A method for expressing human tumor necrosis factor-alpha in alfalfa

Abstract

This invention provides a recombinant transmembrane human tumor necrosis factor expressed in alfalfa. ‑α This method belongs to the field of biotechnology. This invention constructs a transmembrane human tumor necrosis factor... ‑α A plant binary expression vector for the gene is used, and then Agrobacterium-mediated transformation of the recipient alfalfa containing this plant binary expression vector is employed to prepare recombinant transmembrane human tumor necrosis factor. This invention utilizes alfalfa as a substrate plant for recombinant human tumor necrosis factor, providing a platform for recombinant transmembrane human tumor necrosis factor production. ‑α This provides an excellent cell system for expression, catering to the market demand for recombinant transmembrane trimeric tumor necrosis factor. ‑α This provides a new solution to the needs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a recombinant transmembrane human tumor necrosis factor expressed in alfalfa. -α The method. Background Technology

[0002] Tumor necrosis factor-α (TNF-α) is a pleiotropic immunocytokine primarily produced by cells of the innate immune system, such as macrophages and NK cells, as well as cells of the adaptive immune system, particularly activated T cells. Its main function is to regulate immune and inflammatory responses, and it has the effect of killing or inhibiting tumor cells. During infection, tissue damage, or immune responses, the level of TNF-α increases, triggering a series of downstream immune responses by binding to its receptors. TNF-α exists in vivo in two forms: a transmembrane precursor protein (tmTNF-α) with a molecular weight of 26 kDa; and a cleaved soluble form (sTNF-α) with a molecular weight of 18 kDa. Both forms of TNF-α can form homotrimers, thereby binding to their receptors (TNFR1 or TNFR2) to activate signaling pathways such as inflammation, apoptosis, and cell proliferation.

[0003] Most of the recombinant human tumor necrosis factor-α currently sold on the market is produced through prokaryotic expression. However, prokaryotic expression of human tumor necrosis factor-α has the following disadvantages: (1) Due to the lack of a sophisticated post-translational modification system, the prokaryotic system does not perform post-translational modification on the human tumor necrosis factor-α protein produced, resulting in incomplete function and all being in monomeric form; (2) Escherichia coli releases endotoxins during cell rupture to purify human tumor necrosis factor-α. The presence of endotoxins not only affects product quality and brings safety risks, but also significantly increases product costs when removing endotoxins. At the same time, most of the recombinant human tumor necrosis factor-α currently on the market is the soluble form (sTNF-α) after cleavage. However, current molecular biology has shown that the transmembrane precursor protein form of TNF-α (hereinafter referred to as tmTNF-α) also plays an important role in signaling pathways such as immune regulation.

[0004] Plants have been studied as a system for expressing and producing drug proteins for nearly thirty years. Compared to single-celled microorganisms, multicellular plant systems are rich in endomembrane systems and various organelles. The glandular trichomes on the plant surface are important sites for metabolite synthesis and storage. This complex spatiotemporal characteristic provides the most suitable environment for the synthesis of different types of enzymes and metabolites, which is beneficial for maintaining protein activity and yield. The complexity of multicellular plant systems also provides an excellent model system for synthetic biology research. Plants are rich in a large number of metabolites, which can directly provide precursors for the synthesis of plant bioactive molecules. Therefore, utilizing plants to synthesize important humanized proteins and naturally occurring bioactive small molecules has become an inevitable path for scientific and technological development.

[0005] Alfalfa is the earliest cultivated, largest planted, and most widely planted perennial legume forage in the world. It is also the main forage crop cultivated in my country. Its characteristics are high yield, good quality, and strong adaptability. It is the most valuable and economically significant cultivated forage and enjoys the reputation of "King of Forage". Compared with other plants, alfalfa has the following advantages: (1) Alfalfa has a high yield, up to 10 tons per mu, which greatly exceeds the yield of commonly used plants such as tobacco and rice; (2) Alfalfa has a long utilization period, up to 7-10 years, and can quickly recover its vitality after being cut. It can grow for many years after a single sowing, which greatly reduces the amount of labor.

