SARS-Cov-2 protein gene and its corn endosperm expression vector

Abstract

The present invention discloses a SARS-Cov-2 protein gene and a corn endosperm expression vector thereof. It is a gene of an RBD protein optimized by corn codons, a signal peptide is added to the N-terminus, a 2XStrepII-3XFlag tag is added to the C-terminus, and a TEV cleavage site is added between the RBD and the tag; or, it is a gene of an S protein optimized by corn codons, a signal peptide is added to the N-terminus, a 2XStrepII-3XFlag tag is added to the C-terminus, the amino acid sequence RRAR is mutated to GSAS, KV is mutated to PP, the C-terminal transmembrane domain of the S protein is deleted, and a fibrin trimerization motif is added. The optimized RBD protein and S protein can be successfully expressed and efficiently purified in corn seeds. This high-yield, plant-based platform shows great potential in basic research applications, diagnostic method development, and vaccine production.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular agriculture, and specifically relates to a SARS-Cov-2 protein gene and a corn endosperm expression vector thereof. Background Art

[0002] Molecular farming is an alternative biomanufacturing method for producing therapeutic proteins using plants. This method mainly involves expressing therapeutic proteins, such as antigens in plants, and then purifying these proteins for pharmaceutical use (Fausther-Bovendo and Kobinger, 2021). The key advantages of using plants for molecular farming are low cost, scalability, safety, and environmental sustainability (Zhu et al., 2022), which greatly reduces the risk of contamination (Ma et al., 2003). Due to the large-scale and low-cost cultivation capacity of plants and their incompatibility with human pathogens, plant-based production is more cost-effective than traditional bioreactor systems, making molecular farming particularly attractive for developing countries (Clark and Maselko, 2020). Plants can also undergo appropriate post-translational modifications and produce different glycans that can enhance immunogenicity (Sander et al., 2019). In 1989, monoclonal antibodies produced in tobacco leaves by transient transfection were the first successful case of molecular agriculture (Hiatt et al., 1989). Although transient expression systems offer the possibility of scalability and rapid production (Diego-Martin et al., 2020), establishing stable transgenic crop lines has long-term advantages, such as reducing production costs and developing edible vaccines. Over the years, a variety of recombinant proteins have been successfully expressed in transgenic plants such as wheat, potato, tomato, soybean, tobacco, rice, and corn (Chikwamba et al., 2002; He et al., 2008; Kwon et al., 2003; Richter et al., 2000; Shin et al., 2003; Vamvaka et al., 2016). These crops are conducive to the expression of plant proteins because of their stable products and suitability for greenhouse cultivation. Among these crops, corn has become an efficient expression system for the production of functional proteins (Murry et al., 1993). Maize has several key advantages, including storage stability, high yield, ease of transformation, and low production costs (Ramessar et al., 2008). The endosperm of maize is particularly suitable as a bioreactor for the production and storage of pharmaceutical proteins or oral vaccines, and the yield of maize kernels exceeds that of other cereal crops. The storage capacity and inherent protein stability of the endosperm can support the production and purification of large amounts of target proteins. In addition, transgenic products accumulated in maize seeds can remain stable for long periods of time without refrigeration, further enhancing their practicality for large-scale production in resource-limited settings (Larrick and Thomas, 2001).

[0003] SARS-CoV-2 is a single-stranded positive-sense RNA virus that encodes four structural proteins: spike protein (S), envelope (E), membrane (M), and nucleocapsid (N) (Masters, 2006). The S protein facilitates viral entry into host cells and contains a cleavage site (RRAR) that may enhance transmissibility. It is a trimeric fusion protein, and each monomer includes two subunits: S1 for receptor attachment and S2 for membrane fusion (Millet and Whittaker, 2015). The receptor binding domain (RBD) within S1 interacts with human angiotensin-converting enzyme 2 (ACE2), which is a key step in host cell entry (Li et al., 2003). Therefore, the S protein, especially its RBD, is the main target of most SARS-Cov-2 vaccines. Expression and purification of SARS-Cov-2 protein in rice and tobacco have been reported, but there has been no research on expressing the protein in corn endosperm, so it is not clear whether SARS-Cov-2 can be expressed in the corn system. Summary of the invention

[0004] The purpose of the present invention is to provide a method for expressing and purifying SARS-Cov-2 protein in corn endosperm, so as to make large-scale production of low-cost vaccines possible.

