Nucleic acid molecule for expressing alkaline phosphatase and application and method thereof

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a nucleic acid molecule for expressing alkaline phosphatase as well as application and a method thereof. According to the invention, the coding sequence of the animal-derived alkaline phosphatase protein bIAP gene is optimized, and the sequence of the nucleic acid molecule of the optimized alkaline phosphatase is as shown in SEQ ID NO.5. According to the technical scheme, the expression level of alkaline phosphatase in tobacco is improved, and the problem that in the prior art, the expression level of animal-derived alkaline phosphatase protein in tobacco is low is solved; the increasing requirements of industrial production and clinical application on the high-quality alkaline phosphatase are met, the production cost of the alkaline phosphatase is remarkably reduced, and a wider application prospect is developed for the alkaline phosphatase in the field of biological medicines.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a nucleic acid molecule for expressing alkaline phosphatase and an application and method thereof. Background Art

[0002] Alkaline phosphatase is an important enzyme found widely in animals, plants, and microorganisms, and plays a crucial physiological role in living organisms. It catalyzes the hydrolysis of monophosphates and the transfer of phosphate groups, and has broad applications in biotechnology and clinical medicine. Among the known naturally occurring alkaline phosphatases, the one with the highest specific activity is that derived from bovine small intestine.

[0003] However, the expression level of alkaline phosphatase in the existing technology is low and the yield is insufficient, which makes it difficult to meet the large-scale application needs in the fields of biotechnology and clinical medicine. The direct extraction of alkaline phosphatase from animal tissues has problems such as limited raw materials and complex extraction processes, and it is impossible to guarantee a large-scale and high-quality supply. Genetic engineering technology provides a new way for the large-scale production of alkaline phosphatase. Common expression systems include Escherichia coli, yeast, insect and mammalian cell lines, but all have certain defects. The recombinant alkaline phosphatase expressed in Escherichia coli lacks glycosylation modification, which is not conducive to downstream coupling applications; the yeast expression product is prone to excessive glycosylation and uneven glycosylation; the expression levels of insect and mammalian cell lines are generally low, making it difficult to achieve industrial production. Therefore, how to efficiently express alkaline phosphatase in cells and increase its yield is a key technical problem that needs to be solved urgently.

[0004] Among the existing technical solutions, patent CN109554369A provides a nucleic acid aptamer that specifically recognizes alkaline phosphatase heterodimers. Although this technology has application value in the detection and purification of alkaline phosphatase, it does not solve the core problem of increasing production. In contrast, the gene coding sequence optimization strategy proposed by patent CN117925657A is more targeted. This patent significantly improves its expression efficiency in mammalian cells by systematically optimizing the ibAP II gene sequence. This technical route shows that rational design and optimization of gene sequences are an effective way to increase the production of recombinant alkaline phosphatase. However, this technology only targets specific subtypes of alkaline phosphatase, and its optimization strategy may not be directly applicable to other types of alkaline phosphatase.

[0005] Based on the current technological status, how to optimize the coding sequence of the alkaline phosphatase gene and improve its recombinant expression level to obtain high-activity and high-yield alkaline phosphatase remains an important technical problem that needs to be solved urgently. Summary of the Invention

[0006] In response to the above-mentioned deficiencies, the present invention provides a technical solution for optimizing the coding sequence of the alkaline phosphatase gene and efficiently expressing alkaline phosphatase in mammalian cells, which can significantly improve the expression level and yield of alkaline phosphatase, and provide strong support for its application in fields such as biotechnology and clinical medicine.

[0007] The technical solution of the present invention is: In one aspect, the present invention provides a nucleic acid molecule for expressing alkaline phosphatase, wherein the sequence of the nucleic acid molecule is shown in SEQ ID NO.5.

[0008] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

[0009] Specifically, the expression vector may be a plant expression vector.

[0010] Preferably, the plant expression vector may be a pCAMBIA series, a pEAQ series or a pGreen / pSoup system.

[0011] Preferably, the plant expression vector may be of the pEAQ series.

[0012] Preferably, the plant expression vector may be pEAQ-HT or pEAQ-HT-DEST1.

[0013] Preferably, the plant expression vector may be pEAQ-HT.

[0014] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or expression vector.

[0015] Specifically, the host cell is a prokaryotic cell or a eukaryotic cell.

[0016] In another aspect, the present invention provides a transgenic organism comprising the aforementioned nucleic acid molecule or expression vector or host cell.

