CN110144367B - A novel antigen display system based on insect virus capsid and its construction method - Google Patents

Abstract

The invention discloses a novel antigen display system based on insect virus capsids and a construction method thereof, wherein the construction method comprises the following steps: 1) Determining the insertion site of the heterologous protein; 2) Successfully constructing a WhNV recombinant virus-like particle VLP library of fusion enhanced green fluorescent protein EGFP; 3) The construction of a recombinant virus particle library of Wuhan nodavium fused with H5 antigen protein of human avian influenza virus; 4) Establishing a Wuhan nodavirus display system; the system comprises 1) a WHan nodavirus WhNV capsid, 2) a fusion protein inserted into the WhNV capsid protein as a heterologous protein, and 3) a linker, wherein small sections of amino acid sequences are respectively added at the N end and the C end of the fusion protein as the linker to be connected with the WhNV capsid. The construction method is easy to implement and simple to operate, and the WhNV heterologous antigen display system successfully established by the construction method lays a good foundation for establishing an influenza VLP vaccine development platform with high efficiency, stability and high universality, and further establishing various vaccines and drug development platforms.

Description

A novel antigen display system based on insect virus capsid and its construction method

technical field

The invention belongs to the technical field of vaccine preparation, and in particular relates to a method for constructing a display system for efficiently displaying human avian influenza virus antigens on the surface of WhNV capsid VLPs, and also relates to a method for efficiently displaying human avian influenza virus on the surface of WhNV capsid VLPs Antigen display system, which will lay the foundation for the research and development of avian influenza vaccines, and the establishment of a stable and efficient WhNV heterologous polypeptide and protein display system will greatly promote the research and development of other new vaccines and antidotes, which has considerable application prospects. Greatly promote the development of biomedicine.

Background technique

The surface display technology of using viral capsid VLPs to display heterologous polypeptides and protein antigens on its surface has become the current international biological Hotspots and frontiers of pharmaceutical research and development. In this field, foreign scientists and pharmaceutical companies have invested a lot of interest and energy [Kushnir N et al. 2012; Garcea RL et al. 2004].

In the past ten years, phage surface display technology has been applied in various fields of molecular biology and promoted the development of life science and biomedicine; There are considerable limitations in other aspects, and it is difficult to serve as an effective vaccine development platform. In contrast, the viral capsid surface display system prepared in a eukaryotic system makes complex post-translational modifications, glycosylation, etc. possible, so as to maintain the correct conformation and immunogenicity of heterologous antigens as much as possible. In this regard, human papillomavirus type 16 (HPV16) VLPs, Johnson Grass Mosaic virus (JGMV) VLPs, HBV core protein VLPs, etc., have been used for surface display of polypeptide antigens and vaccine development [Sadeyen JR et al. 2003; Saini M et al. 2003; Storni T et al. 2004], in 2008, Canadian scientists used the VLPs of papaya mosaic virus (PapMV) to successfully display the 2-24 amino acid peptide (M2e ), and proved that it has a good immune protection against the lethal challenge of virulent strains of mice, and may become a new method for the development of broad-spectrum influenza virus vaccines [Denis J et al. 2008]. There are limitations. In most cases, only a few to tens of amino acid peptides of heterologous antigens can be displayed, thus affecting its complete immunogenicity and lacking in producing a lasting immune effect[López- Macías C et al. 2012].

