Method for stably improving valence of horse radish peroxidase labeled streptavidin

Abstract

The invention relates to the technical field of biology, in particular to a method for stably improving the titer of horse radish peroxidase labeled streptavidin. According to the method provided by the invention, streptavidin with different histidine label lengths is designed, and then the streptavidin is coupled with horse radish peroxidase oxidized by sodium periodate, so that high-titer horse radish peroxidase labeled streptavidin can be obtained. The invention also discloses application of the enzyme-labeled conjugate in a magnetic particle platform streptavidin-biotin system, and experimental results prove that the method provided by the invention can be used for stably preparing the enzyme conjugate with high titer and low background, and is widely applied in the streptavidin-biotin system.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for stably improving the titer of horseradish peroxidase-labeled streptavidin. Background Art

[0002] Horseradish peroxidase is a glycoprotein with a molecular weight of 44 KDa. It is composed of a colorless enzyme protein and a dark brown iron porphyrin. It is widely distributed in the plant kingdom and is found in high concentrations in horseradish. It is easy to extract, relatively cheap, stable, heat-resistant and resistant to organic solvents. After coupling with antigens or antibodies, its activity is rarely lost. Therefore, it is the most widely used labeling enzyme in immunology. Streptavidin is widely used due to its tight affinity for biotin binding. The system has applications in many fields such as protein science, nanotechnology and catalysis. Biomacromolecules such as proteins or nucleic acids can be specifically captured and coupled by streptavidin after being modified with biotin. Therefore, it is often used in studies such as immobilization of macromolecules, biolabeling, biodetection and cell sorting.

[0003] At present, horseradish peroxidase-labeled streptavidin on the market has batch-to-batch differences in potency, and the potency is relatively low, ranging from 1:3K to 1:10K, which to a certain extent affects the batch-to-batch stability of the detection platform reagents. Secondly, the purity of streptavidin causes nonspecific adsorption, which will make the detection background value higher and affect the detection quality. Summary of the invention

[0004] In view of this, the present invention provides a preparation method for stably improving the titer of horseradish peroxidase-labeled streptavidin, which involves the construction, expression and purification of streptavidin, and the preparation of horseradish peroxidase-labeled streptavidin by labeling, and applying the enzyme-labeled conjugate in a streptavidin-biotin system to verify its titer and background.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides streptavidin, the C-terminus of which is connected to a histidine tag via a flexible linker.

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

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

[0009] In some specific embodiments of the present invention, the streptavidin has:

[0010] (1) an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3; or

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

[0012] (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (1) or (2);

[0013] The plurality is 2 to 5.

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

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

[0016] (4) a nucleotide sequence as shown in any one of SEQ ID NOs: 4 to 6; or

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

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

[0019] The several number is 2 to 15.

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

[0021] The purification comprises:

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

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

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

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

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

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

[0028] In some specific embodiments of the present invention, the horseradish peroxidase in the preparation method of the above-mentioned horseradish peroxidase-labeled streptavidin is activated, and the activation concentration is 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 the horseradish peroxidase to the oxidant in the preparation method of the above-mentioned 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 method for preparing horseradish peroxidase-labeled streptavidin is carried out in a light-proof environment at a temperature 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, and the time is 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 in the preparation method of the above-mentioned horseradish peroxidase-labeled streptavidin is a protein containing 0, 3, and 6 histidine tags, respectively; the activation concentration of the horseradish peroxidase is 8-32 mg / ml; the oxidant is sodium periodate; the molar ratio of the horseradish peroxidase to the oxidant is 1:(100-250); the oxidation is carried out in a light-proof environment at a temperature of 2-25°C and a time of 1-2 h; the feed ratio of the streptavidin to the horseradish peroxidase is 1:(1-10); the temperature of the coupling reaction is 2-25°C and the time is 2-24 h; and the post-treatment of the coupling reaction is dialysis or diafiltration.

[0032] The invention also provides the use of the horseradish peroxidase-labeled streptavidin in a streptavidin-biotin system.

[0033] The invention also provides the use of the streptavidin or the streptavidin labeled with horseradish peroxidase in preparing a detection kit.

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

[0035] In some specific embodiments of the present invention, the detection kit used above is a human immunodeficiency virus antibody and antigen (p24) combined detection kit.

