Method for stabilizing the horseradish peroxidase-labeled streptavidin titer

Abstract

The present application relates to the technical field of biology, and particularly relates to a method for stably improving horseradish peroxidase-labeled streptavidin titer. The method provided by the present application is to design streptavidin with different histidine tag lengths, and then couple with sodium periodate-oxidized horseradish peroxidase, so that high-titer horseradish peroxidase-labeled streptavidin can be obtained. The present application also discloses application of the enzyme-labeled conjugate in a magnetic microsphere platform streptavidin-biotin system. Experimental results prove that the method provided by the present application can stably prepare high-titer and low-background enzyme-labeled conjugate, and the enzyme-labeled conjugate can be widely applied in the streptavidin-biotin system.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for stably increasing the titer of horseradish peroxidase-labeled streptavidin. Background Technology

[0002] Horseradish peroxidase is a glycoprotein with a molecular weight of 44 kDa, composed of a colorless enzyme protein and a dark brown iron porphyrin. Widely distributed in the plant kingdom, it is abundant in horseradish. Due to its ease of extraction, relatively low price, and stable properties (resistant to heat and organic solvents), its activity is minimally affected after conjugation with antigens or antibodies. Therefore, it is the most widely used labeling enzyme in immunology to date. Streptavidin is widely used due to its tight biotin-binding affinity. This system has applications in protein science, nanotechnology, and catalysis. Biotin-modified biomolecules such as proteins or nucleic acids can be specifically captured and conjugated by streptavidin, thus it is commonly used for macromolecule immobilization, biolabeling, biodetection, and cell sorting research.

[0003] Currently, horseradish peroxidase-labeled streptavidin on the market exhibits batch-to-batch potency variations and relatively low potency, ranging from 1:3K to 1:10K, which to some extent affects the batch-to-batch stability of the detection platform reagents. Secondly, the purity of streptavidin can cause non-specific adsorption, leading to higher background values ​​and impacting detection quality. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing horseradish peroxidase-labeled streptavidin with stable and improved potency, involving the construction, expression and purification of streptavidin, as well as the preparation of horseradish peroxidase-labeled streptavidin, and the application of the enzyme-labeled conjugate in the streptavidin-biotin system to verify its potency and background.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides streptavidin, whose C-terminus is linked to a histidine tag via a flexible linker.

[0007] In some specific embodiments of the present invention, the flexible linker of the above-mentioned streptavidin has (GGGGS) characteristics. n sequence.

[0008] In some specific embodiments of the present invention, the histidine tag of the above-mentioned streptavidin consists of 3 or 6 histidines.

[0009] In some specific embodiments of the present invention, the above-mentioned streptavidin has the following characteristics:

[0010] (1) An amino acid sequence as shown in any of SEQ ID NO: 1-3; or

[0011] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0012] (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2);

[0013] The number of items is 2 to 5.

[0014] The present invention also provides a nucleic acid molecule encoding the above-mentioned streptavidin.

[0015] In some specific embodiments of the present invention, the above-mentioned nucleic acid molecules have:

[0016] (4) A nucleotide sequence as shown in any of SEQ ID NO: 4~6; or

[0017] (5) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (4), and whose function is the same as or similar to that of (4); or

[0018] (6) A nucleotide sequence that is at least 90% homologous to the nucleotide sequence shown in (4) or (5);

[0019] The number of items is 2 to 15.

[0020] The present invention also provides a method for preparing the above-mentioned streptavidin, comprising: expressing the above-mentioned nucleic acid molecule, harvesting the protein, purifying the protein, and obtaining the streptavidin;

[0021] The purification includes:

[0022] (i) Purification by inclusion body denaturation and renaturation; and / or

[0023] (ii) At least one of hydrophobic chromatography, affinity chromatography or ion exchange chromatography.

[0024] This invention also provides streptavidin labeled with horseradish peroxidase.

[0025] The present invention also provides a method for preparing the horseradish peroxidase-labeled streptavidin, comprising coupling horseradish peroxidase with the above-mentioned streptavidin.