[0017] Specifically, the transgenic organisms include but are not limited to transgenic plants, transgenic animals or transgenic microorganisms.

[0018] Preferably, the transgenic organism may be a transgenic plant.

[0019] Preferably, the transgenic plant is selected from tobacco, soybean, rice, barley, corn, rye, oats, sugar beet, Beta vulgaris subspecies, parsnip, bean, pea, cowpea, mung bean, chickpea, peanut, lentil, alfalfa, rocket, mustard, lettuce, Brassica juncea, potato, sweet potato, cassava, wheat or spelt.

[0020] Preferably, the transgenic plant may be tobacco.

[0021] Preferably, the tobacco includes but is not limited to: Nicotiana benthamiana, Nicotiana tabacum, Nicotiana truncatula, Nicotiana truncatula or Nicotiana rustica.

[0022] Preferably, the tobacco may be Nicotiana benthamiana.

[0023] In another aspect, the present invention provides a method for preparing the aforementioned transgenic organism, comprising introducing the aforementioned nucleic acid molecule or expression vector or host cell into the organism.

[0024] Specifically, the organism includes but is not limited to plants, animals or microorganisms.

[0025] Preferably, the organism may be a plant.

[0026] Preferably, the plant is selected from tobacco, soybean, rice, barley, corn, rye, oat, sugar beet, Beta vulgaris, parsnip, bean, pea, cowpea, mung bean, chickpea, peanut, lentil, alfalfa, rocket, mustard, lettuce, Brassica juncea, potato, sweet potato, cassava, wheat or spelt.

[0027] Preferably, the plant may be tobacco.

[0028] Preferably, the tobacco includes but is not limited to: Nicotiana benthamiana, Nicotiana tabacum, Nicotiana truncatula, Nicotiana truncatula or Nicotiana rustica.

[0029] Preferably, the tobacco may be Nicotiana benthamiana.

[0030] In another aspect, the present invention provides use of the aforementioned nucleic acid molecule, expression vector, host cell, or transgenic organism in the preparation of alkaline phosphatase.

[0031] In another aspect, the present invention provides use of the aforementioned nucleic acid molecule, expression vector, host cell, or transgenic organism in increasing the production of alkaline phosphatase.

[0032] The beneficial effects of the present invention are: (1) The expression level and production of alkaline phosphatase in mammalian cells were significantly improved, providing strong support for its application in biotechnology and clinical medicine.

[0033] (2) It avoids the disadvantages of extracting and purifying alkaline phosphatase from animal tissues, such as limited raw materials, complex processes, biosafety and ethical issues.

[0034] (3) It provides a new technical route for the large-scale production and industrial application of alkaline phosphatase. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The results of western blot detection are shown in Figure 2. Note: M: Marker; Anti-His: protein tag.

[0036] Figure 2 The figure shows the hIAP protein domain prediction and subcellular localization results. CK is the sup1300-EGFP empty vector; hIAP is the sup1300-hIAP-EGFP fusion protein expression vector; mCherry is pCAMBIA1300-35S-ER-mCherry; EGFP is green fluorescence; Bright field is bright field; Merge is overlay; microscope: 10x magnification, scale bar: 20 μm.

[0037] Figure 3 The white domain prediction and subcellular localization results of bIA, where CK is the sup1300-EGFP empty vector; bIAP is the sup1300-bIAP-EGFP fusion protein expression vector; mCherry is p2300-35S-H2B-mCherry+pCAMBIA1300-35S-ER-mCherry; EGFP is green fluorescence; Bright field is bright field; Merge is overlay, microscope is 10x magnification, and the scale bar is 20 μm.

[0038] Figure 4 This is the result of Coomassie Brilliant Blue staining.

[0039] Figure 5 The diagram shows the enzyme activity results of each group.

[0040] Figure 6 This is the spectrum of pEAQ-HT.

[0041] Figure 7 This is the skeleton map of sup1300-EGFP. DETAILED DESCRIPTION

[0042] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0043] Example 1 1.1 Methods for constructing alkaline phosphatase expression gene and expression vector The human and bovine intestinal AP genes are hIAP and bIAP Gene. hIAPThe full-length CDS sequence of the gene is 1584 bp, encoding a total of 528 amino acids (SEQ ID NO. 1); bIAP The full-length CDS sequence of the gene is 1605 bp, encoding a total of 535 amino acids (SEQ ID NO. 2).