In recent years, FHV in the family Nodamuraviridae has become a new type of efficient surface display system and vaccine drug development platform widely recognized internationally due to its unique capsid space structure characteristics [Destito G et al. 2009; VenterPA et al. 2008 ; Manayani DJ et al. 2007]. Nodamura virus is a single-stranded positive-sense RNA virus with a full-length genome of about 4.6 kb, including RNA1 (3.1 kb) and RNA2 (1.5 kb). Among them, RNA2 encodes the capsid protein precursor Pro α. During the assembly process, 180 copies of Pro α are assembled into a regular icosahedral symmetrical structure with T = 3, and become mature infectious virus particles after self-cleavage [Venter PA et al. al. 2008], the Nodamura virus capsid has a central β-barrel structure whose antiparallel strands are connected by several surface-exposed stem-loops of different sizes and shapes. This β-barrel structure is highly conserved in all Nodamura viruses, but the stem-loop structure (loop) on the surface is very different and has strong flexibility. Therefore, the Nodamura virus capsid is able to tolerate the insertion of larger heterologous polypeptides or intact proteins into these stem-loop structures, and neither the viral capsid protein nor the structural characteristics of the inserted heterologous polypeptide or protein will be greatly affected, Thus, it can be efficiently assembled into VLPs displaying antigens in their native conformation. At the same time, the antigens displayed on the surface of its VLPs are arranged in a high titer (180 copies of capsid protein) and highly ordered on the surface of the icosahedral symmetrical spherical particles. This feature enables antigens to efficiently bind to B cell receptors, thereby inducing more rapid and intense B cell proliferation and antibody production. The FHV capsid protein contains 6 stem-loop structures, and the heterologous polypeptides successfully fused in these stem-loops and displayed on the surface of its VLPs include HIV envelope protein gp41, hepatitis B virus surface antigen HBsAg124-147 fragment, hepatitis C Viral E1 protein 315-328 fragment, FLAG protein tag, neurotransmitter NPY, influenza virus HA antigen A helical domain, etc. [Destito G et al. 2009; Venter PA et al. 2008; Bachmann MF et al. 1993; Bachmann MF et al. 1997; Peng M et al. 2005; Schiappacassi M et al. 1997; Buratti E et al. 1996; Schneemann A et al.2012], while complete proteins (or domains) successfully displayed include The immunoglobulin-like domain (14 kDa) of the omega virus (NωV) capsid protein and the human anthrax virulence receptor ANTXR2 (20 kDa) [Manayani DJ et al. , "PLoS Pathogens" and other authoritative international academic journals, and obtained a number of US and international patents. Among them, the use of FHV VLPs to display the anthrax virulence receptor ANTXR2 and the A-helical domain of the influenza virus HA antigen is an important or breakthrough in the development of anthrax antidotes, highly effective vaccines, and broad-spectrum influenza vaccines [Manayani DJ et al. 2007; Schneemann A et al. 2012], demonstrated the important value of the Nodamura virus capsid display system as an efficient and highly versatile vaccine and drug development platform.

Contents of the invention

The purpose of the present invention is to provide a method for constructing a novel antigen display system based on insect virus capsid, which is easy to implement and easy to operate.

Another object of the present invention is to provide a novel antigen display system based on insect virus capsid. The advantages of this system are: (1) The main component of the system is the Wuhan Nodamura virus WhNV genome, which has a simple structure ( 4.6kb), which is convenient for genetic manipulation in vitro. At present, the applicant has established a WhNV transgene operating system, which can be directly used for the construction of heterologous polypeptide and protein display systems; (2) The use of WhNV VLP for vaccine development has the advantages of Higher biological safety; (3) Compared with the traditional protein surface display system, the WhNV capsid protein display system has a higher heterologous protein copy number (180 copies), which makes the vaccine based on this system relatively Compared with the traditional vaccine preparation method, it can cause a stronger immune response, thus greatly improving its medical value; (4) Compared with the FHV protein display system, the WhNV capsid protein has a larger capacity and a smaller stem-loop structure region. wide, the measurement results show that the stem-loop domain of WhNV capsid protein contains about 24 insertable sites, which provides a broad space for screening high-efficiency vaccines and functional neutralizing antidotes; (5) WhNV is where the inventors The virus discovered by the research laboratory independently in China has its complete and independent intellectual property rights. Compared with the FHV display system mastered by the United States, the WhNV display system is more suitable for the needs of China's public health and national defense security; (6) Wuhan Nodamura virus ( WhNV) has obvious advantages in many aspects as a heterologous polypeptide and protein display system.

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

A method for constructing a novel antigen display system based on an insect virus capsid, the steps of which are:

(1) Determination of the insertion site of the heterologous protein: analyze the structure of the WhNV capsid protein according to bioinformatics methods, find out the position area of the β-barrel structure and the surface stem-loop structure loop, and determine that the loop is suitable for inserting the heterologous polypeptide and potential sites of proteins;

(2) Construction of the WhNV recombinant virus-like particle (VLP) library fused with enhanced green fluorescent protein (EGFP): add a short amino acid sequence as a linker to both ends of the insertion site in the Loop region, and cut the site and other genetic modification methods to construct a vector that can be easily operated, and insert EGFP into the insertable sites of all Loop regions in the WhNV capsid protein through the linker sequence and the linker sequence-free way to construct two fusions The WhNV recombinant VLP library of the method, through immunofluorescence observation, sucrose density gradient centrifugation, electron microscope observation and other methods, preliminarily screened out the WhNV recombinant VLP library that can not only display the correct conformation of EGFP, but also maintain the correct assembly of virions, using EGFP as a reporter gene, Obtain a stable system that can form virus-like particles without affecting its structure and antigenicity after inserting heterologous proteins;

