Fusion protein, method for producing ferritin from plants and application

Abstract

The invention relates to the field of plant gene functions, in particular to a fusion protein, a method for producing ferritin from plants and application. The invention provides a fusion protein. The fusion protein comprises endoplasmic reticulum chaperonin, ferritin and endoplasmic reticulum retention signal peptide from an N terminal to a C terminal, researches find that efficient expression and stable accumulation of ferritin in plant cells are realized by fusing specific signal peptide and an endoplasmic reticulum retention sequence in a target gene, the design not only improves the expression level of ferritin in the plant cells, but also improves the expression level of ferritin in the plant cells through the processing and modification effects of the endoplasmic reticulum. The stability and the biological activity of the ferritin are enhanced. Meanwhile, due to the existence of the endoplasmic reticulum retention sequence, ferritin is effectively retained in the endoplasmic reticulum, and degradation and interference in cytoplasm are avoided, so that the target protein yield is increased.

Description

Technical Field

[0001] This invention relates to the field of plant gene function, and more specifically to a fusion protein, a method for producing ferritin from plants, and its applications. Background Technology

[0002] Ferritin, a major intracellular iron storage protein, possesses a unique cage-like structure with an outer diameter of 12 nanometers and an inner diameter of 8 nanometers, assembled from 24 subunits. This structure endows ferritin with the ability to encapsulate various drug molecules, such as chemotherapeutic drugs, alkaloids, and antibiotics, thereby achieving efficient drug delivery and targeted release. Ferritin's excellent biocompatibility, low toxicity, and inherent tumor-targeting ability make it an ideal nanomedicine platform. Furthermore, ferritin is a novel ligand for transferrin receptor 1 (TfR1). With its natural tumor-targeting ability (mediated by the TfR1 receptor) and blood-brain barrier penetration capability, ferritin shows unique advantages in cancer treatment, such as for the treatment of brain tumors like glioma and breast cancer brain metastases, as well as some neurological diseases that are difficult to treat with traditional drugs that penetrate the blood-brain barrier.

[0003] Despite its significant application potential, ferritin's large-scale production remains limited by traditional animal-derived extraction processes. Currently, commercial ferritin is primarily extracted from horse spleen, resulting in low content and the risk of contamination by animal pathogens. The yield from animal-derived extraction processes is constrained by the supply and extraction efficiency of horse spleen, making it difficult to meet the demands of large-scale production. Expressing recombinant ferritin in E. coli through genetic engineering also carries the risk of microbial toxin contamination, adversely affecting the purity and safety of the ferritin.

[0004] Plant bioreactors, as an alternative production platform, avoid the limitations of animal-derived extraction and microbial fermentation, offering advantages such as being green, safe, and efficient. However, different target proteins may be suited to different plant bioreactors. For example, some proteins may be expressed efficiently in plants like tobacco, Arabidopsis thaliana, or rice, while their expression efficiency is lower in other plants. Therefore, when selecting a plant bioreactor, the characteristics of the target protein and its compatibility with the plant host must be considered to optimize production efficiency and product quality. Furthermore, to enable large-scale production of target proteins from plants and ensure their physiological activity, some studies have reported the use of elements or functional proteins that enhance protein expression in DNA constructs containing the target protein. These include introducing promoters designed for specific tissues such as seeds, leaves, or roots of specific plants to increase the accumulation of the target protein and simplify the purification process; and targeting organelle sequences, such as chloroplasts, endoplasmic reticulum, vacuoles, and cytoplasm, to localize the target protein to specific organelles, thereby improving its stability and expression level. However, these strategies are often limited by the adaptability of the target protein, the plant species, and the type of organelle selected, and may introduce complex regulatory mechanisms that affect the physiological activity and function of the target protein. Currently, no studies have reported the successful large-scale expression of ferritin via plant bioreactors while ensuring that the physiological activity and function of the target protein are not affected. Summary of the Invention

[0005] Therefore, the first technical problem to be solved by the present invention is to provide a fusion protein, a method for producing ferritin from plants, and its application, thereby achieving efficient expression and stable accumulation of ferritin in plant cells.

[0006] Therefore, the present invention provides the following technical solution:

[0007] This invention discloses a fusion protein comprising, from the N-terminus to the C-terminus, endoplasmic reticulum chaperone protein, ferritin, and endoplasmic reticulum retention signal peptide.

[0008] This invention reveals that although ferritin targets chloroplasts, chloroplasts are less efficient than the endoplasmic reticulum (ER) in ferritin folding, modification, transport, and integration with other metabolic pathways. Therefore, a targeting sequence for the ER was chosen, specifically an ER chaperone protein and an ER retention signal peptide. This invention, by fusing an ER chaperone protein and an ER retention signal peptide into a ferritin-containing fusion protein, not only improves the expression level of ferritin in plant cells but also enhances its stability and bioactivity through the processing and modification effects of the ER. Simultaneously, due to the presence of the ER retention sequence, ferritin is effectively retained in the ER, avoiding degradation and interference in the cytoplasm, thus achieving efficient expression and stable accumulation of ferritin in plant cells. Furthermore, after the fusion protein expresses ferritin in the targeted endoplasmic reticulum (ER), the resulting target protein typically lacks an ER chaperone protein at its N-terminus. This is because ER chaperone proteins primarily play an auxiliary role in protein synthesis and folding, helping nascent peptide chains fold and assemble correctly. During ferritin synthesis, ER chaperone proteins may interact with ferritin to assist its folding and maturation, but they dissociate after ferritin maturation. Conversely, after the fusion protein expresses ferritin in the targeted ER, the resulting target protein typically retains an ER retention signal peptide at its C-terminus. This ER retention signal peptide plays a crucial role in the retention of ferritin within the ER.

[0009] In some embodiments, the endoplasmic reticulum chaperone protein assists in the correct folding and modification of ferritin in the endoplasmic reticulum of plant cells, enhancing the stability and biological activity of ferritin, thereby achieving the expression and accumulation of ferritin in plant cells. Further, the endoplasmic reticulum chaperone protein is selected from at least one of heavy chain binding protein (Bip protein), protein disulfide isomerase (PDI protein), calreticulin (CRT protein), Golgi intermediate chamber 53 (ERGIC-53 protein), and endoplasmic reticulum DnaJ homolog (ERd protein). In a preferred embodiment, the endoplasmic reticulum chaperone protein is selected from Bip protein, and the nucleotide sequence encoding Bip protein is shown in SEQ ID NO.3. This invention has found that Bip protein is particularly suitable for ferritin; Bip protein can efficiently assist in the correct folding of ferritin, prevent its mis-aggregation, and more efficiently achieve the efficient expression and stable accumulation of ferritin in plant cells.