[0006] Currently, plant expression systems are considered promising alternatives to animal cell and microbial cultures for large-scale production of recombinant proteins. Production methods using plants as a chassis for recombinant protein production can be found in, for example, Chinese patents CN 107827975 and CN 106554971, and US patents 5750871 and 5565347. However, there are currently no reports on the use of plants to express recombinant human tumor necrosis factor. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a recombinant human tumor necrosis factor expressed in alfalfa, its encoding gene, and a preparation method. This method yields a transmembrane form of human tumor necrosis factor (tmTNF-α), and results show that tmTNF-α exists in alfalfa in a trimer form. This provides a new solution to the problems currently existing in the recombinant tumor necrosis factor α industry.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a recombinant human tumor necrosis factor. -α The preparation method thereof, wherein the preparation method utilizes a compound containing human tumor necrosis factor -α After gene transformation of the recipient plant alfalfa, the resulting genetically transformed positive plants were then cultured to produce the human tumor necrosis factor.-α .

[0010] Specifically, the human tumor necrosis factor -α The amino acid sequence is shown in SEQ ID No: 1.

[0011] Preferably, the human tumor necrosis factor -α The amino acid sequence is also fused with a signal peptide sequence, such as the PR1b signal peptide sequence; specifically, the PR1b signal peptide coding sequence is added to its N-terminus.

[0012] More preferably, the human tumor necrosis factor -α The amino acid sequence also incorporates a KDEL sequence, and further incorporates a tag sequence, such as a 6xHis sequence, specifically in the human tumor necrosis factor. -α The amino acid sequence is fused with 6xHis and KDEL coding sequences at the C-terminus, and the more specific fused amino acid sequence is shown in SEQ ID NO: 4.

[0013] Preferably, the human tumor necrosis factor... -α The nucleotide sequence of the gene was codon-optimized based on alfalfa. More preferably, the human tumor necrosis factor... -α The nucleotide sequence of the gene is shown in SEQ ID NO: 3.

[0014] Furthermore, the preparation method also includes: processing human tumor necrosis factor in genetically transformed positive plants. -α Identification of gene insertions;

[0015] And / or the presence of recombinant human tumor necrosis factor in genetically transformed positive plants -α Protein identification.

[0016] Specifically, it is through the presence of human tumor necrosis factor. -α Agrobacterium-mediated transformation of recipient plants using plant binary expression vectors for genes.

[0017] Preferably, it contains human tumor necrosis factor. -α The plant binary expression vector for the gene is pCambia1300-35S.

[0018] Furthermore, it also includes the preparation of the human tumor necrosis factor from isolated and cultured samples. -α Steps 。

[0019] Specifically, it is obtained by extracting total plant protein.

[0020] This invention also provides the preparation method described above for obtaining human tumor necrosis factor. -α Genes exist in the form of trimers.

[0021] The beneficial effects of this invention are as follows: This invention utilizes regionalized expression in plant cells to increase the expression of human tumor necrosis factor in alfalfa, and obtains human tumor necrosis factor in trimeric form. -α This invention provides a solution to the current problems of limited sources and high prices of human tumor necrosis factor (TNF). Alfalfa is a substrate plant for recombinant protein synthesis, with in-depth molecular biology and genomics research and a mature gene editing and genetic transformation system, providing an excellent cell system for recombinant human tumor necrosis factor expression. This invention utilizes alfalfa as a substrate plant for recombinant human tumor necrosis factor synthesis, providing an excellent cell system for recombinant human tumor necrosis factor expression and offering a new solution to the market demand for recombinant tmTNF-α. Attached Figure Description

[0022] Figure 1 This is in Embodiment 2 of the present invention pC1300-rTNF-α A map of plant binary expression vectors.

[0023] Figure 2 These are photos of the positive seedlings after genetic transformation in Example 3 of this invention before and after flowering. In the left picture (alfalfa before flowering) and the right picture (alfalfa after flowering), the plant on the left is a wild-type plant, and the plant on the right is a representative plant of positive genetic transformation.

[0024] Figure 3 This is a diagram showing the identification results of alfalfa resistant seedlings in Example 3 of this invention; Figure 3 In this diagram, M represents the DNA molecule marker, numbers 1-7 represent the PCR identification results of 7 resistant seedlings, and number 8 represents the PCR identification result of the negative control.