[0044] Using homologous recombination technology, human and bovine intestinal AP genes were hIAP and bIAP The hIAP / bIAP-pEAQ-HT recombinant plasmids were constructed into the pEAQ-HT plant expression vector to obtain transient expression of tobacco. hIAP (SEQ ID NO. 3) and bIAP The target gene fragments of SEQ ID NO.4 were cloned into the vector pEAQ-HT ( Figure 6 The bIAP / hIAP-pEAQ-HT recombinant plasmid vector was obtained by cleaving the double enzyme cutting sites AgeI / SmaI of the pEAQ-HT vector (stored in the laboratory of Beijing Institute of Life Sciences).

[0045] Human intestinal alkaline phosphatase ( hIAP )'s protein and DNA sequences are as follows: SEQ ID NO.1: MQGPWVLLLLGLRLQLSLGVIPAEEENPAFWNRQAAEALDAAKKLQPIQKVAKNLILFLGDGLGVPTVTATRILKGQKNGKLGPETPLAMDRFPYLALSKTYNVDRQVPDSAATATAYLCGVKANFQTIGLSAAARFNQCNTTRGNEVISVMNRAKQAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADMPASARQEGCQDIATQLISNMDIDVILGGGRKYMFPMGTPDPEYPADASQNGIRLDGKNLVQEWLAKHQGAWYVWNRTELMQASLDQSVTHLMGLFEPGDTKYEIHRDPTLDPSLMEMTEAALRLLSRNPRGFYLFVEGGRIDHGHHEGVAYQALTEAVMFDDAIERAGQLTSEEDTLTLVTADHSHVFSFGGYTLRGSSIFGLAPSKAQDSKAYTSILYGNGPGYVFNSGVRPDVNESESGSPDYQQQAAVPLSSETHGGEDVAVFARGPQAHLVHGVQEQSFVAHVMAFAACLEPYTACDLAPPACTTDAAHPVAASLPLLAGTLLLLGASAAP; SEQ ID NO.3: Protein and DNA sequences of bovine intestinal alkaline phosphatase (bIAP): SEQ ID NO.2: MQGACVLLLLGLQLQLSLGLIPVEEEDPAFWNCQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYKTIGVSAAARYNQCNTTSGNEVTSVMNRAKKAGKAVGVVTTSRVQHASPAGAYAHTVNRNWYSDADLPADAQMNGCQDIATQLVYNMDIDVILGGGRMYMFPEGTPDPEYPYDVNQTGVRKDKRNLVQEWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLQEMTEVALRVLSRNPRGFYLFVEGGRIDHGHHEGKAYMALTDTVMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPSKALDSKSYTSILYGNGPGYALGGGSRPDVNDSTSEDPSYQQQAAVPLASETHGGEDVAVFARGPQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPTTATSIPDAAHLAASPPPLALLAGAMLLLLAPTLY; SEQ ID NO.4:

[0046] 1.2 Specific methods and conditions for tobacco transfection Transient genetic transformation of hIAP / bIAP-pEAQ-HT recombinant plasmid into tobacco Selection of Nicotiana benthamiana: In the experiment, the best injection period was when Nicotiana benthamiana had grown to 5-7 true leaves. At this time, the tobacco leaves were young and tender, in good growth condition, and protein expression was best.

[0047] Agrobacterium activation and injection into Nicotiana benthamiana: Positive single colonies obtained after transformation into GV3101 (psoup-p19 background, Shanghai Weidi Biotechnology, CAT#: AC1003) competent Agrobacterium cells were propagated for approximately 12 hours in LB (liquid LB + 50 mg / L kanamycin + 50 mg / L rifampicin). The culture was centrifuged at 5000 rpm for 6 minutes at room temperature, and the supernatant LB containing the resistance was removed as much as possible. The culture was then resuspended in 10 mM MES-KOH pH 5.6, 10 mM MgCl2, 200 μM acetosyringone, and the OD600 was adjusted to 0.8-1.0. The culture was incubated in the dark for 2 hours before injection into Nicotiana benthamiana.

[0048] 1.3 Specific methods for extracting and purifying alkaline phosphatase from tobacco tissue 1.3.1 Protein extraction and protein immunoblotting Samples were taken on the third day after injection of Nicotiana benthamiana and protein expression was detected by Western Blot (WB).

[0049] The specific detection methods are as follows: (a) Protein extraction and SDS-PAGE preparation The following protein extraction processes were all performed on ice Add 600 μL of protein lysis buffer to 0.1 g of tobacco leaves, grind and mix thoroughly, and then lyse on ice for 20 minutes. Then centrifuge at 4°C and 13,000 rpm for 20 minutes. Take the supernatant and transfer it to a new centrifuge tube. Centrifuge again for 20 minutes. The supernatant is the protein extract, which is packaged and stored in a -80°C refrigerator.

