

[0051] MHHHHHHENLYFQGMGQYIKANSKFIGITERRRRPRRGSRSAPSSAD

[0052] ANFRVLSQQLSRLNKTLA

[0053] AGRPTINHPTFVGSERCKPGYTFTSITLKPPKIDRGSYYGKRLLLPDSVTEY

[0054] DKKLVSRIQIRVN

[0055] PLPKFDSTVWVTVRKVPASSDLSVAAISAMFADGASPVLVYQYAASGVQA

[0056] NNKLLYDLSAMRADI

[0057] GDMRKYAVLVYSKDDALETDELVLHVDVEHQRIPTSGVLPVGGGGSLPETG*

[0058] SEQ ID NO.2:

[0059] ATGCACCATCATCATCATCACgagaatctttattttcagggcATGggccagtatattaaggccaa ctccaaatttatcgggattaccgagcgtcgacgtcgtccgcgtcgtggttcccgctccgccccctcctccgcggatgctaactttagagtcttgtcgcagcagctttcgcgacttaataagacgttagcagctggtcgtccaactattaaccacccaacctttgtagggagtgaacgctgtaaacctgggtacacgttcacatctatcaccctaaagccaccaaaaatagaccgtgggtcttattatggtaaaaggttgttattacctgattcagtcacggaatatgataagaaacttgtttcgcgcattcaaattcgagttaatcctttgccgaaatttgattcaaccgtgtgggtgacagtccgtaaagttcctgcctcttcggacttatccgttgccgccatttctgctatgtttgcggacggagcctcaccggtactggtttatcagtacgctgcatctggagtccaagctaacaacaaactgttgtatgatctttcggcgatgcgcgctgatataggcgacatgagaaagtacgccgtcctcgtgtattcaaaagacgatgcactcgagacagacgagttagtacttcatgttgacgtcgagcaccaacgtattcccacatctggggtgctcccagttGGTGGAGGAGGTTCTCTGCCAGAAACCGGTTAG。

Claims

1. A CMV virus-like particle for producing a VLP recombinant vaccine, characterized in that, The CMV virus-like particle clones the CMV capsid gene of cucumber mosaic virus (CMV) modified with C4SLPETG at the C-terminus into a prokaryotic expression vector to obtain a recombinant expression vector. The recombinant expression vector is then transfected into Escherichia coli BL21(DE3), and the recombinant E. coli BL21(DE3) is expressed. The amino acid sequence of the C4SLPETG-modified CMV is SEQ ID NO.

1. The method for preparing the CMV virus-like particles includes the following steps: (1) The his-CMV-G4SLPETG gene synthesis fragment was cloned into the pET28a prokaryotic expression vector to obtain the positive recombinant plasmid his-CMV-G4SLPETG-pET28a; (2) Transform Escherichia coli BL21(DE3) with the recombinant plasmid that has been correctly sequenced in step (1) to obtain the recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21; (3) The recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21 was cultured and IPTG was added to induce expression to obtain CMV virus-like particles. The positive recombinant plasmid his-CMV-G4SLPETG-pET28a was obtained by cloning the nucleotides shown in SEQ ID NO.2 into the NdeI and BamhI sites of the prokaryotic expression vector pET28a via homologous recombination.

2. The CMV virus-like particle as described in claim 1, characterized in that, The recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21 contains the positive recombinant plasmid his-CMV-G4SLPETG-pET28a.

3. A method for preparing CMV virus-like particles for producing VLP recombinant vaccines, characterized in that, Includes the following steps: (1) The his-CMV-G4SLPETG gene synthesis fragment was cloned into the pET28a prokaryotic expression vector to obtain the positive recombinant plasmid his-CMV-G4SLPETG-pET28a; (2) Transform Escherichia coli BL21(DE3) with the recombinant plasmid that has been correctly sequenced in step (1) to obtain the recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21; (3) The recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21 was cultured and IPTG was added to induce expression to obtain CMV virus-like particles. The positive recombinant plasmid his-CMV-G4SLPETG-pET28a was obtained by cloning the nucleotides shown in SEQ ID NO.2 into the NdeI and BamhI sites of the prokaryotic expression vector pET28a via homologous recombination.

4. The method for preparing CMV virus-like particles for producing VLP recombinant vaccines according to claim 3, characterized in that, The recombinant expression strain his-CMV-G4SLPETG-pET28a-BL21 contains the positive recombinant plasmid his-CMV-G4SLPETG-pET28a.

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