[0025] Figure 4 This is a diagram showing the Coomassie brilliant blue staining results of recombinant human tumor necrosis factor in positively transformed alfalfa in Example 4 of this invention. Figure 4 The rightmost band is the protein marker, the four lanes adjacent to the marker are the total protein of wild-type alfalfa, and the remaining lanes are the total protein of positive plants identified by PCR. The red box indicates the recombinant human tumor necrosis factor in the total protein.

[0026] Figure 5 This is a Western blot result of recombinant human tumor necrosis factor in positively transformed alfalfa in Example 4 of this invention. Figure 5 The rightmost band is the protein marker, the four lanes adjacent to the marker are the total protein of wild-type alfalfa, and the remaining lanes are the total protein of positive plants identified by PCR. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it.

[0028] Furthermore, unless otherwise specified, the experimental methods in the specific embodiments disclosed below are all conventional methods; and unless otherwise specified, the materials and reagents used in the specific embodiments disclosed below are all commercially available.

[0029] Example 1: Codon Optimization of Human Tumor Necrosis Factor

[0030] Mature in the human body TNF-α The gene contains 702 bp (including the stop codon, GC=61%). Based on the codon preference in alfalfa, the PR1b signal peptide coding sequence was added to its N-terminus, and the 6xHis and KDEL coding sequences were added to its C-terminus to obtain the recombinant human tumor necrosis factor-α expressed in alfalfa, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0031] > SEQ ID NO: 1

[0032] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGV ELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL.

[0033] The amino acid sequence of the recombinant human tumor necrosis factor in this embodiment includes:

[0034] a) PR1b signal peptide sequence, the amino acid sequence of which is shown in SEQ ID NO: 2;

[0035] > SEQ ID NO: 2

[0036] MGFFLFSQMPSFFLVSTLLLFLIISHSSHA.

[0037] b) The fusion sequence of recombinant human tumor necrosis factor TNF-α, 6xHis and KDEL, with the amino acid sequence shown in SEQ ID NO: 4.

[0038] > SEQ ID NO: 4

[0039] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDN QLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALHHHHHHKDEL.

[0040] Among them, based on the codon preference in alfalfa, the optimized rTNF-α The gene's base sequence is shown in SEQ ID NO: 3, GC=49%.

[0041] > SEQ ID NO: 3

[0042] .

[0043] rTNF-α Genes are based on alfalfa codon bias in human tumor necrosis factor. TNF-α Based on the gene sequence, it is artificially designed (i.e. obtained after codon optimization) and encodes the same amino acid sequence.

[0044] Example 2 TNF-α Gene overexpression vector pC1300-rTNF-α Construction

[0045] Will rTNF-α The base sequence was sent to the gene synthesis company, and rTNF-α Gene synthesis to pCambia 1300-35S The vector contains HindIII and BamHI, and is named […]. pC1300-TNF-α Plant binary expression vectors (i.e., containing) rTNF-α Plant binary expression vectors for genes), specifically pC1300-rTNF-α Plant binary expression vectors (see) Figure 1 .

[0046] In other words, the human tumor necrosis factor coding sequence with optimized codons, along with the PR1b signal peptide coding sequence and the 6xHis and KDEL coding sequences, were then constructed into a plant binary expression vector through gene synthesis.

[0047] Example 3 pC1300-rTNF-α Alfalfa genetic transformation using plant binary expression vectors

[0048] I. Preparation of Agrobacterium competent cells

[0049] 1) Take out the Agrobacterium strain (LBA4404) stored at -80℃, dip a small amount of bacterial solution into the sterilized inoculation loop, streak it on a YEB solid medium plate containing 20 mg / L rifampicin, and incubate at 28℃ for 18 h.