[0005] A SARS-Cov-2 protein gene, which is a gene of an RBD protein optimized by corn codons, with a signal peptide added to the N-terminus, a 2XStrepII-3XFlag tag added to the C-terminus, and a TEV cleavage site added between the RBD and the tag, and the nucleotide sequence thereof is shown in SEQ ID NO: 1 in the sequence listing;

[0006] Alternatively, it is a gene of the S protein that has been optimized by corn codons, with a signal peptide added to the N-terminus, a 2XStrepII-3XFlag tag added to the C-terminus, the amino acid sequence RRAR mutated to GSAS, KV mutated to PP, the C-terminal transmembrane domain of the S protein deleted, and a fibrin trimerization motif added, and its nucleotide sequence is shown in SEQ ID NO: 2 in the sequence table.

[0007] The amino acid sequence of the optimized RBD protein is shown in SEQ ID NO: 3 in the sequence listing.

[0008] The amino acid sequence of the optimized S protein is shown in SEQ ID NO: 4 in the sequence listing.

[0009] The vector containing the SARS-Cov-2 protein gene connects the optimized RBD protein gene or the optimized S protein gene into the pCAM3300 vector backbone, and uses the corn 19kDa a-Zein promoter to drive expression.

[0010] The method for constructing the vector of the SARS-Cov-2 protein gene uses the pCAM3300 vector as a transgenic vector, connects the sequence shown in SEQ ID NO: 1 or the sequence shown in SEQ ID NO: 2 into the pCAM3300 vector, and replaces the ubiquitin promoter in the pCAM3300 vector with the corn 19-kDa a-Zein promoter.

[0011] The SARS-Cov-2 protein gene is used in constructing a transgenic plant expressing the SARS-Cov-2 protein.

[0012] The transgenic plants are transgenic corn plants.

[0013] Beneficial effects of the present invention: The present invention successfully expressed and purified RBD and S proteins from corn endosperm by optimizing the amino acid sequences and gene sequences of RBD and S proteins. The purified RBD protein was recognized by the ACE2 receptor in an enzyme-linked immunosorbent assay, confirming its biological activity. RBD and S proteins can be successfully expressed and efficiently purified in corn seeds, proving for the first time the feasibility of using corn endosperm to produce recombinant drug proteins. This high-yield, plant-based platform shows great potential in basic research applications, diagnostic method development, and vaccine production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The RBD construct was optimized for maize codons;

[0015] In the figure, a is a vector map of the pZein::RBD-2XStrepII-3XFlag construct based on the pCAM3300 vector backbone; the purple box represents the signal peptide; the green box represents the 2XStrepII-3XFlag tag; RB: T-DNA right border; Nos: terminator; LB: T-DNA left border; b is a western blot analysis of crude protein extracts from different corn lines expressing the pZein::RBD-2XStrepII-3XFlag construct; c is the detection of protein using Flag antibody, NS: nonspecific band; Line 1 to Line 7 represent different transgenic corn lines.

[0016] Figure 2 To purify the receptor binding domain (RBD) from maize kernels;

[0017] In the figure, a is the SDS-PAGE analysis of the purified RBD protein expressed in corn kernels, with the molecular weight (kDa) marked on the left; b is the western blot analysis of the purified RBD protein using the Flag antibody; input: total protein; flow-through: protein not bound to the magnetic beads; elution: protein bound to the magnetic beads and eluted; c is the ability of the crude seed extract containing RBD to bind to ACE2 determined by enzyme-linked immunosorbent assay (ELISA); the figure represents the binding efficiency, the y-axis represents the absorbance, and the x-axis represents a series of dilution concentrations; Line 1, Line 4 and Line 5 represent 3 independent transgenic corn lines; negative control: PBST containing 1% bovine serum albumin (BSA); the purified RBD protein was prepared by two serial dilutions starting from 0.8 ng / ml.

[0018] Figure 3 The S protein construct was codon-optimized for maize;

[0019] In the figure, a is a schematic diagram of the maize codon-optimized recombinant S protein; the construct includes amino acid substitutions (RRAR replaced by GGAS, KV replaced by PP), removal of the transmembrane domain, and addition of a fibrin trimerization (FD) motif to promote trimerization; SP: signal peptide; green box: 2XStrepII-3XFlag tag; b is the cloning of the maize codon-optimized S protein coding sequence into the pCAM3300 destination vector driven by the pZein promoter, containing a 2XStrepII-3XFlag tag for purification; key features include RB (right border), Nos (terminator) and LB (left border).