[0010] In some embodiments, the endoplasmic reticulum (ER) retention signal peptide assists in the transport of ferritin and other metabolic pathways, promoting the targeting and retention of ferritin in the ER, thereby achieving the expression and accumulation of ferritin within plant cells and the ER. Further, the ER retention signal peptide is selected from at least one of HDEL or KDEL proteins. In a preferred embodiment, the ER retention signal peptide is selected from HDEL protein, and the nucleotide sequence encoding the HDEL protein is shown in SEQ ID NO. 4. This invention has found that HDEL protein is particularly suitable for ferritin, and can better cooperate with Bip protein. HDEL protein can efficiently assist in the transport and retention of ferritin in the ER, and facilitate other metabolic pathways within the ER, thus achieving more efficient expression and stable accumulation of ferritin in plant cells.

[0011] This invention discloses a biomaterial comprising any one of the following:

[0012] (1) A nucleic acid molecule encoding the fusion protein; optionally, the nucleic acid molecule is DNA or RNA;

[0013] (2) Expression cassette, recombinant vector, recombinant microorganism or transgenic cell line expressing the fusion protein;

[0014] (3) Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing the nucleic acid molecules described in (1);

[0015] (4) Recombinant vectors, recombinant microorganisms or transgenic cell lines containing the expression cassettes described in (2) or (3);

[0016] (5) Host cells containing the recombinant vector described in (2) or (3) or (4);

[0017] (6) Plant cells or plant bodies containing the recombinant vector described in (2) or (3) or (4) or the host cell described in (5).

[0018] In some embodiments, the 5' end of the nucleic acid molecule encoding the fusion protein further includes at least one of a promoter and a 5'UTR element. In a preferred embodiment, the 5' end of the nucleic acid molecule encoding the fusion protein includes a promoter, said promoter being a 35S promoter. This invention has found that, compared to other common promoters (such as the NOS promoter), the 35S promoter is a strong promoter for cauliflower mosaic virus (CaMV), which can be used across species in plants, and is particularly suitable for driving efficient transcription of the ferritin-encoding gene, thereby increasing ferritin expression levels. In a preferred embodiment, the 5' end of the nucleic acid molecule encoding the fusion protein further includes a 5'UTR element, said 5'UTR element being located between the promoter and the coding gene of the fusion protein. The 5'UTR (5' untranslated region) is the 5' untranslated region of eukaryotic gene mRNA, generally derived from highly expressed genes, and can enhance mRNA stability and translation efficiency. This invention has found that setting a 5'UTR element at the 5' end of the fusion protein can help to efficiently transcribe the ferritin coding gene and improve the ferritin expression level.

[0019] In some embodiments, the 3' end of the nucleic acid molecule encoding the fusion protein further includes at least one of a 3'UTR element, a terminator, a gene silencing repressor element, and a CaMV poly(A)signal. In a preferred embodiment, the 3' end of the nucleic acid molecule encoding the fusion protein includes a terminator, which is a 35S terminator. This invention has found that, compared to other common terminators (NOS terminators), the 35S terminator, being a CaMV terminator, can efficiently terminate the transcription process, especially suitable for ensuring the integrity and stability of the transcription product of the ferritin-encoding gene, which is beneficial for subsequent translation and thus improves ferritin expression levels. In a preferred embodiment, the 3' end of the nucleic acid molecule encoding the fusion protein further includes a 3'UTR element, which is located between the coding gene of the fusion protein and the terminator. In a preferred embodiment, the 3' end of the nucleic acid molecule encoding the fusion protein further includes a gene silencing repressor element, which is located at the 3' end of the terminator. This invention has found that, compared to not adding gene silencing repressor elements or other common gene silencing repressor elements, selectively adding the P19 element, which is derived from tomato yellow leaf curl virus and is an RNA interference repressor, can inhibit plant RNA silencing mechanisms, and is particularly suitable for more stable expression of the ferritin gene in plant cells. In a preferred embodiment, the 3' end of the nucleic acid molecule encoding the fusion protein also includes CaMV poly(A), which is the polyadenylated nucleotide signal of cauliflower mosaic virus, guiding the addition of a polyadenylated nucleotide tail to the 3' end of the mRNA, thereby enhancing mRNA stability and translation efficiency.

[0020] In some embodiments, the initial vector for the recombinant vector is selected from PEAQ-HT vector, pBI121 vector, and pCAMBIAC2300S vector. In a preferred embodiment, the initial vector is selected from PEAQ-HT vector. As mentioned above, this invention has found that placing a 35S promoter at the 5' end and a 35S terminator and gene silencing repressor element P19 at the 3' end of the nucleic acid molecule encoding the fusion protein, through this combination, can significantly improve the expression level of ferritin. The PEAQ-HT vector includes a 35S promoter, a 35S terminator, and a gene silencing repressor element P19, while the pBI121 vector contains a NOS promoter and a NOS terminator, but lacks the gene silencing repressor element P19. The pCAMBIAC2300S vector has a 35S promoter and a 35S terminator, but lacks the gene silencing repressor element P19. Therefore, the preferred embodiment is to select the PEAQ-HT vector. Furthermore, the PEAQ-HT vector also possesses the following advantages: A CPMV-based supertranslation system enhances protein expression through 5'UTR and 3'UTR, enabling efficient expression of exogenous genes in a short time. The vector is simple: non-essential sequences are removed, reducing interference and allowing for more focused target gene expression. It is easy to operate: containing multiple cloning sites and Gateway recombination sites facilitates target gene insertion and allows N- or C-terminal histidine tag fusion, facilitating protein purification and detection. It enhances stability: containing the P19 gene silencing repressor protein encoding gene, inhibiting plant RNA silencing mechanisms, increasing mRNA stability, and making the target gene more stably expressed. It is compatible with multiple cloning technologies: supporting restriction enzyme digestion and Gateway recombination, among other methods. It has multi-gene expression capabilities: allowing the construction of multiple expression cassettes in a single vector, enabling simultaneous expression of multiple genes, which is helpful for studying gene interactions and multi-gene engineering. It integrates technological advantages: combining the rapid and high expression of viruses, the efficient transfection of Agrobacterium, and the low-cost production and diverse synthetic capabilities of plants, including post-translational modifications.

[0021] In some embodiments, the host cell may be Agrobacterium.

[0022] In some embodiments, the plant is selected from tobacco, Arabidopsis thaliana, soybean, tomato, lettuce, rice, wheat, or corn. In a preferred embodiment, the plant is tobacco; in a more preferred embodiment, the tobacco is Nicotiana benthamiana or cultivated tobacco. This invention has found that Nicotiana benthamiana increases ferritin expression levels.

[0023] This invention discloses a method for producing ferritin from plants, comprising the step of culturing the plant cells or plant body. The plant is selected from tobacco, Arabidopsis thaliana, soybean, tomato, lettuce, rice, wheat, or corn. In a preferred embodiment, the plant is tobacco; in a more preferred embodiment, the tobacco is Nicotiana benthamiana or cultivated tobacco. This invention has found that ferritin expression levels are increased in Nicotiana benthamiana.