[0050] 2) After the culture is completed, pick a single colony and place it in 5 mL of YEB liquid medium containing 20 mg / L rifampicin (Rif), and culture at 28°C and 200 rpm for 16-24 h (24 h in this example) to obtain the activated bacterial solution;

[0051] 3) Inoculate the activated bacterial culture into 50 mL of YEB liquid medium containing 20 mg / L rifampicin at a volume ratio of 1:100, and incubate at 28°C and 200 rpm until OD reaches the target value. 600 = Approximately 0.5;

[0052] 4) Transfer the cultured bacterial solution to a pre-cooled 50mL centrifuge tube, centrifuge at 4℃ and 5000rpm for 10min, and discard the supernatant;

[0053] 5) Add 10 mL of pre-cooled 0.1 M CaCl2 aqueous solution, gently suspend the cells, place on ice for 20 min, then centrifuge at 4 °C and 5000 rpm for 5 min, and discard the supernatant;

[0054] 6) Add 4 mL of pre-cooled 0.1 M CaCl2 aqueous solution containing 15% glycerol, and gently suspend;

[0055] 7) Agrobacterium suspension was dispensed into sterile Eppendorf tubes, 100 μL per tube, and frozen at -80°C to obtain Agrobacterium competent cells.

[0056] II. Plasmid transformation of Agrobacterium competent cells

[0057] 1) Remove Agrobacterium competent cells from -80℃ and place them on ice. After they thaw, add 2 μL of the prepared... pC1300-rTNF-α The plant binary expression vector was mixed thoroughly using a pipette.

[0058] 2) Place in an ice bath for 30 minutes, then in liquid nitrogen for 5 minutes;

[0059] 3) Water bath at 37℃ for 5 minutes;

[0060] 4) Incubate on ice for 5 minutes, then add 800 mL of LB liquid culture medium;

[0061] 5) After incubating at 28℃ and 200rpm for 3 hours with shaking, the mixture was then plated onto LB agar plates containing 50μg / mL kanamycin.

[0062] 6) Incubate at 28℃ until single colonies form, to obtain the product containing... rTNF-α Agrobacterium, a plant binary expression vector for the gene, was stored in a -70°C freezer.

[0063] III. Agrobacterium-mediated genetic transformation of alfalfa

[0064] 1) Cultivation of aseptic alfalfa seedlings

[0065] 11) Place alfalfa seeds in a 2mL centrifuge tube, soak in 70% ethanol for 1 minute, and rinse 2-3 times with sterile water (3 times in this example).

[0066] 12) Soak the seeds in a 20% sodium hydroxide solution with 1 drop of Tween 20 for about 15 minutes (shaking occasionally during this time), and rinse 5 times with sterile water;

[0067] 13) Inoculate alfalfa seeds onto germination medium (MS medium or 1 / 2 MS medium, both with a pH of 5.8; MS medium with a pH of 5.8 is used in this example), with about 3 seeds per bottle. Place the seeds in a light incubator at 25±1℃ and 2000 lux for 16 h / d of light to germinate for about one month to obtain sterile alfalfa seedlings for later use.

[0068] 2) Cultivation of Agrobacterium tumefaciens in bacterial suspension

[0069] 21) Use an inoculation loop to inoculate the contents stored in a -70°C freezer. rTNF-α Agrobacterium, the plant binary expression vector of the gene, was inoculated on LB medium containing 1.0% agar and cultured in a constant temperature incubator at 28°C for 2 days;

[0070] 22) After incubation in the incubator, pick a single colony and place it in LB liquid medium. Incubate overnight at 28°C with shaking at 200 rpm until the bacterial concentration reaches OD500. 600The value is approximately 0.5-0.6, yielding Agrobacterium bacterial suspension;

[0071] 3) Infection

[0072] Cut tender leaves of sterile alfalfa seedlings into 0.8cm×0.8cm pieces, place them in a sterile beaker, add cultured Agrobacterium tumefaciens solution, vacuum for 10 minutes, and gently shake at room temperature for 3 hours to infect the alfalfa seedlings.

[0073] 4) Co-cultivation

[0074] After infection is complete, discard the bacterial solution, immediately remove the leaves, blot the surface bacterial solution on sterile filter paper, place them in co-culture medium, and incubate in the dark at 25°C for 2 days;

[0075] The co-culture medium was MS medium supplemented with 4.5 µM 2,4-D, 0.9 µM kinetin, 30 g / L sucrose, and 7.5 g / L agar.

[0076] 5) Filtering

[0077] After co-culture, the leaves were inoculated onto the selection medium and cultured at 25°C under light. Subculture was performed every 2 weeks for approximately 3-4 weeks.