[0026] In some specific embodiments of the present invention, the preparation method of the above-mentioned horseradish peroxidase-labeled streptavidin includes: oxidizing horseradish peroxidase with an oxidant, then coupling the above-mentioned streptavidin with the oxidized horseradish peroxidase, and performing ultrafiltration or dialysis on the product to obtain the horseradish peroxidase-labeled streptavidin.

[0027] In some specific embodiments of the present invention, the oxidant in the above-described method for preparing horseradish peroxidase-labeled streptavidin is sodium periodate.

[0028] In some specific embodiments of the present invention, the horseradish peroxidase in the above-described method for preparing horseradish peroxidase-labeled streptavidin is activated at an activation concentration of 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 22 mg / ml, 24 mg / ml, 26 mg / ml, 28 mg / ml, 30 mg / ml, or 32 mg / ml.

[0029] In some specific embodiments of the present invention, the molar ratio of horseradish peroxidase to the oxidant in the above-described method for preparing horseradish peroxidase-labeled streptavidin is 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:210, 1:220, 1:230, 1:240, or 1:250.

[0030] In some specific embodiments of the present invention, the oxidation in the above-described method for preparing horseradish peroxidase-labeled streptavidin is carried out in a light-protected environment at temperatures of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C for times of 1 h, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, 100 min, 105 min, 110 min, 115 min, or 2 h.

[0031] In some specific embodiments of the present invention, the streptavidin labeled with horseradish peroxidase in the above-mentioned method for preparing streptavidin is a protein containing 0, 3, or 6 histidine tags, respectively; the activated concentration of horseradish peroxidase is 8-32 mg / ml; the oxidant is sodium periodate; the molar ratio of horseradish peroxidase to the oxidant is 1:(100-250); the oxidation is carried out in a light-protected environment at a temperature of 2-25°C for 1-2 h; the feed ratio of streptavidin to horseradish peroxidase is 1:(1-10); the coupling reaction is carried out at a temperature of 2-25°C for 2-24 h; and the post-treatment of the coupling reaction is dialysis or washing.

[0032] This invention also provides the application of the above-mentioned horseradish peroxidase-labeled streptavidin in the streptavidin-biotin system.

[0033] This invention also provides the application of the above-mentioned streptavidin or horseradish peroxidase-labeled streptavidin in the preparation of detection kits.

[0034] In some specific embodiments of the present invention, the detection kit described above is a kit for detecting HIV.

[0035] In some specific embodiments of the present invention, the detection kit described above is a combined detection kit for human immunodeficiency virus antibodies and antigens (p24).

[0036] The present invention also provides a reagent kit comprising:

[0037] (a) the above-mentioned streptavidin; or

[0038] (b) The above-mentioned nucleic acid molecules; or

[0039] (c) The above-mentioned horseradish peroxidase-labeled streptavidin.

[0040] The method of the present invention has the following effects:

[0041] The histidine tags of varying lengths constructed in this invention significantly improve protein purity without affecting protein structure or biological activity. Specifically, during protein purification, the addition of 3 M NaCl exposes hydrophobic groups embedded within the protein molecule, increasing surface hydrophobicity and the likelihood of histidine tag exposure. This facilitates the attachment of the target protein to the column during affinity chromatography, removing most of the contaminating proteins. Furthermore, since the amino acids involved in the streptavidin reaction are primarily located at the C-terminus, the fused histidine tags constructed in this invention expose the C-terminal lysine residue on the outer surface, increasing the coupling rate with horseradish peroxidase during labeling and thus enhancing the titer of the labeled product. In summary, this invention addresses the shortcomings of existing horseradish peroxidase-labeled streptavidin by designing streptavidin with histidine tags of varying lengths to stably improve the titer of horseradish peroxidase-labeled streptavidin while maintaining a low background level to reduce background interference. This invention can be widely applied in streptavidin-biotin systems. This invention is simple to operate, low in cost, and can significantly reduce background values ​​in HIV combined detection kits. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0043] Figure 1 The image shows purified streptavidin. The left image shows SA01 (boiled and unboiled samples), and the right image, from left to right, shows SA02 and SA03 (both unboiled samples).