[0024] This invention provides the use of the aforementioned fusion protein, the aforementioned biomaterial, or the aforementioned method for producing ferritin from plants in the delivery of a drug or the preparation of a product for delivering a drug.

[0025] The technical solution of this invention has the following advantages:

[0026] 1. This invention provides a fusion protein comprising, from the N-terminus to the C-terminus, an endoplasmic reticulum chaperone protein, ferritin, and an endoplasmic reticulum retention signal peptide. This invention has found that by fusing a specific signal peptide and an endoplasmic reticulum retention sequence into a target gene, efficient expression and stable accumulation of ferritin in plant cells are achieved. This design not only improves the expression level of ferritin in plant cells but also enhances its stability and biological activity through processing and modification by the endoplasmic reticulum. Simultaneously, due to the presence of the endoplasmic reticulum retention sequence, ferritin is effectively retained in the endoplasmic reticulum, avoiding degradation and interference in the cytoplasm, thereby increasing the yield of the target protein.

[0027] 2. This invention provides a method for producing ferritin from plants, utilizing plant cells or plant bodies for culture. This invention selects a promoter applicable to a wide range of plant species and combines it with the endoplasmic reticulum (ER) localization signal Bip and the retention sequence HDEL to construct a fusion gene, Bip-Ferritin-CO-HDEL. This design not only improves the expression level of ferritin in plant cells but also enhances its stability and biological activity through ER processing and modification. Simultaneously, due to the presence of the ER retention sequence, ferritin is effectively retained in the ER, avoiding degradation and interference in the cytoplasm, thereby increasing the yield of the target protein.

[0028] Furthermore, the plant selected is *Nicotiana benthamiana*, which possesses advantages such as high biomass, short growth cycle, ease of scaling up, and short transient conversion cycle. By introducing the ferritin gene into *Nicotiana benthamiana* cells and utilizing an Agrobacterium-mediated transient expression system, a large amount of plant material expressing ferritin can be obtained in a short time. Therefore, using *Nicotiana benthamiana* as a substrate holds promise for achieving efficient heterologous synthesis of ferritin, opening up a new pathway for ferritin production.

[0029] Furthermore, the method of this invention avoids the limitations of traditional animal-derived extraction and microbial fermentation, and has advantages such as being green, safe, and efficient, providing a new approach for the large-scale production of ferritin. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 These are the spectra of the three recombinant vectors in Embodiment 1 of the present invention;

[0032] Figure 2 This is the result of detecting the relative expression level of the Ferritin gene in tobacco leaf samples transiently transformed by three recombinant vectors in Example 3 of the present invention;

[0033] Figure 3 This is the Western blot result of the Ferritin protein level detection in tobacco leaf samples transiently transformed by three recombinant vectors in Example 3 of the present invention.

[0034] Figure 4 This is the extraction and purification result of Ferritin protein in tobacco leaf samples transiently transformed by the PEAQ-HT-Ferritin (original) recombinant vector in Example 4 of this invention; the left figure shows the result of Western blot detection of the target protein; the right figure shows the SDS-PAGE electrophoresis result of each eluent; TP represents the total protein in the supernatant, 20mM E represents the eluent collected by elution with 20mM Wash buffer, and so on; CK represents the control group;

[0035] Figure 5 This is the result of quantification of Ferritin protein in Tobacco Benzoinus in Example 5 of the present invention; the left figure is the SDS-PAGE of the concentrated proteins (20mM Ferritin and 50mM Ferritin) after using 2μg, 4μg, 6μg, 8μg, and 10μg BSA standards and 20mM and 50mM eluents from Example 4; the right figure is the BSA standard curve;

[0036] Figure 6 This is the transmission electron microscopy (TEM) negative staining observation result of Ferritin protein and horse spleen ferritin in tobacco Benzoin in Example 6 of the present invention; the left image is the TEM negative staining observation result of horse spleen ferritin (HSF), and the right image is the TEM negative staining observation result of Ferritin protein (F) extracted from tobacco Benzoin.

[0037] Figure 7 The figure shows the zeta potential measurement results of Ferritin protein and horse spleen ferritin in tobacco Benzoate in Example 6 of this invention; HSF in the figure represents horse spleen ferritin, and F represents Ferritin protein extracted from tobacco Benzoate.

[0038] Figure 8 These are the ultraviolet and fluorescence spectra of Ferritin protein and horse spleen ferritin in *Nicotiana benthamiana* in Example 6 of this invention; the left figure shows the ultraviolet spectrum results, and the right figure shows the fluorescence spectrum results.

[0039] Figure 9 This is the positive identification result of the Ferritin-targeted ER transgenic line mRNA level in Example 7 of the present invention;

[0040] Figure 10 This is the positive identification result of the Ferritin-targeted ER transgenic line protein level in Example 7 of the present invention;

[0041] Figure 11 The results are observed by excitation lamp illumination after the recombinant vectors constructed by the three vectors in Example 8 of this invention are transformed into Tobacco Benzovia. Detailed Implementation

[0042] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0043] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0044] Information concerning biomaterials in the following embodiments includes:

[0045] Tobacco variety: Benedictine tobacco, provided by the National Tobacco Gene Research Center;

[0046] Vector: PEAQ-HT vector, provided by the National Tobacco Gene Research Center;

[0047] The pBI121 vector was provided by the National Tobacco Gene Research Center.

[0048] pCAMBIAC2300S vector was provided by the National Tobacco Gene Research Center;

[0049] DH5α Escherichia coli competent cells were purchased from Qingke Biotechnology Co., Ltd.

[0050] GV3101 Agrobacterium competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0051] Primer synthesis and DNA sequencing were provided by Beijing BGI Genomics Co., Ltd.

[0052] Experimental reagents:

[0053] 2×Phanta Max Master MixP515 high-fidelity enzymes were purchased from Novizan Biotechnology Co., Ltd.

[0054] AgeI and XhoI restriction endonucleases were purchased from the NEB website;

[0055] C112 seamless cloning reagent was purchased from Novizan Biosciences Co., Ltd.;

[0056] Easy The Quick Gel Extraction Kit was purchased from TransGen Biotech Inc.

[0057] The plant RNA rapid extraction kit was purchased from Beijing Codon Biotechnology Co., Ltd.

[0058] The reverse transcription kit was purchased from TaKaRa.

[0059] Perfect Green qPCR Super Mix was purchased from Beijing TransGen Biotech Co., Ltd.

[0060] Sure PAGE TM The Tris-MES-SDS Running Buffer was purchased from Nanjing Genscript Biotech Co., Ltd.