[0050] The protein lysis buffer formula is shown in Table 1: Table 1 Protein lysis buffer formula

[0051] Note: The two protease inhibitors in Table 1 need to be added immediately before use.

[0052] (b) Sample preparation and electrophoresis 30 μL of supernatant protein was aspirated, 10 μL of 4× LDS Sample Buffer (Thermo Fisher Scientific, Cat. No. NP0008) was added, vortexed to mix, and then denatured in a 100°C water bath for 10 min.

[0053] Select a 10% (v / v) concentration, 12-well precast gel (SurePAGE™, Bis-Tris, GenScript, Cat. No. M00665). Remove the comb vertically, with the short side facing inward. Add 1× Tris–MES-SDS Running Buffer (Tris–MES-SDS Running Buffer Powder, GenScript, Cat. No. M00677) to cover the short side. Load 10 μL of protein sample and 10 μL of prestained protein marker (Thermo Fisher Scientific, Cat. No. 26616). Connect the power supply and run the gel. Initially maintain a constant voltage of 80 V for 10 minutes, then adjust the voltage to 200 V. The running buffer should stop when it reaches the bottom edge of the gel, which should take approximately 25 minutes.

[0054] (c) Membrane transfer and protein blocking Activate a PVDF membrane (Beyotime, 0.45 μm, Cat. No. FFP33) in methanol for 1 minute. Remove the short plate, cut off the bottom stained section and the top unstained section, and place it on a sandwich plate: bottom black - (foam - filter paper - glue (gently remove bubbles, top side up) - PVDF membrane (remove bubbles) - filter paper - foam) - top black. Close the plates, black facing black (outside) and white facing red (inside). Add transfer buffer (Beyotime, Cat. No. BL315B) to cover the transfer apparatus and cycle at 400 mA for 25 minutes. Transferring generates heat; place crushed ice around the transfer chamber or add an ice pack directly to the chamber.

[0055] After transfer, push up and remove the device. Remove the PVDF membrane with the black side facing down and place it with the front side facing up in 5% (v / v) skim milk powder blocking buffer. Block at 50-60 rpm at room temperature for 2 hours. Use 1xTBST (biosharp, Cat. No. P0572) as the solvent to prepare the 5% (v / v) skim milk powder.

[0056] (d) Antibody incubation Discard the blocking solution and rinse three times with 1× TBST (10 min each). Transfer to an incubation box containing the primary antibody (diluted 1:5000 in blocking solution) and incubate for 2 hours at room temperature on a slow rocker (50-60 rpm). Wash the membrane three times with 1× TBST (10 min each). Incubate the membrane with HRP-conjugated secondary antibody (horseradish peroxidase-conjugated antibody, diluted 1:8000 in blocking buffer) for 40 minutes at room temperature on a shaker (50-60 rpm). After incubation, wash the membrane three times with 1× TBST (10 minutes each) to remove any residual secondary antibody.

[0057] (e) Development analysis Prepare the chemiluminescent detection substrate working solution by mixing two chemiluminescent substrate developers (Thermo Fisher Scientific, Catalog No. YL382880) in a 1:1 ratio. Soak the membrane in the developer solution in the dark for 1 minute to develop the color. Develop the membrane using a developer, selecting the optimal exposure time.

[0058] Test results such as Figure 1 As shown, both bIAP and hIAP proteins are 6xHis-tagged proteins with a molecular weight of approximately 0.8 kDa. The 80 kDa dimeric form of the alkaline phosphatase protein is approximately 55-60 kDa in the reduced state.

[0059] 1.3.2 Protein purification process Purify AP protein using Ni-NTA agarose purification resin: Remove 200 g of injected tobacco leaves from a -80°C freezer and add 800 mL of plant protein lysis buffer (3-5 mL per 1 g of leaves). Mix thoroughly using a wall disrupter and lyse on ice for 30 minutes (add protease inhibitors PMSF and Coktail). Centrifuge the lysed proteins at 15,000 rpm for 20 minutes at 4°C, and collect the supernatant.