[0044] Figure 2 Horseradish peroxidase-labeled streptavidin is shown in HPLC analysis. Green represents SA01-HRP, red represents SA02-HRP, and blue represents SA03-HRP. Detailed Implementation

[0045] This invention discloses a method for preparing horseradish peroxidase-labeled streptavidin with stable and enhanced potency. Those skilled in the art can refer to this method and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0046] To address the shortcomings of commercially available horseradish peroxidase-labeled streptavidin titers and background values, a novel horseradish peroxidase marker and its preparation method are proposed. The specific technical solution is as follows:

[0047] I. Construction of streptavidin with different histidine lengths:

[0048] 1. The sequence of the unfused histidine-tagged streptavidin is shown in SEQ ID NO: 1;

[0049] 2. This invention adds a histidine tag to the C-terminus of the aforementioned streptavidin, adding 3 and 6 histidine residues respectively, and linking them with a hydrophobic flexible peptide linker. This fully exposes the tag while preserving the native structure of the streptavidin protein, which is beneficial for correct protein folding and facilitates subsequent purification. The specific sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3;

[0050] 3. The amino acid sequence was reverse-translated into a nucleotide sequence, codons were optimized according to the codon preference of E. coli, and the sequence was sent to Shanghai Sangon Biotech for synthesis and ligated into the pET30a vector;

[0051] 4. The recombinant plasmids were transformed into BL21(DE3) competent cells to construct prokaryotic expression engineered strains, which were named BL21-pET30a-SA01, BL21-pET30a-SA02, and BL21-pET30a-SA03, respectively.

[0052] II. Streptavidin Expression and Purification

[0053] 1. Induction and expression

[0054] Pick a single colony and inoculate it into LB medium containing kanamycin, then incubate at 37°C until OD500. 600 When the concentration reaches 0.8-0.1, add IPTG to a final concentration of 0.2 mM for induction, continue culturing for 4-6 h, and collect the bacterial pellet by centrifugation.

[0055] 2. Inclusion body changes and renaturation

[0056] After sonication and washing, the precipitate was dissolved in 6 M guanidine hydrochloride and slowly added dropwise to pre-cooled PBS for dilution and refolding. The mixture was stirred slowly at 4°C for 2-4 h, and the refolded supernatant was collected by centrifugation.

[0057] 3. Column Chromatography

[0058] The refolded supernatant was purified by various chromatographic methods, including hydrophobic chromatography, affinity chromatography, and ion exchange, to remove octamers and other impurities as much as possible, ultimately achieving a protein purity of over 95%.

[0059] 4. Marking

[0060] The streptavidin antigen was replaced with 0.01 M CB pH 9.6 buffer, and the labeling concentration was adjusted. Simultaneously, horseradish peroxidase was oxidized with an oxidant, causing the hydroxyl groups on the glycosylated horseradish peroxidase to be oxidized to aldehyde groups. Then, the streptavidin antigen was coupled with the oxidized horseradish peroxidase. The reacted sample was subjected to 300 kDa membrane ultrafiltration or dialysis to remove small molecules, yielding the streptavidin-labeled horseradish peroxidase.

[0061] 5. Perform the test using an HIV combined test kit.