[0020] Figure 4 To express and purify S protein from corn kernels;

[0021] In the figure, a is the western blot analysis of the corn codon-optimized S protein expressed in transgenic plants; the sample was detected with Flag antibody; Line1-Line6 represent different transgenic corn strains; NS: non-specific band; b is the western blot analysis of the purified corn codon-optimized S protein; Input: total protein; Flow-through: protein not bound to the purified magnetic beads; Elution: protein successfully bound to the magnetic beads; the expected molecular weight of the codon-optimized S protein is 146 kDa. DETAILED DESCRIPTION

[0022] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0023] Example 1 Vector construction and corn transformation

[0024] First, the RBD protein was expressed in S1. To ensure strong and specific endosperm expression, the maize 19kDa a-Zein promoter was used to drive expression. To ensure its correct modification, the inventors added an ER signal peptide at the 5' end. To facilitate purification and detection, a 2XStrepII-3XFlag tag was added to its C-terminus, and a TEV cleavage site was added between the RBD and the tag to achieve tag-free purification. In order to maximize the expression of the RBD protein, an optimized RBD protein sequence was synthesized based on codon preference and constructed into the pCAM3300 vector. In order to achieve endosperm-specific expression, the ubiquitin promoter in the pCAM3300 vector was replaced with the maize 19-kDa a-Zein promoter ( Figure 1 a). After sequence verification, the vector pZein::RBD-2XStrepII-3XFlag was introduced into Agrobacterium, and the final constructed vector was introduced into Agrobacterium tumefaciens after verification, and then transformed into the maize inbred line KN5585. In order to study the expression pattern of RBD in different tissues, the inventors collected different plant tissues, including nodes, tassels, leaves, filaments, bracts and seeds, extracted total proteins from these tissues, and performed protein blotting (western blot) analysis using Flag antibodies. The results showed that RBD was mainly expressed in seeds and weakly expressed in stem nodes and bracts ( Figure 1 b). Further analysis of the grains of seven independent T2 transgenic lines showed that six of them successfully expressed RBD ( Figure 1 c).

[0025] Secondly, in order to obtain the uncut full-length S protein, the S1 / S2 cleavage site (RRAR) was mutated to GSAS. In addition, the SARS-Cov-2 S protein was modified by deleting the C-terminal transmembrane domain (to achieve efficient expression), introducing two proline substitutions (to stabilize the S2 fusion mechanism), and adding a fibrin trimerization motif (to ensure proper trimer formation). The gene encoding the maize codon-optimized S protein sequence was cloned into the vector driven by the maize endosperm-specific promoter Zein and fused to the 2XStrepII-3XFlag tag at its C-terminus ( Figure 3 ). After sequence verification, the construct was introduced into maize via Agrobacterium-mediated transformation.

[0026] Example 2 Western blot analysis and ELISA

[0027] Protein extraction and purification: Maize kernels were harvested 30 days after pollination and ground into fine powder under liquid nitrogen to prepare corn protein extracts. Protein extraction buffer (100 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 5% glycerol, 1% TritonX-100, protease inhibitor [Cat#1183617001, Millipore Sigma]) was added to the powder of corn kernels (2 μl of protein buffer extraction buffer was added per g of tissue). The samples were gently mixed and centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was collected and filtered through a 0.45-μm filter to obtain total protein extracts. The supernatant was then mixed with MagStrep Strep-Tactin XT beads (IBA-lifesciences) at 4°C for 30 min, and the proteins were affinity purified according to the manufacturer's instructions.

[0028] Western blot analysis: SDS-PAGE was performed on 10% polyacrylamide gels in SDS buffer (25mM Tris, 250mM glycine, 0.1% SDS) at 200V for 35min. For western-blot experiments, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) by semi-dry transfer (12V, 25min). The membranes were incubated with TBST (TBS containing 0.05% Tween-20) containing 5% skim milk for 2h at room temperature, incubated with Flag antibody (1:5000 dilution) (Cat#F1804, Millipore Sigma) for 1.5h at room temperature, and then washed 5 times with TBST for 10min each. The membranes were then incubated with HRP-conjugated secondary antibody (1:1000) (Cat#31458, ThermoFisher) at room temperature for 1h and then washed 5 times with TBST for 10min each. Immunoblotting was performed with Amersham ECL (Report medicine RPN2105, Cytiva) detection reagent and imaging was performed with Fujifilm LAS-3000 imager.

[0029] ELISA: Coaster 96-well plates (Corning) were coated with 10 mg / ml ACE2 (Cat#287935, Abcam) in bicarbonate buffer (pH=9.6) and incubated overnight at 4°C. After incubation, the coating buffer was discarded and the plates were washed three times with PBST (carbonate buffer containing 0.05% Tween-20). Blocking was performed with PBST containing 1% BSA at 37°C for 1 h to prevent nonspecific binding. After blocking, plant-produced RBD proteins were incubated at 37°C for 2 h. In this step, PBST containing 1% BSA was used as a negative control. After washing three times with PBST, the plates were incubated with Flag antibody (1:1000) at room temperature for 2 h. The plates were washed again and incubated with HRP-conjugated secondary antibody (1:5000) (Cat#AP308P, Millipore Sigma) at 37°C for 1 h. After the final washing step, color was developed using TMB substrate (Reportmedicine, G7431, Promega), and the absorbance was measured at 450 nm using a microplate reader.