[0060] Take a Ni-NTA prepacked column (5mL filler) (Shanghai Biotech, Cat. No.: C600793) and let the storage buffer flow out by gravity. Equilibrate the column with two column volumes of equilibration solution and slowly discharge at a flow rate of 0.5-1mL / min. Add the protein extract to the column, then wash the impurity protein with 5 column volumes of washing solution, collect the flow-through, and repeat until the baseline at the absorbance at 280nm is flat. Finally, elute the His-tagged protein on the column with two column volumes of elution solution and repeat twice. See Table 2 for the reagent formula in the purification experiment: Table 2 Protein extract formula

[0061] Note: Two protease inhibitors need to be added immediately upon use, and PMSF inhibitor needs to be replenished every 20 minutes.

[0062] The formula of the balance solution is shown in Table 3: Table 3 Balance solution formula

[0063] The formula of the cleaning solution is shown in Table 4: Table 4: Cleaning liquid formula

[0064] The eluent formula is shown in Table 5: Table 5 Eluent formula

[0065] Effect Example 1 1. Subcellular localization analysis of recombinant hIAP / bIAP proteins To further explore the distribution of hIAP / bIAP proteins in cells, tobacco sup1300-bIAP-EGFP ( Figure 7 sup1300-EGFP was stored in the laboratory of Beijing Institutes of Life Sciences) and sup1300-hIAP-EGFP fusion protein expression vector.

[0066] First, bioinformatics methods were used to perform preliminary protein localization predictions. For example, based on the amino acid sequence of the target protein, the online tool Cell-PLoc (Cellular Localization of Proteins, http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2 / ) was used to predict the subcellular localization of bIAP / hIAP proteins. Subsequently, empty sup1300-bIAP-EGFP and sup1300-EGFP plasmids were co-injected with Agrobacterium expressing nuclear localization markers (p2300-35S-H2B-mCherry) and membrane localization markers (pCAMBIA1300-35S-ER-mCherry), respectively. Empty sup1300-hIAP-EGFP and sup1300-EGFP plasmids were co-injected with an endoplasmic reticulum localization marker (pCAMBIA1300-35S-ER-mCherry), respectively, and transformed into Nicotiana benthamiana plants. After culturing under weak light conditions for 12 hours, the cells were transferred to light culture. After culturing for 48 hours, the protein fluorescence localization was observed under a laser confocal microscope.

[0067] Protein domain prediction and subcellular localization results showed that ( Figure 2-Figure 3), hIAP protein is localized in the endoplasmic reticulum, and bIAP protein is localized in the nuclear membrane.

[0068] 2. Recombinant bIAP protein yield The BSA standard protein gradient loading amount was 1μg, 2μg, 3μg, 4μg, 5μg, 6μg, and 7μg. The imidazole concentrations used for elution of the purified protein were 200mM, 300mM, 400mM, and 500mM, respectively, and the protein was retained by centrifugation using a 30KDa ultrafiltration tube. The retained protein was electrophoresed at a 10% (V / V) concentration on a 12-well precast protein gel (SurePAGE™, Bis-Tris, GenScript, Cat. No. M00665). The gel was then stained with Coomassie Brilliant Blue. The staining results are shown in the figure. Figure 4 ImageJ software was used to calculate the BSA standard gradient protein grayscale value to create a standard curve. The protein grayscale value was then substituted into the standard curve to calculate the protein yield per 200g fresh tobacco leaf sample. The protein yield before sequence optimization was 2.6μg / g, and the protein yield after sequence optimization was 6μg / g, an increase of approximately 56.7% compared to the protein yield before optimization.

[0069] Modified DNA sequence of bovine intestinal alkaline phosphatase (bIAP) (SEQ ID NO.5): Human intestinal alkaline phosphatase (hIAP) modified DNA sequence (SEQ ID NO.6):

[0070] 3. Verification of in vitro enzyme activity of optimized recombinant bIAP protein (1) AP demetallization to eliminate endogenous metal interference Purified AP was mixed with 10 mM EDTA (final concentration 5 mM) and incubated at 4°C for 1 hour. EDTA was removed by dialysis using an ultrafiltration centrifuge tube (10 kDa cutoff) and replaced with metal-free Tris-HCl buffer (pH 9.0) (Solarbio, Product No. T1160).

[0071] (2) Group design of enzyme activity verification reaction system Each group was set up with 4 replicates, and the total reaction volume was 100 μL. The specific grouping and treatment conditions are shown in Table 6.

[0072] Table 6 Grouping and treatment conditions

[0073] Commercial AP (Sigma-Aldrich, P7640) concentration: 100 μg / mL; Final concentration of substrate pNPP (p-nitrophenyl phosphate): 5 mM; Reaction buffer: Tris-HCl (pH 9.0) with a final concentration of 50 mM; Final ZnCl2 concentration: 1 mM; Final MgCl2 concentration: 1 mM; Final EDTA concentration: 5 mM.