(3) Construction of the WhNV recombinant VLP library fused with amino acids 50-204 of the human avian influenza virus H5 antigenic protein: on the basis of preliminary screening of suitable candidate insertion sites using EGFP in the step (2), combined with biological Informatics analysis, further screening of the insertable site, similarly insert the 50-204 amino acid domain of the human avian influenza virus H5 antigen protein into the insertable site through the linker sequence and the method not mediated by the linker sequence, to construct the fusion human and poultry The WhNV recombinant VLP library of the 50-204 amino acid domain of the influenza virus H5 antigen protein, using electron microscopy and immunoreactivity experiments to screen out correctly folded and highly immunoreactive VLPs;

(4) Establishment of WhNV display system: Combined with the results in step (3), optimize the expression system of recombinant VLP by exploring different cell lines, culture media and culture conditions, and immunize the optimized recombinant VLP Reactivity experiments, and preliminary evaluation of it as a possible new vaccine, through the screening of the above step (2) and the above step (3), further determine the insertable sites on the WhNV Loop, combined with the optimized recombinant VLP Expression system, to establish a stable WhNV heterologous polypeptide and protein display system.

The linker sequence in the step (2) and step (3) is "Gly4-Ser-Gly4".

Through the technical measures of the above steps, the most critical step is step 1: the position of the stem-loop on the surface of the WhNV capsid protein was predicted, and 5 insertion sites were selected; the linker peptide was improved to make the screening of EGFP protein smooth The virus VLPs that can display the 50-204 amino acid domain of the human avian influenza virus H5 antigen protein were obtained on the surface, and the recombinant VLP library fused with the human avian influenza virus H5, N1 and M1 antigens was constructed, and the virus VLPs that could display the human avian influenza virus H5, N1 and M1 antigens were screened. Influenza virus antigens, and recombinant VLPs that can maintain the correct assembly of VLPs.

A novel antigen display system based on insect virus capsid, including: (1) Wuhan Nodamura virus WhNV capsid, (2) fusion protein inserted into WhNV capsid protein as a heterologous protein, (3) linker linker, It is characterized in that a small segment of amino acid sequence is respectively added to the N-terminal and C-terminal of the fusion protein as a linker to connect with the WhNV capsid.

Further, the fusion protein is enhanced green fluorescent protein EGFP.

Further, the amino acid sequence of the small segment is "Gly4-Ser-Gly4".

Compared with the prior art, the present invention has the following advantages and effects:

(1) Established a surface display technology based on WhNV capsid VLPs to display heterologous polypeptides and protein antigens on its surface. The present invention uses the WhNV capsid protein stem-loop domain to contain about 24 insertable sites , provides a broad space for screening high-efficiency vaccines; using insect virus VLPs as a display system has no biological safety problems and is highly feasible; compared with traditional VLP surface display systems, WhNV capsid display systems have higher heterogeneous Antigen copy number (180~540 copies), and can display a larger complete protein or domain, which makes the vaccine based on this system can cause a stronger immune response than the traditional vaccine preparation method, thus greatly Improve its medical value;

(2) The use of recombinant WhNV VLPs to display human avian influenza virus antigens will hopefully lead to the development of highly efficient and safe H5N1 human avian influenza virus VLPs vaccines; the use of insect cells to prepare and produce VLPs vaccines can overcome the avian influenza epidemic’s need for the production of traditional vaccines. threats to embryo production;

(3) The insect cell expression system is safe, does not contain mammalian pathogenic microorganisms, is non-carcinogenic, and is suitable for the production of vaccines;

(4) On the basis of the successful research and development of H5N1 vaccine, the research and development of new influenza virus strain vaccine can be completed in a relatively short period of time, and a general influenza vaccine research and development platform can be established;

(5) The establishment and optimization of the WhNV heterologous peptide and protein antigen display system has laid a solid foundation for its application in various fields such as vaccine and targeted drug development.

