Expression method of animal-derived alkaline phosphatase protein in tobacco
By using a binary vector system and Ni-NTA metal ion affinity chromatography technology in tobacco, the nuclear membrane localization and efficient expression of animal-derived alkaline phosphatase protein were achieved, solving the problem of low expression level, increasing yield and reducing costs.
Patent Information
- Application Number
- CN202510975351.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-15
- Publication Date
- 2025-09-19
AI Technical Summary
In the existing technology, the expression level of animal-derived alkaline phosphatase protein in eukaryotic cells is low, which is difficult to meet the needs of industrial production. Direct expression in tobacco may lead to problems such as low expression level and protein instability.
By using a binary vector system such as pEAQ-HT in tobacco, optimizing the physiological response window and purification method, the nuclear membrane localization and efficient expression of animal-derived alkaline phosphatase protein were achieved, and Ni-NTA metal ion affinity chromatography was used for purification.
It achieves high-level expression of animal-derived alkaline phosphatase protein, increases yield, reduces production costs, and provides a new technical route for industrial production.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering, and particularly relates to a method for expressing animal-derived alkaline phosphatase protein in tobacco. Background Art
[0002] Alkaline phosphatase (AP) is an important industrial enzyme widely used in diagnostic reagents, biosensors, and other fields. Currently, the ALP used in industrial production is primarily extracted from bovine intestine, which presents challenges such as low yield, high cost, and biosafety risks. Genetic engineering technology has provided a new approach for large-scale production of ALP, but prokaryotic expression systems suffer from limitations such as a lack of glycosylation and low activity. While eukaryotic expression systems, such as mammalian cell lines, can address glycosylation issues, expression levels are generally low, making it difficult to meet the demands of industrial production. As an important economic crop with a mature genetic transformation system, tobacco is an ideal model plant for plant bioreactor research. Using tobacco to express animal-derived ALP proteins has the potential to overcome the limitations of prokaryotic expression systems, achieving high-level expression and accurate glycosylation, providing a new technical route for industrial production. However, there are no reports on expressing animal-derived ALP proteins in tobacco. Due to significant differences between animal-derived ALP proteins and plant-derived proteins, direct expression in tobacco can result in low expression levels and protein instability.
[0003] Animal-derived alkaline phosphatase proteins have important physiological functions. Efficient expression in tobacco and localization to specific organelles would provide a powerful tool for studying their role in tobacco. Localizing animal-derived alkaline phosphatase at the nuclear membrane offers distinct advantages over targeting the endoplasmic reticulum (ER), primarily in terms of functional regulation, isolation and purification, and cytocompatibility. The nuclear membrane, a double-layered membrane structure of the cell nucleus, shares continuity with the ER, but its unique protein composition and relatively independent microenvironment provide more controlled conditions for exogenous protein expression. First, nuclear membrane localization reduces the risk of excessive modification caused by protein secretion into the ER lumen or Golgi apparatus, particularly preventing plant-specific glycosylation from interfering with the functional properties of animal proteins. Second, nuclear membrane proteins are often directly anchored to the membrane via their transmembrane domains. This localization simplifies subsequent purification procedures. For example, enrichment of nuclear membrane fractions by differential centrifugation combined with membrane fractionation is more efficient than extracting soluble proteins from the ER lumen. In addition, nuclear membrane expression may reduce the burden on the cell secretion system and avoid endoplasmic reticulum stress response caused by excessive accumulation of exogenous proteins, thereby improving the survival rate and expression stability of host cells.
[0004] However, nuclear membrane localization also presents technical challenges for specific targeting. Compared with the universally applicable signal peptides for the endoplasmic reticulum (such as the KDEL sequence), nuclear membrane localization signals are less well-studied, requiring precise design of fusion tags (such as the transmembrane domain of the nuclear membrane proteins LBR or Emerin) to ensure correct localization. Furthermore, due to the limited space at the nuclear membrane, overexpression may interfere with the function of the nuclear pore complex, affecting key physiological processes such as nucleocytoplasmic transport. Therefore, in practical applications, a balance must be struck between expression efficiency and cellular adaptability. Promoter optimization (such as the use of inducible promoters) and the rational selection of nuclear membrane targeting elements can achieve efficient expression with low toxicity. Overall, nuclear membrane localization is a theoretically more promising strategy, but further optimization of the targeting system and expression regulation are needed to fully realize its advantages.