[0074] (3) Enzyme activity reaction process (a) Preincubation: bIAP protein was premixed with different metal ions / EDTA in buffer and incubated at 37°C for 10 minutes.

[0075] (b) Start the reaction: pNPP (final concentration 5 mM) was added, and the plates were immediately transferred to a 96-well plate and incubated at 37°C for 10 minutes.

[0076] (c) Termination reaction: The reaction was terminated by adding 50 μL of 1 M NaOH.

[0077] (d) Detect absorbance: The absorbance of the samples at 405 nm (OD405) was measured using a NanoDrop spectrophotometer, and the blank control value was deducted from the calculated results.

[0078] (e) Data processing Enzyme activity calculation: Enzyme activity (IU) = △OD405 / min × V total / ε×d×V 酶 ; Where ε (molar extinction coefficient) = 18,000 M -1 cm -1 (p-nitrophenol); V total : reaction volume (L); d: optical path (cm), usually 0.6 cm for a 96-well plate.

[0079] The results are as follows Figure 5 As shown, the addition of zinc and magnesium ions to the positive control commercial AP increased enzyme activity, while the addition of the metal ion chelator EDTA significantly decreased it. The purified bIAP exhibited enzymatic activity when magnesium ions were added alone, but not when zinc ions were added alone. Furthermore, the simultaneous addition of magnesium and zinc ions significantly increased enzymatic activity. The addition of EDTA significantly inhibited the enzymatic activity of the experimental bIAP, maintaining activity levels consistent with those of the control group. These results indicate that bIAP activity is primarily dependent on zinc ions compared to magnesium ions, and that simultaneous addition of magnesium and zinc ions increases enzymatic activity.

[0080] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A nucleic acid molecule expressing alkaline phosphatase, characterized in that The sequence of the nucleic acid molecule is shown in SEQ ID NO.

5.

2. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 1.

3. The expression vector according to claim 2, characterized in that The expression vector is a plant expression vector.

4. The expression vector according to claim 3, characterized in that The plant expression vector is a pCAMBIA series, a pEAQ series or a pGreen / pSoup system.

5. The expression vector according to claim 4, characterized in that The plant expression vector is pEAQ series.

6. The expression vector according to claim 5, characterized in that The plant expression vector is pEAQ-HT or pEAQ-HT-DEST1.

7. The expression vector according to any one of claims 3 to 6, characterized in that The plant expression vector is pEAQ-HT.

8. A host cell, characterized in that The method comprises the nucleic acid molecule according to claim 1 or the expression vector according to any one of claims 2 to 6.

9. The host cell according to claim 8, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

10. A genetically modified organism, characterized in that The method comprises the nucleic acid molecule according to claim 1, the expression vector according to any one of claims 2 to 6, or the host cell according to claim 8 or 9.

11. The transgenic organism according to claim 10, characterized in that The transgenic organism is a transgenic plant, a transgenic animal or a transgenic microorganism.

12. The transgenic organism according to claim 11, characterized in that The transgenic organism is a transgenic plant.

13. The transgenic organism according to claim 12, characterized in that The transgenic plant is selected from tobacco, soybean, rice, barley, corn, rye, oats, sugar beet, Beta vulgaris subspecies, parsnip, bean, pea, cowpea, mung bean, chickpea, peanut, lentil, clover, rocket, mustard, lettuce, Brassica juncea, potato, sweet potato, cassava, wheat or spelt.

14. The transgenic organism according to claim 13, characterized in that The transgenic plant is tobacco.

15. The transgenic organism according to claim 14, characterized in that The tobacco is Nicotiana benthamiana, common tobacco, forest tobacco, flowering tobacco or yellow tobacco.

16. The transgenic organism according to claim 15, characterized in that The tobacco is Nicotiana benthamiana.

17. The method for preparing a transgenic organism according to any one of claims 8 to 16, characterized in that: The method comprises introducing the nucleic acid molecule according to claim 1, the expression vector according to any one of claims 2 to 6, or the host cell according to claim 8 or 9 into an organism.

18. Use of the nucleic acid molecule according to claim 1, the expression vector according to any one of claims 2 to 6, the host cell according to claim 8 or 9, or the transgenic organism according to any one of claims 10 to 16 in the preparation of alkaline phosphatase.

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