[0061] The trichrome prestained protein molecular weight standards (8–180 kDa) were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0062] Western PCR rapid transfer buffer and PVDF membrane were purchased from Beyotime Biotechnology Co., Ltd.

[0063] Protein-free rapid blocking solution (1×) and TBST (10×, pH 7.4) were purchased from Shanghai Yamei Biomedical Technology Co., Ltd.; Nonfat-Dried Milk was purchased from Beijing Kulaibo Technology Co., Ltd.

[0064] Anti-His Mouse mAb, Goat Anti-Mouse lgG, and HRP were purchased from TransGen Biotech Inc.

[0065] The instruments are all commonly used in molecular biology and genetic engineering laboratories.

[0066] Table 1. MS medium:

[0067]

[0068]

[0069] Table 2. MSO liquid culture medium:

[0070]

[0071] Table 3, washing buffer:

[0072]

[0073] Table 4. Differentiation culture medium:

[0074]

[0075] Table 5. Rooting medium:

[0076]

[0077] Example 1: Construction of Ferritin Expression Vector

[0078] This embodiment provides a method for constructing a ferritin expression vector, including the following steps:

[0079] (1) Obtaining the target gene

[0080] The Ferritin gene and its codon-optimized sequence based on tobacco codon preference were obtained commercially. The nucleotide sequence of the Ferritin gene (original) is shown in SEQ ID NO.1. To facilitate purification and detection, a coding gene encoding 8 histidines was placed downstream of the Ferritin gene (original). The codon-optimized sequence (Ferritin-CO) is shown in SEQ ID NO.2. Similarly, to facilitate purification and detection, a coding gene encoding 8 histidines was placed downstream of the Ferritin-CO gene.

[0081] (2) Synthesis of the fusion gene (Bip-Ferritin-CO-HDEL)

[0082] In step (1), the target gene is fused upstream and downstream with the nucleotide sequences of the endoplasmic reticulum chaperone protein sequence (Bip) (as shown in SEQ ID NO.3) and the endoplasmic reticulum retention signal peptide sequence (HDEL) (as shown in SEQ ID NO.4), respectively, specifically including the following steps:

[0083] Specific amplification primers were designed for the nucleotide sequences of Bip and HDEL and cloned upstream and downstream of the target gene (Ferritin-CO) in step (1);

[0084] The primer sequences are as follows:

[0085] Upstream primer Bip-F (SEQ ID NO.5):

[0086] 5'-CTGCCCAAATTCGCGACCGGTATGGCTCGCTCGTTTGGAG-3';

[0087] Downstream primer BiP(Ferritin-CO)-R (SEQ ID NO.6):

[0088] 5'-TCTCACTTGAGACTCTAACTTCGTAGCCTCTTCTATTGCAG-3';

[0089] Upstream primer Ferritin-CO-F (SEQ ID NO.7):

[0090] 5'-GAGGCTACGAAGTTAGAGTCTCAAGTGAGACAACAATTCTCTAAG-3';

[0091] Downstream primer Ferritin-CO-HDEL-R (SEQ ID NO.8):

[0092] 5'-AACCAGAGTTAAAGGCCTCGAGTCAAAGTTCATCGTGATGATGATGATGATGATGATGAGAACC-3';

[0093] The PCR reaction in a 50 μl system included: upstream primer (a mixture of Bip-F and Ferritin-CO-F), 10 μM per primer, 2 μL; downstream primer (a mixture of BiP(Ferritin-CO)-R and Ferritin-CO-HDEL-R), 10 μM per primer, 2 μL; template DNA (Ferritin-CO), 100 ng, 2 μL; 2×PhantaMax MasterMixP515 high-fidelity enzyme, 25 μL; ddH2O, 20 μL.

[0094] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 32 cycles; and a final extension at 72℃ for 5 min.

[0095] After PCR, agarose gel electrophoresis was performed, followed by gel recovery. The PCR amplification product was purified according to the gel recovery kit instructions to obtain the target fusion gene.

[0096] (3) Carrier linearization

[0097] The PEAQ-HT vector was linearized using AgeI and XhoI restriction endonucleases.

[0098] The 50 μL enzyme digestion reaction consisted of: 1 μL each of AgeI and XhoI; 1 μg of PEAQ-HT; and 10× rCutSmart. TM Buffer, 5 μL; add ddH2O to bring the total to 50 μL.

[0099] The enzyme digestion reaction conditions were: 37℃ digestion for 15 min, followed by heat inactivation at 65℃ for 20 min.

[0100] (4) Connect sequences homologous to both ends of the linear vector at both ends of the fusion gene.

[0101] Specific primers are designed based on the target gene in step (1) or the fusion gene obtained in step (2). The primers contain homologous sequences at both ends of the linearized vector to meet the requirements of seamless cloning. Primers designed based on the target gene Ferritin gene (original) are shown as PEAQ-Ferritin-F and PEAQ-Ferritin-R; primers designed based on the target gene Ferritin gene (codon optimized) are shown as PEAQ-Ferritin-CO-F and PEAQ-Ferritin-CO-R; primers designed based on the fusion gene Bip-Ferritin-CO-HDEL are shown as PEAQ-Ferritin-CO-ER-F and PEAQ-Ferritin-CO-ER-R. The designed primer sequences are as follows:

[0102] PEAQ-Ferritin-F(SEQ ID NO.9):

[0103] 5'-CTGCCCAAATTCGCGACCGGTATGGAAAGCCAAGTGCGTCA-3';

[0104] PEAQ-Ferritin-R(SEQ ID NO.10):

[0105] 5'-AACCAGAGTTAAAGGCCTCGAGTTAATGATGGTGATGGTGATGGTGATGG-3';

[0106] PEAQ-Ferritin-CO-F (SEQ ID NO.11):

[0107] 5'-CTGCCCAAATTCGCGACCGGTATGGAGTCTCAAGTGAGACAACAATTCT-3';

[0108] PEAQ-Ferritin-CO-R(SEQ ID NO.12):

[0109] 5'-AACCAGAGTTAAAGGCCTCGAGTTAGTGATGATGATGATGATGATGAGAACC-3';

[0110] PEAQ-Ferritin-CO-ER-F (SEQ ID NO.13):

[0111] 5'-CTGCCCAAATTCGCGACCGGTATGGCTCGCTCGTTTGGAG-3';

[0112] PEAQ-Ferritin-CO-ER-R(SEQ ID NO.14):

[0113] 5'-AACCAGAGTTAAAGGCCTCGAGTCAAAGTTCATCGGTGATGATGATGATGATG-3';

[0114] The designed primers are used to perform PCR amplification of the fusion gene sequence (or target gene) to obtain the fusion gene (or target gene) sequence with homologous arms;

[0115] The PCR reaction system includes: a 50 μl PCR reaction system comprising: upstream primers, each with a concentration of 10 μM, 2 μL; downstream primers, each with a concentration of 10 μM, 2 μL; template DNA, 100 ng, 2 μL; 2×PhantaMax MasterMixP515 high-fidelity enzyme, 25 μL; ddH2O, 20 μL.