[0062] The sequences involved in this application are as follows:

[0063] SEQ ID NO: 1:

[0064] MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS;

[0065] SEQ ID NO: 2:

[0066] MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGGGGSHHH;

[0067] SEQ ID NO: 3:

[0068] MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGGGGSHHHHHH;

[0069] The optimized codon sequence encoding the polypeptide shown in SEQ ID NO: 1:

[0070] ATGGCTGAAGCTGGTATCACGGGCACCTGGTACAATCAACTGGGCTCCACCTTCATCGTTACTGCGGGTGCAGATGGCGCCCTGACGGGTACCTACGAAAGCGCAGTAGGCAACGCGGAATCCCGTTACGTTCTGACCGGTCGTTACGACTCTGCTCCGGCAACCGACGGCTCTGGTACGGCTCTGGGTTGGACGGTTGCGTGGAAAAACAACTACCGCAATGCACACTCCGCCACTACCTGGTCTGGTCAGTACGTAGGTGGCGCAGAAGCGCGTATTAACACCCAGTGGCTGCTGACTAGCGGCACTACGGAAGCCAACGCTTGGAAGTCCACCCTGGTCGGCCACGACACTTTCACCAAAGTTAAACCGTCCGCAGCGTCT (SEQ ID NO: 4);

[0071] Codon-optimized sequence encoding the polypeptide shown in SEQ ID NO: 2:

[0072] ATGGCGGAAGCAGGCATTACTGGCACTTGGTACAATCAGCTGGGCAGCACCTTTATTGTGACCGCTGGTGCCGATGGCGCACTGACCGGCACCTACGAATCCGCTGTGGGTAACGCAGAAAGCCGTTACGTGCTGACCGGTCGCTACGACTCTGCTCCGGCGACTGATGGTTCTGGTACCGCACTGGGCTGGACCGTCGCTTGGAAAAACAACTACCGCAACGCCCACTCCGCCACGACCTGGTCCGGTCAGTACGTTGGTGGCGCAGAAGCACGTATCAATACCCAGTGGCTGCTGACCTCTGGTACCACCGAGGCAAATGCTTGGAAGTCCACCCTGGTTGGCCACGACACCTTCACCAAGGTGAAACCGTCCGCAGCATCCGGCAGCGGTGGTGGTGGCTCTCATCACCAC (SEQ ID NO: 5);

[0073] Codon-optimized sequence encoding the polypeptide shown in SEQ ID NO: 3:

[0074] ATGGCGGAAGCAGGCATTACGGGCACTTGGTACAACCAGCTGGGCTCTACCTTCATTGTTACCGCGGGTGCAGATGGTGCACTGACTGGCACCTACGAAAGCGCCGTAGGTAATGCGGAATCCCGCTACGTGCTGACCGGCCGTTATGATTCTGCGCCGGCAACTGATGGTTCTGGCACTGCACTGGGCTGGACCGTTGCGTGGAAAAACAAC TACCGCAATGCGCACAGCGCTACCACTTGGTCTGGTCAGTATGTAGGCGGCGCAGAAGCGCGCATCAACACCCAAATGGCTGCTGACCTCTGGTACCACCGAAGCTAACGCGTGGAAATCTACTCTGGTTGGTCACGACACTTTCACTAAGGTTAAACCGTCCGCAGCATCTGGTTCTGGTGGCGGTGGCTCTCATCACCACCATCATCAC (SEQ ID NO: 6).

[0075] The potency of the enzyme-labeled avidin described in this application is characterized by its dilution factor; that is, the higher the dilution factor, the higher the potency of the enzyme-labeled avidin.

[0076] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0077] The present invention will be further illustrated below with reference to the embodiments.

[0078] Example

[0079] 1. Construct streptavidin with different histidine lengths

[0080] This invention fuses histidine tags of different lengths to the C-terminus of streptavidin, specifically adding 3 and 6 histidine residues to the C-terminus, respectively, and links them with a hydrophobic flexible peptide linker, as shown in SEQ ID NO: 2 and SEQ ID NO: 3. The amino acid sequences are reverse-translated into nucleotide sequences, and codon optimization is performed according to the codon preference of *E. coli*, as shown in SEQ ID NO: 4~SEQ ID NO: 6 above. The recombinant plasmid is synthesized using a whole-genome synthesis method and directly synthesized into the pET-30a vector.