[0078] The screening medium was MS medium supplemented with 4.5 µM 2,4-D, 0.9 µM kinetin, 30 g / L sucrose, 7.5 g / L agar, 30 mg / L hygromycin (Hyg), and 300 mg / L cephalosporin antibiotics (Cef).

[0079] 6) Rooting

[0080] After co-culturing until shoots differentiate, cut off the robust resistant shoots and inoculate them into rooting medium, then continue light culture at 25°C (e.g., Figure 2 (as shown in Figure D in the diagram).

[0081] The rooting medium was MS medium, supplemented with 30 mg / L hygromycin (Hyg) and 300 mg / L cephalosporin antibiotic (Cef).

[0082] 7) Obtaining resistant seedlings

[0083] Once the resistant seedlings have developed a well-developed root system and are growing well, they are placed indoors for a week to harden off before transplanting. The seedlings are thoroughly watered three times before being managed in the field to obtain resistant seedlings. At the same time, wild alfalfa is planted as a blank control.

[0084] IV. Identification of Resistant Seedlings

[0085] 1) Plant genomic DNA extraction (CTAB method)

[0086] 11) Take plant tissues from 7 resistant seedlings (the plant tissues in this example are leaves), place the corresponding plant tissues into 2mL centrifuge tubes, add 2 sterile steel balls, freeze in liquid nitrogen, and grind in a ball mill.

[0087] 12) Add 500 μL of 2×CTAB extraction buffer to each container, incubate at 65°C for 20 min, and invert and mix several times during the incubation period;

[0088] 13) Add 800 μL of phenol, chloroform and isopropanol in a volume ratio of 25:24:1 respectively, mix by inverting, centrifuge at 12000 rpm for 10 min, and transfer the upper aqueous phase to a new 2 mL centrifuge tube.

[0089] 14) Add 0.6 times the volume of isopropanol to the obtained aqueous phase and precipitate at -20℃ for more than 30 minutes;

[0090] 15) After precipitation, centrifuge at 12,000 rpm for 10 min, discard the supernatant, and wash twice with 75% ethanol;

[0091] 16) Dry the residual alcohol at room temperature, dissolve it in 50 μL of distilled water to obtain the corresponding plant genomic DNA, and store at -20℃.

[0092] 2) PCR method for identifying resistant seedlings

[0093] To save on primer design costs and the stability of PCR conditions, this invention uses a marker gene on a vector for identification (i.e., to identify whether plant genomic DNA contains hygromycin). Utilizing... pC1300-rTNF-α The plant binary expression vector was used as a positive control. The specific reaction system for PCR identification is as follows:

[0094] Table 1. Summary of reaction systems for PCR identification

[0095]

[0096] The reaction procedure for PCR identification was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 30 sec, for a total of 28 cycles; 72℃ extension for 5 min.

[0097] In this invention, we obtained a total of 7 resistant seedlings. After PCR identification, 5 of them were positive, meaning they were positive alfalfa transformations. The identification results are as follows: Figure 3 As shown.

[0098] Example 4: Western blot identification of tmTNF-α protein in genetically transformed alfalfa plants

[0099] The protein expression levels of four positively transformed alfalfa plants (i.e., genetically transformed positive alfalfa plants) were identified using SDS-PAGE and Western blot methods, and wild-type alfalfa was used as a negative control.

[0100] S1, total plant protein extraction and SDS-PAGE

[0101] S11, Total Plant Protein Extract

[0102] Take 50-100 mg of fresh leaves and place them in 2 ml centrifuge tubes. Add steel balls, freeze quickly in liquid nitrogen, shake for 1 min on a tissue homogenizer, then add 500 μL of protein extraction buffer, shake for 15 sec, let stand for 5 min, and then centrifuge at 4 °C for 12000 g × 15 min. Take the corresponding supernatant.

[0103] The protein extraction buffer was 50 mM Tris-HCl with a pH of 7.5 and the cOmpleteULTRA protease inhibitor (Roche) was added.

[0104] S12. Determining protein concentration using the Bradford method.