[0116] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 32 cycles; and a final extension at 72℃ for 5 min.

[0117] After PCR, agarose gel electrophoresis was performed, followed by gel recovery. The PCR amplification product was purified according to the gel recovery kit instructions to obtain the fusion gene sequence with homologous arms.

[0118] (5) Seamless cloning

[0119] The fusion gene (or target gene) with homologous arms in step (4) is mixed with the PEAQ-HT fragment of the linearized vector after enzyme digestion in step (3). The fusion gene (or target gene) is then linked to the linearized vector using seamless cloning technology to form a complete expression vector.

[0120] Seamless cloning reaction in a 10 μl system includes: target fragment, 200 ng, 2 μL; linearization vector, 100 ng, 5 μL; 5×CE II Buffer, 2 μL; Exnase II, 1 μL;

[0121] The seamless cloning reaction conditions are: react at 37°C for 30 min, then cool to 4°C or immediately place on ice to cool;

[0122] The ligation products were transformed into competent *E. coli* cells. Positive clones were identified and screened to obtain three recombinant plasmids for expressing Ferritin. The recombinant plasmid expressing Ferritin, prepared using only the original Ferritin gene as the target gene, was named PEAQ-HT-Ferritin(original). The recombinant plasmid expressing Ferritin, prepared using only codon-optimized Ferritin as the target gene, was named PEAQ-HT-Ferritin-CO. The recombinant plasmid expressing Ferritin, prepared using the fusion gene, was named PEAQ-HT-Bip-Ferritin-CO-HDEL, and its nucleotide sequence is shown in SEQ ID NO. 19. Maps of the three recombinant vectors are shown below. Figure 1 As shown.

[0123] Example 2 Instantaneous conversion of Tobacco Benzoin

[0124] The three recombinant vectors expressing Ferritin obtained in Example 1 were mixed with GV3101 Agrobacterium competent cells, and the vectors were introduced into the Agrobacterium cells by heat shock, specifically including the following steps:

[0125] Remove Agrobacterium competent cells from -80℃ and thaw them on ice; add 100-200 ng of recombinant vector to 50 μL of competent cells, mix well, and incubate on ice for 5 min; incubate in liquid nitrogen for 5 min, heat shock at 37℃ for 5 min, incubate on ice for 5 min, and add 700 μL of antibiotic-free LB liquid medium.

[0126] Incubate at 28℃ and 200rpm for 2h, centrifuge the bacterial suspension at 4500rpm for 2min, remove the supernatant, obtain the bacterial suspension, and spread it evenly on LB solid plates (containing Kan (50μg / mL) and Rif (50μg / mL)), and incubate upside down at 28℃ for 36 to 48h until single colonies are formed;

[0127] Single colonies were picked, expanded, and then the bacterial culture was subjected to PCR identification to identify the correct positive clone strains.

[0128] Select positive clones and inoculate them into LB liquid medium (containing Kan (50 μg / mL) and Rif (50 μg / mL)) and incubate at 28°C and 200 rpm with shaking until OD. 600nm The value was 0.8; the bacterial culture was centrifuged at 4500 rpm for 3 min, the supernatant was discarded, and the bacterial cells were resuspended in osmotic buffer MMA (20 mM MgCl2, 20 mM MES, 2-morpholine ethanesulfonic acid) and 200 μM acetosytingone to OD. 600nm After being incubated in the dark for 2-3 hours, the cultured Agrobacterium was injected into *Nicotiana benthamiana* leaves to transiently transform them. This utilized the natural infectivity of Agrobacterium to achieve transient expression of Ferritin protein in *Nicotiana benthamiana*. The injection method was standard: using a 1mL sterile syringe (needle removed), the Agrobacterium culture was drawn up and gently injected into the lower epidermis of the tobacco leaf until a water-soaked appearance appeared, indicating successful injection. The injected tobacco plants were then placed in a light incubator at 25°C with a 16-hour light / 8-hour dark cycle for 3-5 days for subsequent experimental observation or protein extraction.

[0129] Example 3: Detection of Ferritin gene transcription level and Ferritin protein expression level

[0130] 1. Real-time quantitative PCR transcription level detection:

[0131] (1) The tobacco leaf samples from Example 2 were flash-frozen in liquid nitrogen on the fifth day after transient transformation, and total RNA was extracted.

[0132] (2) Using the extracted RNA as a template, cDNA synthesis reaction was carried out using reverse transcriptase;

[0133] (3) Using the synthesized cDNA as a template and Actin as an internal reference gene, specific quantitative primers for Ferritin gene were designed and real-time fluorescence quantitative PCR reaction was performed.

[0134] The quantitative primer sequences for Ferritin-CO-ER (representing the fusion gene Bip-Ferritin-CO-HDEL), Ferritin, and Ferritin-CO are as follows:

[0135] Q-Ferritin-F (SEQ ID NO.15): 5'-CGGGCCTGTTTCTGTTTGATC-3';

[0136] Q-Ferritin-R (SEQ ID NO. 16): 5'-GCTGTTCCGCCACATACCAC-3';

[0137] Q-Ferritin-CO-F (or Q-Ferritin-CO-ER-F) (SEQ ID NO. 17):

[0138] 5'-ATGAGCAGGTGAACAAGGAGAT-3';

[0139] Q-Ferritin-CO-R (or Q-Ferritin-CO-ER-R) (SEQ ID NO. 18):

[0140] 5'-TCAGGAGCAGAAATAGAAGTAAGC-3'.

[0141] Table 6. Real-time quantitative PCR reaction systems for the Ferritin, Ferritin-CO, and Ferritin-CO-ER genes:

[0142]

[0143] Table 7. Real-time quantitative PCR reaction conditions for the Ferritin, Ferritin-CO, and Ferritin-CO-ER genes:

[0144] The Ct values ​​of the Ferritin gene (or Ferritin-CO gene, Ferritin-CO-ER gene) and the internal reference gene were calculated to assess its transcriptional level in tobacco leaves.