[0081] 2. Induced expression

[0082] 1) The recombinant plasmids were transformed into BL21(DE3) competent cells to construct prokaryotic expression engineered strains, which were named BL21-pET30a-SA01, BL21-pET30a-SA02, and BL21-pET30a-SA03, respectively.

[0083] 2) Select single colonies of uniform shape and size and inoculate them into 20 ml of LB (containing Kan+) medium for overnight culture;

[0084] 3) Subsequently, the seed culture was inoculated into 1 L LB liquid medium and incubated at 37°C for 2–4 h, OD 600 When the concentration reaches 0.8-1, add IPTG to a final concentration of 0.2 mM for induction, continue culturing for 4-6 h, and collect the bacterial pellet by centrifugation.

[0085] 3. Inclusion body mutation, refolding purification, and purification

[0086] 1) The bacterial pellet was resuspended in 50 mM Tris + 5 mM EDTA pH 8.0 buffer at a ratio of 1:20 and stirred until there were no obvious bacterial clumps.

[0087] 2) Perform ultrasonic disruption under ice bath conditions: 300 W for 3 seconds, with a 3-second interval, 10 min / time, for a total of 2 times, followed by centrifugation at 4℃ for 20 min, and collect the precipitate;

[0088] 3) The precipitate was resuspended and washed 1-2 times with buffer solution 0 mM Tris + 5 mM EDTA pH 8.0, and the bacterial precipitate was collected by centrifugation.

[0089] 4) Dissolve the precipitate with 6 M guanidine hydrochloride, stir at low temperature (4°C or ice bath) until no obvious bacterial clumps are visible, the solution is yellow and gelatinous, and collect the supernatant after centrifugation;

[0090] 5) Slowly add the supernatant to pre-cooled PBS for dilution and refolding, stir slowly at 2-8℃ for 3 h, and centrifuge at 4℃ to collect the supernatant;

[0091] 6) Add 3 M NaCl to the refolded supernatant and pass it through a hydrophobic column (Butyl S), equilibrate with starting buffer (20 mM PBS + 3 M NaCl, pH 7.4), and elute with buffer (20 mM PBS, pH 7.4). Collect the protein at the elution peak. Continue passing the supernatant through an affinity column (Ni), equilibrate with starting buffer (20 mM PBS, pH 7.4), and elute with buffer (20 mM PBS + 1 M imidazole, pH 7.4). Collect the protein at the elution peak. Replace the eluted protein with buffer to 20 mM PB, pH 7.4, and pass it through an ion-exchange column (DEAE), equilibrate with starting buffer (20 mM PB, pH 7.4), collect the flow-through, and perform SDS-PAGE gel electrophoresis to detect the protein (electrophoresis results are shown in the image). Figure 1 As shown, HPLC analysis is as follows Figure 2 (As shown). The collected protein was replaced with ultrafiltration buffer to 50 mM MTTricine pH 9.0 buffer, and the protein was concentrated to 20 mg / ml for storage.

[0092] 4. Mark

[0093] 1) Dissolve horseradish peroxidase in ultrapure water, then add freshly prepared periodic acid solution and mix at 2-8°C in the dark for 60 min to activate the horseradish peroxidase. Add ethylene glycol to the activated horseradish peroxidase solution, mix well, and incubate at 2-8°C in the dark for 30 min to terminate the activation reaction. Dialyze the activated horseradish peroxidase to 10 mM acetate pH 4.4 buffer, repeating the dialyze 3-4 times.

[0094] 2) Dialyze the streptavidin antigen to 0.01 M CB pH 9.6 buffer beforehand, add 4 times the mass of horseradish peroxidase, and place the mixture in a water bath at 2-8℃ for 22-26 h. The next day, add freshly prepared NaBH4 and let it stand at 2-8℃ for another 2 h.

[0095] 3) Place the above liquid into a dialysis bag and in 0.067 M PBS pH 6.8 buffer. Dialyze 3-5 times at 4°C. After unpacking the dialysis bag, accurately measure the volume, add an equal volume of glycerol, and mix well to obtain the labeled streptavidin.