[0005] Therefore, how to efficiently express animal-derived alkaline phosphatase protein in tobacco and localize it to specific organelles is a key 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 method for expressing animal-derived alkaline phosphatase protein in tobacco, which achieves the localization of bIAP protein to the nuclear membrane and high expression of alkaline phosphatase protein using only conventional methods in the prior art.
[0007] The technical solution of the present invention is: In one aspect, the present invention provides a method for expressing an animal-derived alkaline phosphatase protein in tobacco, the expression method comprising the following steps: S1. Constructing an animal-derived alkaline phosphatase protein gene into a plant expression vector to obtain a recombinant plasmid; S2. Transfecting tobacco with the recombinant plasmid from step S1, and sampling during the physiological response window to obtain a sample; S3, the sample in step S2 is lysed with a protein lysis buffer to obtain a supernatant; S4. The supernatant of step S3 is purified to obtain alkaline phosphatase protein; The gene is a bovine intestinal alkaline phosphatase protein gene.
[0008] Specifically, the plant expression vector in step S1 includes but is not limited to: a binary vector system, a single vector system, an RNAi vector or a CRISPR / Cas vector.
[0009] Preferably, the plant expression vector can be a binary vector system.
[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] Specifically, the tobacco in step S2 includes but is not limited to: Nicotiana benthamiana, common tobacco, forest tobacco, flowering tobacco or yellow tobacco.
[0015] Preferably, the tobacco in step S2 may be Nicotiana benthamiana.
[0016] Preferably, the Nicotiana benthamiana described in step S2 can be selected at the 5-7 true leaf stage.
[0017] Specifically, the physiological response window period in step S2 can be 3-5 days, specifically 3 days, 3.1 days, 3.2 days, 3.3 days, 3.3 days, 3.4 days, 3.5 days, 3.6 days, 3.7 days, 3.8 days, 3.9 days, 4 days, 4.1 days, 4.2 days, 4.3 days, 4.3 days, 4.4 days, 4.5 days, 4.6 days, 4.7 days, 4.8 days, 4.9 days, 5 days and any other value within the range.
[0018] Preferably, the physiological response window period in step S2 may be 3-4 days.
[0019] Preferably, the physiological response window period in step S2 may be 3-3.5 days.
[0020] Preferably, the physiological response window period in step S2 may be 3 days.
[0021] Specifically, the purification method in step S4 is preferably such that the physiological response window period in step S2 can be 3-4 days. Affinity chromatography.
[0022] Preferably, the purification method in step S4 includes but is not limited to: metal ion affinity chromatography, bioaffinity chromatography, covalent affinity chromatography or hydrophobic affinity chromatography.
[0023] Preferably, the purification method in step S4 can be metal ion affinity chromatography.
[0024] Preferably, the metal ion affinity chromatography is Ni-NTA metal ion affinity chromatography.
[0025] Preferably, the core of the Ni-NTA metal ion affinity chromatography is to achieve specific adsorption and elution of the target protein through imidazole competitive binding.
[0026] The beneficial effects of the present invention are: By using tobacco as an expression host and localizing the bIAP protein to the nuclear membrane, the problem of low expression levels of animal-derived alkaline phosphatase proteins in eukaryotic cells is solved, high-level expression is achieved, and a new technical means is provided for industrial production. The method of the present invention is simple and easy to operate, can effectively increase the yield of animal-derived alkaline phosphatase proteins, reduce production costs, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The results of western blot detection are shown in Figure 2. Note: M: Marker; Anti-His: protein tag.
[0028] 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.
[0029] 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.
[0030] Figure 4 This is the result of Coomassie Brilliant Blue staining.
[0031] Figure 5 The diagram shows the enzyme activity results of each group.
[0032] Figure 6 This is the spectrum of pEAQ-HT.
[0033] Figure 7 This is the skeleton map of sup1300-EGFP. DETAILED DESCRIPTION
[0034] 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.