[0105] The protein concentration in the supernatant was adjusted to 1 μg / μL to obtain the corresponding protein solutions.

[0106] Take 21 μL of protein solution, then add 7 μL of 4 X sample loading buffer, mix well, then boil the protein at 95 °C for 8 min, and finally load the sample for SDS-PAGE (protein gel electrophoresis).

[0107] The 4X sample loading buffer was 250 mM Tris–HCl with a pH of 6.8, and contained 8% (w / v) sodium dodecyl sulfate (SDS), 0.2% (w / v) bromophenol blue, 40% (v / v) glycerol, and 20% (v / v) β-mercaptoethanol.

[0108] S2, Western blot (protein blotting)

[0109] After SDS-PAGE, the protein on the protein gel is transferred to the PVDF membrane using the wet transfer method. The specific operation method is as follows:

[0110] S21. Prepare 1X transfer buffer; wherein, 1X transfer buffer is prepared by mixing 100mL of 10X transfer buffer, 200mL of methanol and 700mL of ddH2O.

[0111] S22. Cut a PVDF membrane that is slightly larger than the protein gel (approximately 9cm*6cm).

[0112] S23. Prepare ice in advance;

[0113] S24. Use tweezers to soak the PVDF membrane in methanol;

[0114] S25. Peel off the gel, gently scrape off the concentrated gel, being careful not to scratch the separating gel;

[0115] S26. Transfer membrane: Place the transfer clamp in the tray containing 1X transfer buffer, black side down (negative electrode), and place the special sponge → filter paper → gel → PVDF membrane → filter paper → sponge in sequence. After removing air bubbles, clamp the transfer clamp and place it in the transfer tank. Pour in 1X transfer buffer, place an ice pack in the tank to cool it down, and apply a constant current voltage of 300mA for 1.5h.

[0116] Coomassie brilliant blue staining results are as follows Figure 4 As shown, the recombinant tmTNF-α expressed in alfalfa in this invention has a molecular weight of approximately 80 kDa ( ). Figure 4 The band within the red border is shown in the image. The estimated molecular weight of tmTNF-α is 26 kDa. To confirm that the band in the Coomassie Brilliant Blue staining was tmTNF-α, we performed a Western blot experiment using a His-tagged antibody as the primary antibody. The results showed that the band in the Coomassie Brilliant Blue staining was indeed tmTNF-α (as shown in the image). Figure 5 Based on the above results, it can be found that recombinant tmTNF-α exists in alfalfa in the form of a trimer.

[0117] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Those skilled in the art can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing recombinant human tumor necrosis factor-α, characterized in that, The preparation method involves using alfalfa, a plant containing the human tumor necrosis factor-α gene, to transform the recipient plant, and then culturing the resulting genetically transformed positive plants to prepare the human tumor necrosis factor-α, which exists in the form of a trimer. The amino acid sequence of the human tumor necrosis factor-α is shown in SEQ ID No: 1, with a PR1b signal peptide coding sequence added to its N-terminus and a KDEL coding sequence integrated at its C-terminus; at the same time, the nucleotide sequence of the human tumor necrosis factor-α gene has been codon-optimized based on alfalfa. The amino acid sequence of the PR1b signal peptide is shown in SEQ ID NO: 2; The optimized nucleotide sequence of the human tumor necrosis factor-α gene is shown in SEQ ID NO:

3.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of the human tumor necrosis factor-α is further fused with a tag sequence.

3. The preparation method according to claim 2, characterized in that, The fused amino acid sequence is shown in SEQ ID NO:

4.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The preparation method further includes: Identification of insertions of the human tumor necrosis factor-α gene in genetically transformed positive plants; And / or identification of recombinant human tumor necrosis factor-α protein in genetically transformed positive plants.

5. The preparation method according to claim 4, characterized in that, It involves Agrobacterium-mediated transformation of recipient plants using a plant binary expression vector containing the human tumor necrosis factor-α gene.

6. The preparation method according to claim 5, characterized in that, The plant binary expression vector containing the human tumor necrosis factor-α gene is the pCambia1300-35S vector.

7. The preparation method according to any one of claims 1 to 3, characterized in that, It also includes the step of preparing the human tumor necrosis factor-α from an isolated culture.

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