[0145] The results are as follows Figure 2 As shown in the figure, the CK group is the control group (untreated Tobacco Benedictine samples). The relative expression levels of Ferritin-1 and Ferritin-2 (parallel experiments) in the figure are 5.8 × 10⁻⁶. 4 5.7×10 4 The relative expression levels of Ferritin-co-1 and Ferritin-co-2 (in parallel experiments) were 8 × 10⁻⁶. 4 9.7×10 4 The relative expression levels of Ferritin-co-ER-1 and Ferritin-co-ER-2 were 1.47 × 10⁻⁶. 5 1.53×10 5 In the figure, *** and **** indicate that, compared with CK, p < 0.001 and p < 0.0001, respectively. As can be seen from the figure, the Ferritin gene or the codon-optimized Ferritin-co gene was successfully transcribed in the transiently transformed Nicotiana benthamiana with the three recombinant vectors. However, compared with the other two groups, the relative expression level of Ferritin-co-ER was significantly higher. This is because the strategy of targeting the endoplasmic reticulum optimizes the intracellular localization of ferritin, placing it in an environment more conducive to synthesis, folding, and modification, and ensures its stable accumulation with the help of the endoplasmic reticulum retention signal, thus resulting in a higher relative expression level than the codon-optimized and original methods.

[0146] 2. Western blot protein level detection:

[0147] Leaf samples of *Nicotiana benthamiana* from Example 2 were taken five days after *Agrobacterium benthamiana* injection, and the protein expression level of the gene in the leaves was detected. The specific method is as follows:

[0148] (1) Weigh 0.1g of tobacco leaves after injection, freeze them quickly with liquid nitrogen, and then grind them on a grinder. The grinding frequency is set to 55HZ and the running time is 90 seconds.

[0149] (2) Add 400 μl of 2×SDS loading buffer, vortex to mix, and boil in boiling water at 100℃ for 10 minutes.

[0150] (3) Centrifuge at 12000 rpm for 10 minutes, aspirate 10 μl of supernatant and load onto a sample, then perform a Sure PAGE. TMProtein gel and Tris-MES-SDS running buffer were used for electrophoresis. SDS-PAGE was used to separate the proteins. The electrophoresis was set to a constant current of 400mA for 25 minutes.

[0151] (4) Activate the PVDF membrane with methanol for 2 minutes, and use wet transfer to transfer the protein on SDS-PAGE onto the PVDF membrane. Set the electrophoresis voltage to 200V and 30 minutes.

[0152] (5) Block the PVDF membrane with Western Blot rapid blocking solution and shake at room temperature for 30 minutes.

[0153] (6) Prepare 30 ml of 5% skim milk solution with TBST to prepare primary and secondary antibody dilution buffer;

[0154] (7) After incubating the PVDF membrane with the primary and secondary antibody dilution buffer for 45 minutes, wash it 4 times with TBST for 10 minutes each time;

[0155] (8) Mix colorimetric solutions A and B in a volume ratio of 1:1, and drop them evenly onto the PVDF membrane. React in the dark for 2 minutes, and observe the experimental results using a gel imaging system.

[0156] The results are as follows Figure 3 As shown in the figure, CK represents untreated Nicotiana benthamiana samples, and PEAQ-HT represents unrecombined empty vectors. Ferritin protein was successfully expressed in Nicotiana benthamiana samples transiently transformed by the three recombinant vectors. In addition, it can be seen from the figure that the Ferritin-co-ER band was significantly wider than the other two groups. This is because targeting the ER (endoplasmic reticulum) increases the expression of ferritin in Nicotiana benthamiana.

[0157] Example 4: Ferritin protein extraction and purification

[0158] (1) Collect 60g of tobacco leaves from the fifth day after injection (in Example 2), and set up a CK group (untreated tobacco leaves from the fifth day after injection). Add 3 times the volume of protein extraction buffer and crush them in a plant cell wall breaker for 3 minutes.

[0159] (2) Transfer the mixture from step (1) to a 50ml centrifuge tube, centrifuge at 4℃ and 15000rpm for 20 minutes, discard the precipitate, repeat the centrifugation once, collect the supernatant (total protein in the supernatant), and discard the precipitate.

[0160] (3) Beads pretreatment: Pipette 4 ml of beads into a centrifuge tube, centrifuge at 500 rpm for 30 seconds, and discard the supernatant. Add 2 ml of extraction buffer, centrifuge at 500 rpm for 30 seconds, centrifuge again and discard the supernatant. Repeat this step twice.

[0161] (4) Add 1 ml of beads (step (3)) to every 50 ml of supernatant from step (2) and place it on a 4℃ rotator for 2 hours;

[0162] (5) Centrifuge the mixture in step (4) at 500 rpm for 3 minutes and keep 3 ml of supernatant for resuspending the beads;

[0163] (6) Wash the PD-10 column twice with washing buffer, gently pipette to resuspend the beads, add them to the PD-10 column, wash the beads with 25 ml of wash buffer twice, and collect the eluent.

[0164] (7) Elute the target protein on the beads in step (6) twice with 20mM Wash buffer, 25ml each time, and collect the eluent.

[0165] (8) Elute the target protein on the beads in step (7) twice with 50mM Wash buffer, 25ml each time, and collect the eluent.

[0166] (9) Finally, wash the beads in step (8) twice with 100mM, 250mM, 500mM and 1M Wash buffer respectively, 25ml each time, and collect the eluent.

[0167] Western blot analysis of protein content in each elution buffer yielded the following results: Figure 4 As shown in the middle left figure, Figure 4 To obtain the results from the *Nicotiana benthamiana* leaves transfected with PEAQ-HT-Ferritin (original) in Example 2, the target protein was collected in 20 mM and 50 mM eluents. The 20 mM and 50 mM eluents containing the target protein were transferred to 10 kDa concentration tubes and centrifuged at 4°C and 4000 rpm for 5 minutes each time, until the 50 ml eluent was concentrated to approximately 5 ml. After concentration, SDS-PAGE electrophoresis was performed for observation. The results are as follows: Figure 4 The concentrated protein was added with an equal volume of 20% glycerol and stored at -80°C.

[0168] Example 5: Quantification of Ferritin Protein in Nicotiana benthamiana using the BSA Standard Curve Method

[0169] (1) Bovine serum albumin (BSA) was used as a standard protein to quantitatively determine the protein content in the sample;

[0170] (2) Prepare BSA standards of different concentrations, and load SDS-PAGE samples at amounts of 2μg, 4μg, 6μg, 8μg, and 10μg.

[0171] (3) Add the concentrated 20mM and 50mM to 1 / 4 volume of protein loading buffer, and the loading volume is 25μl;

[0172] (4) After electrophoresis, stain with Coomassie Brilliant Blue for 30 minutes, and destain with destaining solution for 10 minutes each time, for a total of three times;

[0173] (5) After the blue background has faded to colorless and transparent and the protein bands are clearly visible, place the gel in a gel imaging system to take pictures and save the experimental data;

[0174] (6) Image J calculates the gray values ​​of each protein band and plots the BSA standard curve;

[0175] (7) Calculate the gray values ​​of 20mM and 50mM protein bands obtained by extracting and purifying exogenous ferritin from tobacco leaf samples instantaneously transformed by the three recombinant vectors in Example 2 according to Example 4, input the data into the BSA standard curve, obtain the protein loading amount, and estimate the yield of exogenous ferritin in Tobacco Benedict.