[0035] 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. hIAP The 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).
[0036] 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).
[0037] 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:
[0038] 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.
[0039] 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.
[0040] 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).
[0041] 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.
[0042] The protein lysis buffer formula is shown in Table 1: Table 1 Protein lysis buffer formula
[0043] Note: Two of the protease inhibitors need to be added immediately before use.
[0044] (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.
[0045] 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.
[0046] (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.
[0047] 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.
[0048] (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.
[0049] (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.
[0050] 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.
[0051] 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.
[0052] Take a Ni-NTA prepacked column (5mL filler) (Shanghai Biotech, catalog number: C600793) and let the storage buffer flow out by gravity. Equilibrate the column with two column volumes of equilibrium 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 of 280nm is flat. Finally, elute the His-tagged protein on the column with two column volumes of eluent, and repeat twice. The reagent formula for the purification experiment is shown in Table 2: Table 2 Protein extract formula
[0053] Note: Two protease inhibitors need to be added immediately after use, and PMSF inhibitor needs to be replenished every 20 minutes.
[0054] The formula of the balance solution is shown in Table 3: Table 3 Balance solution formula
[0055] The formula of the cleaning solution is shown in Table 4: Table 4: Cleaning liquid formula
[0056] The eluent formula is shown in Table 5: Table 5 Eluent formula
[0057] 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-GFP fusion protein expression vector.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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):
[0062] 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).
[0063] (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.
[0064] Table 6 Grouping and treatment conditions
[0065] 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.
[0066] (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.
[0067] (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.
[0068] (c) Termination reaction: The reaction was terminated by adding 50 μL of 1 M NaOH.
[0069] (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.
[0070] (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.
[0071] 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.
[0072] 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 method for expressing animal-derived alkaline phosphatase protein in tobacco, characterized in that: The expression method comprises the following steps: S1. Constructing an animal-derived alkaline phosphatase protein gene into a plant expression vector to obtain a recombinant plasmid; S2, transfecting tobacco with the recombinant plasmid from step S1, and sampling during the physiological response window to obtain a sample; S3, the sample in step S2 is lysed with a protein lysis buffer to obtain a supernatant; S4. The supernatant of step S3 is purified to obtain alkaline phosphatase protein; The gene is a bovine intestinal alkaline phosphatase protein gene.
2. The expression method according to claim 1, characterized in that The plant expression vector in step S1 is a binary vector system, a single vector system, an RNAi vector or a CRISPR / Cas vector.
3. The expression method according to claim 2, characterized in that The plant expression vector is a binary vector system.
4. The expression method 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 method according to claim 4, characterized in that The plant expression vector is pEAQ series.
6. The expression method according to claim 5, characterized in that The plant expression vector is pEAQ-HT or pEAQ-HT-DEST1.
7. The expression method according to claim 6, characterized in that The plant expression vector is pEAQ-HT.
8. The expression method according to claim 7, characterized in that The tobacco in step S2 is Nicotiana benthamiana, common tobacco, forest tobacco, flowering tobacco or yellow tobacco.
9. The expression method according to claim 8, characterized in that The tobacco described in step S2 is Nicotiana benthamiana.
10. The expression method according to claim 9, characterized in that In step S2, the Nicotiana benthamiana is selected from the stage of 5-7 true leaves.
11. The expression method according to claim 10, characterized in that The physiological response window period in step S2 is 3-5 days.
12. The expression method according to claim 11, characterized in that The physiological response window period in step S2 is 3-4 days.
13. The expression method according to claim 12, characterized in that The physiological response window period in step S2 is 3 days.
14. The expression method according to any one of claims 1 to 13, characterized in that The purification method in step S4 is affinity chromatography.
15. The expression method according to claim 14, characterized in that The purification method in step S4 is metal ion affinity chromatography, bioaffinity chromatography, covalent affinity chromatography or hydrophobic affinity chromatography.
16. The expression method according to claim 15, characterized in that The purification method in step S4 is metal ion affinity chromatography.
17. The expression method according to claim 16, characterized in that The metal ion affinity chromatography is Ni-NTA metal ion affinity chromatography.