[0030] The inventors used Strep-Tactin XT magnetic beads to purify the recombinant RBD protein, and recovered an average of 31.36 mg of recombinant protein per gram of fresh tissue, indicating that the expression and purification were effective. The expression and purification of the recombinant RBD protein were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein blotting analysis (western blot). Figure 2 a) and western blot ( Figure 2 b) The expected molecular weight of the RBD protein was observed to be approximately 38 kDa. To test whether the RBD protein purified from corn endosperm is biologically active and can bind to ACE2, the inventors performed ELISA tests using purified RBD protein and corn kernel extracts, as well as bovine serum albumin (BSA) and non-transgenic corn kernels as negative controls. The inventors found that the plant-produced RBD protein showed a higher signal than the negative control and wild-type protein. In addition, the signal weakened as the concentration of RBD protein decreased, confirming the specific, concentration-dependent interaction between RBD and ACE2 ( Figure 2 c). These results confirm the structural integrity and in vitro binding activity of the RBD protein produced in maize endosperm and suggest that maize seeds are suitable for developing a scalable production platform.

[0031] The inventors used Strep-Tactin XT magnetic beads to purify the recombinant S protein, and proved through western blot experiments with Flag antibodies that 3 of the 6 independent transgenic lines could express the S protein ( Figure 4 a). Among the six transgenic lines, line 6 had the highest protein accumulation and was therefore selected for further analysis. The recombinant S protein was purified from 15 g of corn kernels using the previous method. Western blot analysis showed that the purified protein was enriched in the immunoprecipitation channel (IP) and a major band was observed at approximately 146 kDa ( Figure 4 b). These results confirmed that the codon-optimized S protein could be successfully expressed and efficiently purified in corn seeds.

[0032] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A nucleotide encoding a SARS-Cov-2 protein, characterized in that: The nucleotide encoding the SARS-Cov-2 protein is a nucleotide obtained by adding a signal peptide to the N-terminus, adding a 2XStrepII-3XFlag tag to the C-terminus, and adding a TEV cleavage site between the RBD and the tag to the gene of the RBD protein optimized by corn codons. The nucleotide sequence of the nucleotide encoding the SARS-Cov-2 protein is shown in SEQ ID NO: 1 in the sequence listing; Alternatively, the nucleotide encoding the SARS-Cov-2 protein is a gene obtained by adding a signal peptide to the N-terminus of the S protein nucleotide optimized by corn codon, adding a 2XStrepII-3XFlag tag to the C-terminus, mutating the amino acid sequence RRAR to GSAS, mutating KV to PP, deleting the C-terminal transmembrane domain of the S protein, and adding a fibrin trimerization motif. The nucleotide sequence of the nucleotide encoding the SARS-Cov-2 protein is shown in SEQ ID NO: 2 in the sequence listing.

2. The nucleotide encoding SARS-Cov-2 protein according to claim 1, characterized in that The amino acid sequence of the protein translated from the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing is shown in SEQ ID NO: 3 in the sequence listing.

3. The nucleotide encoding SARS-Cov-2 protein according to claim 1, characterized in that The amino acid sequence of the protein translated from the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing is shown in SEQ ID NO: 4 in the sequence listing.

4. A vector comprising the nucleotide encoding the SARS-Cov-2 protein according to claim 1, characterized in that: The nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide sequence shown in SEQ ID NO: 2 is connected to the pCAM3300 vector backbone, and the corn 19kDa a-Zein promoter is used to drive the expression of the nucleotide encoding the SARS-Cov-2 protein.

5. A method for constructing a vector comprising the nucleotide encoding the SARS-Cov-2 protein according to claim 1, characterized in that: The pCAM3300 vector is used as a transgenic vector, the sequence shown in SEQ ID NO: 1 or the sequence shown in SEQ ID NO: 2 is connected to the pCAM3300 vector, and the ubiquitin promoter in the pCAM3300 vector is replaced with a corn 19-kDa a-Zein promoter to drive the expression of the nucleotide encoding the SARS-Cov-2 protein.

6. Use of a vector comprising the nucleotide encoding the SARS-Cov-2 protein according to claim 1 in constructing transgenic corn expressing the SARS-Cov-2 protein.

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