[0176] The results are as follows Figure 5 As shown, the estimated yields of exogenous ferritin in *Nicotiana benthamiana* were Ferritin (0.03 mg / g FW), Ferritin-CO (0.08 mg / g FW), and Ferritin-CO-ER (0.11 mg / g FW).

[0177] Example 6 Physicochemical characterization of tobacco-derived Ferritin protein

[0178] 1. TEM electron microscopy observation with negative staining:

[0179] (1) Dilute the protein sample (the 20mM concentrated sample of exogenous ferritin extracted and purified from the tobacco leaf sample transiently transformed by the PEAQ-HT-Ferritin (original) expression vector in Example 2 according to Example 4) to 0.02-0.05 mg / ml with Tris-HCl buffer (20 mmol / L, pH 7.0);

[0180] (2) Use a copper mesh with a support film and treat the copper mesh with a glow discharge instrument for 20-45 seconds to enhance the hydrophilicity of the copper mesh.

[0181] (3) Use 2% uranium acetate solution to stain the sample in step (1);

[0182] (4) Pipette 5 μl of sample onto the copper grid, use filter paper to absorb the excess solution from the side, air dry, and then observe on the instrument.

[0183] (5) TEM microscopy was performed on the ferritin sample at 80kV using a Hitachi H-7650 electron microscope.

[0184] The results are as follows Figure 6 As shown, the transmission electron microscopy negative staining observation results of Ferritin protein and horse spleen ferritin in tobacco Benzoate show that the tobacco-derived ferritin presents uniform white granules with a similar granule morphology to horse spleen ferritin, and its particle size is close to the theoretical diameter of Ferritin, which is 12 nm.

[0185] 2. Zeta potential measurement:

[0186] The zeta potential of ferritin samples was detected using the Zeta potential analyzer advance from Malvern, UK.

[0187] Measurements were taken at 25°C, and the samples were tested in triplicate.

[0188] All experimental data results are averaged.

[0189] The results are as follows Figure 7 As shown, by comparing the zeta potentials of Ferritin protein in Nicotiana benthamiana and horse spleen ferritin, it was concluded that both horse spleen ferritin and tobacco-derived ferritin carry a negative charge on their surface, and the magnitudes of the negative charges are similar, indicating that tobacco-derived ferritin has similar stability to horse spleen ferritin.

[0190] 3. UV and fluorescence spectral analysis of ferritin samples:

[0191] The measurements were performed using a UV-2550PC ultraviolet-visible spectrometer at an experimental temperature of 25℃.

[0192] The sample was diluted 3-fold with pH 7.5, 20mM Tris-HCl buffer;

[0193] Place the ferritin sample into a 1 mL cuvette, use Tris-HCl buffer (pH 7.5, 20 mM) as a blank control, and set the scanning wavelength range between 200-600 nm.

[0194] The intrinsic fluorescence emission spectrum of the sample was determined using a Cray Eclipse fluorescence spectrophotometer.

[0195] Ferritin samples were diluted 3-fold with 20mM Tris-HCl buffer at pH 7.5.

[0196] The excitation wavelength was set to 290 nm, and the widths of the excitation and emission slits were set to 10 nm. The emission spectrum was scanned in the range of 300-500 nm.

[0197] The results are as follows Figure 8 As shown, the ultraviolet and fluorescence spectra of ferritin protein and horse spleen ferritin in tobacco Benzoate indicate that the iron ion content in tobacco-derived ferritin is lower than that in natural horse spleen ferritin, which is more conducive to drug loading.

[0198] Furthermore, since the N-terminus of the target protein obtained after PEAQ-HT-Bip-Ferritin-CO-HDEL expresses ferritin in the target endoplasmic reticulum usually does not contain endoplasmic reticulum chaperone proteins, and the endoplasmic reticulum retention signal peptide is a short peptide that does not affect ferritin, it can be concluded that the ferritin expressed by PEAQ-HT-Bip-Ferritin-CO-HDEL has a comparable physicochemical characterization to that expressed by the PEAQ-HT-Ferritin (original) expression vector in Nicotiana benthamiana.

[0199] Example 7: Preparation of stably inherited Ferritin materials

[0200] The *Agrobacterium* bacterial culture with ER-targeting Ferritin from Example 2 was expanded and cultured until OD... 600nm At a concentration of 0.8, Agrobacterium was resuspended in MSO liquid medium and adjusted to OD. 600nmThe concentration was 0.8; the gene was stably transformed into Nicotiana benthamiana using Agrobacterium-mediated leaf disc method; the specific experimental steps are as follows: (1) Culture of sterile seedlings: Take an appropriate amount of Nicotiana benthamiana seeds and put them into a 1.5ml centrifuge tube. Disinfect with 10% sodium hypochlorite solution for 10 minutes. Carefully aspirate the sodium hypochlorite solution from the centrifuge tube using a pipette, being careful not to aspirate the seeds. Wash the seeds 4 times with sterile water and dry them on sterile filter paper. Sow the seeds on MS medium with a sterile toothpick and place them in a plant culture box with 25℃, 16h light / 8h darkness conditions for culture. About 3 weeks after sowing, when the Nicotiana benthamiana reaches the cross stage, transfer it to a tissue culture bottle on MS solid medium and continue to culture it in a plant culture box for one month for later use. (2) Preparation of bacterial suspension: Positive colonies were picked from the Agrobacterium tumefaciens plate of the target gene and inoculated into 10 ml of 50 μg / ml Kan and 50 μg / ml Rif LB liquid medium and cultured overnight at 28℃ and 200 rpm in a shaker until the OD600 reached 0.8. Under room temperature conditions, the bacterial suspension was centrifuged at 5000 rpm for 10 minutes, the supernatant was discarded, and the bacterial cells were resuspended in MSO liquid medium and diluted to about 0.5 for stable transformation of Tobacco Benzoenta. (3) Infection of Tobacco Benzoenta with Agrobacterium tumefaciens: Leaves of sterile Tobacco Benzoenta seedlings were cut off in a clean bench. The relatively flat leaf parts were cut into leaf discs about the size of a little fingernail and placed in sterile Petri dishes for later use. To prevent the leaf discs from wilting, sterile water was added to the Petri dishes to keep them moist. The leaf discs were transferred into the Agrobacterium tumefaciens resuspension for 10 minutes of infection, and the plates were shaken continuously to ensure that the leaf discs were in full contact with the Agrobacterium tumefaciens. After infection, the leaf disc surface was dried with filter paper, and the leaf disc was placed flat in MS culture dish with the front side facing up. The culture dish was sealed with sealing film, wrapped with aluminum foil and placed in a 25℃ incubator for dark culture for 2 days. (4) Differentiation and screening culture of tobacco: The leaves after dark culture were taken out, and the surface bacteria were washed with sterile water. Then, they were washed twice with sterile water containing 400mg / ml carbenicillin for 10min and 5min respectively. They were washed once more with sterile water. After the leaf disc was taken out, the water was dried with filter paper, and the leaf disc was placed flat in the differentiation medium with the front side facing up. It was placed in a plant culture incubator with 25℃, 16h light / 8h darkness conditions for culture. During the period, the leaf differentiation status was observed and fresh differentiation medium was replaced in time. (5) Rooting culture of resistant shoots: After the leaves are cultured in the differentiation medium for 3 weeks, the small shoots can be cut off and transferred to the rooting medium. They are then placed in a plant culture box with 25℃, 16h light / 8h darkness conditions for cultivation. Rooting can be seen one week after being transferred to the rooting medium. After another week of growth, the rooted tobacco seedlings can be taken out of the tissue culture bottle, the solid culture medium on the roots can be washed with water, and then they can be transferred to the soil substrate (3 / 4 volume of nutrient soil and 1 / 4 volume of vermiculite) for growth. A layer of plastic wrap is covered to keep the soil moist. After the seedlings have grown for 3 days, the plastic wrap is removed and they are cultured in a plant culture room with 25℃, 16h light / 8h darkness conditions.