Description of drawings

Fig. 1 is a schematic diagram of predicting the three-dimensional structure of the core region of a capsid protein of Wuhan Nodamura virus WhNV.

Fig. 2 is a schematic view of a fluorescent microscope observing virus-like particles fused with EGFP.

Fig. 3 is a schematic view of a fluorescent microscope observing virus-like particles fused with EGFP with a linker.

Fig. 4 is a schematic diagram of predicting the structure of a CP protein inserted with an antigen of an avian influenza virus.

Fig. 5 is a schematic diagram of eukaryotic expression and sucrose density gradient centrifugation purification of a wild-type and recombinant WhNV capsid protein.

Fig. 6 is a schematic diagram of electron microscope observation of an intercalated VLPs. The recombinant capsid protein samples were negatively stained with phosphotungstic acid and observed under an electron microscope, and it was found that the recombinant capsid protein could be correctly assembled into inserted VLPs.

Detailed ways

Example 1:

A method for constructing a novel antigen display system based on an insect virus capsid, the steps of which are:

1. Prediction of the three-dimensional structure of the WhNV capsid protein of Wuhan Nodamura Virus: Using the Protein Data Bank (PDB for short), swi (SWISS-MODEL is a service for predicting the tertiary structure of proteins, which uses the method of homology modeling Realize the prediction of the tertiary structure of an unknown sequence, using a series of working software to extract the simulated structure information of the protein query sequence from the classic Brookhaven protein structure database (Brookhaven PDB), and use the known structure with protein similarity The molecular model of the unknown structural protein established by the protein, specifically four steps of template selection, target sequence template sequence comparison, modeling, and energy minimization) and other prediction methods, to predict the three-dimensional structure of the WhNV capsid protein, After fitting the predicted results, it was found that similar to FHV, the WhNV capsid protein has a β-barrel structure composed of 7 β-sheet structures, and simulated the three-dimensional structure of the WhNV capsid protein (Figure 1). The location of the stem-loop was determined, and five loops that could be used as potential heterologous protein insertion sites were preliminarily analyzed.

As shown in Figure 1, Loop 1 includes amino acids 127-134 of Wuhan Nodamura capsid protein, Loop 2 includes 143-155, Loop 3 includes 179-185, Loop 4 includes 188-199, and Loop 5 includes 207-214 bit. These regions provide a large number of potential foreign protein insertion sites, providing a basis for screening recombinant VLPs with high immunogenicity.

2. Use immunofluorescence technology to screen the WhNV VLP library fused with enhanced green fluorescent protein (EGFP): on the basis of the predicted insertable sites in step (1), use the method of inserting EGFP to initially screen out 5 ( Loop1131-132, Loop 2 148-149, Loop 3 180-181, Loop 4 191-192, Loop 5 209-210) can make the inserted protein display its native conformation site.

As shown in Figure 2, fluorescent signals can be detected when EGFP is inserted into the amino acid 131-132 region of Loop1 and the amino acid 191-192 region of Loop 4, while the fluorescence signals of other EGFP-fused VLPs are almost undetectable, indicating that insertion These sites affect the structure and function of EGFP (showing green fluorescence).

3. To preliminarily test the role of linker peptides that can improve stem-loop flexibility and insertion efficiency in antigen display. Using highly flexible linker peptide sequences can further improve the flexibility and insertion capacity of the stem-loop structure , to help the heterologous antigen display its correct conformation.

With this design, by adding linker sequences at both ends of EGFP, EGFP can obtain more free stretching space on the surface of VLPs, so that it can fold correctly and exhibit stronger activity.

A peptide of an amino acid sequence (Gly4-Ser-Gly4) was added as a linker at the N-terminal and C-terminal of EGFP (Figure 3 A), and then inserted into 148-149 of Loop 2, 180-181 of Loop 3 and 209-210 of Loop 5. In Figure 2, these virus-like particles without the linker cannot produce fluorescence, but after the linker is added, the inventors found that 148-149 inserted into Loop 2 and 209-210 of Loop 5 can produce fluorescence through fluorescence electron microscope observation Signal, among which the signal at position 148-149 of Loop 2 is the strongest (Fig. 3B).

Therefore, using the linker sequence can effectively maintain the natural conformation of the heterologous protein on the surface of WhNV VLPs. This has laid a good foundation for the subsequent application and optimization of the linker peptide in the WhNV VLPs display system.