[0096] 5. Application of horseradish peroxidase-labeled streptavidin

[0097] The Human Immunodeficiency Virus Antibody and Antigen (p24) Combined Detection Kit (Magnetic Microparticle Chemiluminescence Assay) utilizes the biotin-avidin system. This kit employs a double-antigen sandwich method and a double-antibody sandwich method for detection. To evaluate the titer and background level of enzyme-labeled avidin, p24 antigen positive controls, negative controls, and negative samples were tested. The titer of enzyme-labeled avidin was adjusted to achieve a p24 antigen positive signal value between 11 and 13 million.

[0098] Table 1 shows the titers of enzyme-labeled antigens constructed differently.

[0099] Table 1: Valence and Background Measurements

[0100]

[0101] As shown in Table 1, the variation in the signal value of the antigen-positive control is as shown in Table 2 when the concentration of enzyme-labeled avidin is increased.

[0102] Table 2

[0103]

[0104] In Table 2, the final antigen-positive control signal value was increased by 11% for SA01-HRP at a dilution ratio of 1:1K compared to 1:2K; by 12% for SA01-HRP at a dilution ratio of 1:0.5K compared to 1:1K; by 23% for SA02-HRP at a dilution ratio of 1:3K compared to 1:6K; by 25% for SA02-HRP at a dilution ratio of 1:1.5K compared to 1:3K; by 31% for SA03-HRP at a dilution ratio of 1:6K compared to 1:12K; and by 33% for SA03-HRP at a dilution ratio of 1:3K compared to 1:6K.

[0105] In summary, the results showed that the horseradish peroxidase-streptavidin prepared without histidine tags had significantly lower titers (Table 1), and the titer increase was not significant with increasing dilution ratios (Table 2). Conversely, the horseradish peroxidase-streptavidin prepared with histidine tags had higher titers (Table 1), especially the horseradish peroxidase-streptavidin prepared with six histidine tags, which showed a significant increase in titer (Table 2).

[0106] SA03-HRP was prepared repeatedly according to the above antigen preparation and labeling process to verify the enzyme titer and background of different batches. The results are shown in Table 3.

[0107] Table 3: Measurement of titer and background of different batches of enzyme-labeled avidin

[0108]

[0109] The results showed that the titers of the five batches of enzyme-labeled antigens ranged from 9K to 12K, with the titer deviation between different batches within 33%. The background of the five batches of enzyme-labeled antigens was low, which avoided interference with the test samples.

[0110] Comparative Example

[0111] Table 4 shows the titers of different batches of enzyme-labeled avidin from manufacturer K. The results show that there are batch-to-batch differences in the titers of different batches of enzyme-labeled antigen from this manufacturer, and the titers are relatively low, ranging from 1:3K to 1:10K.

[0112] Table 4: Enzyme potency of different batches from manufacturer K

[0113]

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Streptavidin, characterized in that, Its C-terminus is connected to a histidine tag via a flexible linker; The flexible linker has (GGGGS) n sequence; The histidine tag consists of 3 or 6 histidine residues; The amino acid sequence of the streptavidin is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

2. A nucleic acid molecule encoding the streptavidin of claim 1.

3. The nucleic acid molecule as described in claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO: 5 or SEQ ID NO:

6.

4. A method for preparing streptavidin according to claim 1, characterized in that, include: Express the nucleic acid molecule according to claim 2 or 3, harvest the protein, purify the protein, and obtain the streptavidin; The purification includes: (i) Purification through inclusion body denaturation and renaturation; and / or (ii) At least one of hydrophobic chromatography, affinity chromatography or ion exchange chromatography.

5. Streptavidin labeled with horseradish peroxidase, characterized in that, The streptavidin is the streptavidin according to claim 1.

6. The method for preparing streptavidin as described in claim 5, characterized in that, This includes conjugating horseradish peroxidase with the streptavidin of claim 1.

7. The use of streptavidin as described in claim 1 or 5 in the preparation of a detection kit.

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