[0201] After screening with resistant culture media (with kanamycin and carboxybenzyl added to the culture media in Table 4 and the differentiation and rooting culture media in Table 5) and positive identification at the RNA and protein levels (as performed in Example 3), stable transformed lines were obtained. The expression levels of the stable transformed lines were detected as in Example 3 to perform positive identification of the stable transformed lines.

[0202] The results are as follows Figure 9 and Figure 10 As shown, Ferritin-co-ER#1 to Ferritin-co-ER#4 represent stable transformed lines obtained from four parallel experiments. As can be seen from the figure, this embodiment successfully obtained Ferritin-co-ER transgenic lines.

[0203] Example 8: Component Selection

[0204] This example investigated the effect of different elements on the expression level of ferritin in the fusion gene (Bip-Ferritin-CO-HDEL). PEAQ-HT, pBI121, and pCAMBIAC2300S vectors were selected for the experiment. The PEAQ-HT vector contains a 35S promoter, a 5'UTR element, a 3'UTR element, a 35S terminator, a gene silencing repressor element P19, and a CaMV poly(A)signal in the 5'→3' direction. The pCAMBIAC2300S vector differs from the PEAQ-HT vector in that it lacks the gene silencing repressor element P19. The pBI121 vector contains a NOS promoter, a 5'UTR element, a 3'UTR element, a NOS terminator, and a CaMV poly(A)signal in the 5'→3' direction. Following the procedure in Example 1, the fusion gene was ligated to the three vectors described above. For ease of observation, the gene encoding UVGFP (green fluorescent protein) was also fused to the C-terminus of the fusion gene.

[0205] The three recombinant vectors obtained above were implemented according to Example 2. Then, tobacco leaf samples five days after transient transformation were observed under an excitation lamp (excitation wavelength of 440-460 nm) for fluorescence observation. The results are as follows. Figure 11 As shown, the CK group consists of untreated tobacco leaves. As can be seen in the figure:

[0206] 1 represents pBI121-Bip-Ferritin-CO-HDEL-UVGFP;

[0207] Representative 2: pCAMBIAC2300S-Bip-Ferritin-CO-HDEL-UVGFP;

[0208] 3 represents: pEAQ-HT-Bip-Ferritin-CO-HDEL-UVGFP.

[0209] As can be seen, the pEAQ-HT-Bip-Ferritin-CO-HDEL-UVGFP group exhibits strong green fluorescence, indicating that pEAQ-HT-Bip-Ferritin-CO-HDEL-UVGFP efficiently expresses and stably accumulates ferritin in plant cells.

[0210] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A fusion protein, characterized in that, The direction from the N-terminus to the C-terminus includes: endoplasmic reticulum chaperone proteins, ferritin, and endoplasmic reticulum retention signal peptides.

2. The fusion protein according to claim 1, characterized in that, The endoplasmic reticulum chaperone protein is selected from at least one of Bip protein, PDI protein, CRT protein, ERGIC-53 protein, and ERd protein. And / or, the endoplasmic reticulum retention signal peptide is selected from at least one of HDEL protein or KDEL protein.

3. The fusion protein according to claim 2, characterized in that, The endoplasmic reticulum chaperone protein is selected from the Bip protein, and the nucleotide sequence encoding the Bip protein is shown in SEQ ID NO.3; And / or, the endoplasmic reticulum-associated retention signal peptide is selected from HDEL protein, the nucleotide sequence of which is shown in SEQ ID NO.

4.

4. A biomaterial, characterized in that, Includes any one of the following: (1) A nucleic acid molecule encoding the fusion protein according to any one of claims 1-3; optionally, the nucleic acid molecule is DNA or RNA; (2) An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line for expressing the fusion protein of claim 1 or 2; (3) Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing the nucleic acid molecules described in (1); (4) Recombinant vectors, recombinant microorganisms or transgenic cell lines containing the expression cassettes described in (2) or (3); (5) Host cells containing the recombinant vector described in (2), (3), or (4); (6) Plant cells or plant bodies containing the recombinant vector described in (2), (3), or (4) or the host cell described in (5).

5. The biomaterial according to claim 4, characterized in that, The 5' end of the nucleic acid molecule encoding the fusion protein of claim 1 or 2 further includes at least one of a promoter and a 5'UTR element; optionally, the promoter is a 35S promoter.

6. The biomaterial according to claim 4 or 5, characterized in that... ， The 3' end of the nucleic acid molecule encoding the fusion protein of claim 1 or 2 further includes at least one of a 3'UTR element, a terminator, a gene silencing repressor element, and CaMV poly(A); optionally, the terminator is a 35S terminator; optionally, the gene silencing repressor element is selected from P19.

7. The biomaterial according to any one of claims 4-6, characterized in that, The initial vector for the recombinant vector is selected from PEAQ-HT vector, pBI121 vector, and pCAMBIAC2300S vector; optionally, the initial vector is selected from PEAQ-HT vector. And / or, the host cell is Agrobacterium.

8. The biomaterial according to any one of claims 4-7, characterized in that, The plant is selected from tobacco, Arabidopsis thaliana, soybean, tomato, lettuce, rice, wheat or corn; optionally, the plant is tobacco; optionally, the tobacco is Nicotiana benthamiana or cultivated tobacco.

9. A method for producing ferritin from plants, characterized in that, include: Cultured using the plant cells or plant body described in claim 4.

10. Use of the fusion protein of claim 1 or 2, the biomaterial of any one of claims 3-8, or the ferritin produced by the method of producing ferritin from plants according to claim 9 in the delivery of a drug or in the preparation of a product for delivering a drug.

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