4. Predict the structure of the WhNV capsid protein fused with H5, N1 and M1 antigens of avian influenza virus, and use bioinformatics methods (homology modeling) to further predict the WhNV capsid fused with one or three antigens of avian influenza virus Three-dimensional structure of protein (CP). The amino acid sequence of the antigen to be inserted was integrated into the amino acid sequence of WhNV CP according to the software (PDB) prediction and the insertion site determined in the previous screening, and the threading method (threading) and homology modeling (swi, ExPDB: template database, Extract high-quality data files from PDB, ExNRL-3D: sequence database, which is the sequence part of ExPDB template database, used for BLASTP2 template search. Loop library: collection of random coil structures obtained from experimental data. Rotamer library rotamer library: used to determine the spatial configuration of non-backbone amino acids) to analyze its overall structure. As shown in Figure 4A-C, in terms of spatial structure, H5, N1 or M1 antigens of avian influenza virus can be inserted into the Loop region of CP (H5 is inserted into 131-132 of Loop1, N1 is inserted into 148-149 of Loop 2, M1 is inserted into 131-132 of Loop1) and keep it folded correctly. In addition, the structure of WhNV CP protein with three avian influenza virus antigens inserted in three different loop regions was also predicted. As shown in FIG. 4D , the CP after simultaneous insertion of H5, N1 and M1 antigens of avian influenza virus can also maintain its correct folding. This prediction result indicates the possibility of simultaneously displaying 2-3 avian influenza virus antigens on the surface of WhNV VLPs, and it is hopeful that this display system can be used to develop highly effective human avian influenza VLPs vaccines.

5. Purification and electron microscope observation of recombinant VLPs inserted with exogenous H5 antigen: the inventor inserted the 50-204 amino acid domain of human avian influenza virus H5 antigen protein (referred to as the H5 antigen core domain) on the surface of WhNV VLPs. The wild-type (uninserted) and WhNV CP proteins inserted with the above antigens were isolated and purified by sucrose density gradient centrifugation, and the band size was consistent with the expected results detected by Western blot (Figure 5). The recombinant CP protein sample was negatively stained with phosphotungstic acid and observed under an electron microscope, and it was found that the recombinant capsid protein could be correctly assembled into inserted VLPs (Figure 6). The above-mentioned VLPs purification and electron microscope observation provided good technical support for the follow-up work.

Through the above technical measures, the following effects are obtained:

1) Optimize the design of the linker sequence to improve the flexibility of the stem-loop and insert efficiency; significantly improve the flexibility and insertion capacity of the stem-loop structure, and help the heterologous antigen display its correct conformation.

2) Construct a recombinant VLP library fused with human avian influenza virus H5, N1 and M1 antigens, and screen out recombinant VLPs that can not only display human avian influenza virus antigens on the surface, but also maintain the correct assembly of VLPs;

3) Structural analysis of WhNV VLPs displaying heterologous polypeptides or proteins using cryo-EM three-dimensional reconstruction technology;

4) Optimizing the preparation and purification conditions of WhNV VLPs displaying human avian influenza virus antigens;

5) Establish a stable WhNV VLPs heterologous antigen display system, making it a new technology platform for efficient and universal vaccine (and drug) development.

Example 2:

A novel antigen display system based on insect virus capsid, including: (1) Wuhan Nodamura virus WhNV capsid, (2) fusion protein inserted into WhNV capsid protein as a heterologous protein, (3) linker linker, It is characterized in that a small segment of amino acid sequence is respectively added to the N-terminal and C-terminal of the fusion protein as a linker to connect with the WhNV capsid.

Further, the fusion protein is enhanced green fluorescent protein EGFP.

Further, the amino acid sequence of the small segment is "Gly4-Ser-Gly4".

Claims (2)

1. A novel antigen display system based on insect virus capsids, comprising: (1) the WhNV capsid of the marhan nodavirus, (2) a fusion protein inserted as a heterologous protein into the WhNV capsid protein, (3) a linker characterized in that: the insertion site of the heterologous protein is 131-132, 148-149 or 209-210 of WhNV capsid protein of the Wuhan nodavirus, and Gly4-Ser-Gly4 small-segment amino acid sequences are respectively added at the N end and the C end of the fusion protein and used as linker to be connected with the WhNV capsid.

2. The antigen display system of claim 1, wherein the fusion protein is an enhanced green fluorescent protein EGFP.

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