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

[0037] (a) streptavidin as above; or

[0038] (b) the above-mentioned nucleic acid molecule; or

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

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

[0041] The different lengths of histidine tags constructed by the present invention have a great effect on improving protein purity, and have no effect on the structure and biological activity of the protein. Specifically, in the protein purification process, 3 M NaCl is added to expose the hydrophobic groups buried inside the protein molecules, so that the surface hydrophobicity is increased, and the probability of histidine tag exposure is increased, which is convenient for the target protein to hang on the column in affinity chromatography and remove most of the impurities. In addition, the amino acids participating in the reaction of streptavidin are mainly located at the C-terminus. The fused histidine tag constructed by the present invention displays the C-terminal lysine on the outer surface while being exposed, which can increase the coupling rate with horseradish peroxidase during labeling, thereby increasing the titer of the marker. In short, the present invention is aimed at the shortcomings of existing horseradish peroxidase-labeled streptavidin, designs streptavidin with histidine tags of different lengths to stably improve the titer of horseradish peroxidase-labeled streptavidin, and maintains a low background to reduce background interference, which can be widely used in streptavidin-biotin systems. The present invention is simple to operate and low in cost, and can significantly reduce the background value of interfering samples in the application of HIV combined detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

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

[0044] Figure 2 Shows horseradish peroxidase-labeled streptavidin analyzed by HPLC, green is SA01-HRP, red is SA02-HRP, and blue is SA03-HRP. DETAILED DESCRIPTION

[0045] The present invention discloses a preparation method for stably improving the titer of horseradish peroxidase-labeled streptavidin. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0046] Aiming at the shortcomings of the titer and background value of horseradish peroxidase-labeled streptavidin on the market, a new horseradish peroxidase marker and its preparation method are proposed. The specific technical solutions are:

[0047] 1. Construction of streptavidin with different histidine lengths:

[0048] 1. The sequence of streptavidin without fusion of histidine tag is shown in SEQ ID NO: 1;

[0049] 2. The present invention adds a histidine tag to the C-terminus of the streptavidin, adds 3 and 6 histidines to the C-terminus respectively, and connects them with a hydrophobic flexible peptide segment (linker), which can fully expose the tag while retaining the natural structure of the streptavidin protein, which is conducive to the correct folding of the protein and facilitates subsequent purification. The specific sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3;

[0050] 3. Reversely translate the amino acid sequence into a nucleotide sequence, optimize the codons according to the codon preference of E. coli, send it to Shanghai Biosynthetics, and connect it to the pET30a vector;

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

[0052] 2. Streptavidin expression and purification

[0053] 1. Induction and expression

[0054] Pick a single colony and inoculate it into LB medium containing kanamycin and culture it at 37℃ until OD 600 When it reached 0.8-0.1, add IPTG with a final concentration of 0.2 mM for induction, continue culturing for 4-6 h, and collect the bacterial precipitate by centrifugation.

[0055] 2. Inclusion body transformation and renaturation

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

[0057] 3. Column chromatography

[0058] The refolded supernatant was purified by hydrophobic chromatography, affinity chromatography, ion exchange and other chromatography methods to remove octamers and other impurities as much as possible, and the final purity of the target protein reached more than 95%.

[0059] 4. Marking

[0060] The streptavidin antigen replaces the buffer to 0.01 M CB pH 9.6 buffer, and the labeling concentration is adjusted. At the same time, the horseradish peroxidase is oxidized with an oxidant to oxidize the hydroxyl group on the glycosylation of the horseradish peroxidase to an aldehyde group. Then the streptavidin antigen and the oxidized horseradish peroxidase are coupled to react. The sample after the reaction is ultrafiltered or dialyzed with a 300 KDa membrane to remove small molecules, thereby obtaining the streptavidin-labeled horseradish peroxidase.

[0061] 5. Tested in HIV combination 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] Codon-optimized 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 TACCGCAATGCGCACAGCGCTACCACTTGGTCTGGTCAGTATGTAGGCGGCGCAGAAGCGCGCATCAACACCCAATGGGCTGCTGACCTCTGGTACCACCGAAGCTAACGCGTGGAAATCTACTCTGGTTGGTCACGACACTTTCACTAAGGTTAAACCGTCCGCAGCATCTGGTTCTGGTGGCGGTGGCTCTCATCACCACCATCATCAC (SEQ ID NO: 6).

[0075] The titer of the enzyme-labeled avidin described in the present application is characterized by its dilution multiple, that is, the higher the dilution multiple, the higher the titer of the enzyme-labeled avidin.

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

[0077] The present invention will be further described below in conjunction with the embodiments.

[0078] Example

[0079] 1. Construction of streptavidin with different histidine lengths

[0080] The present invention fuses histidine tags of different lengths at the C-terminus of streptavidin, that is, 3 and 6 histidines are added to the C-terminus, respectively, and connected with a hydrophobic flexible peptide segment (linker), and the specific sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3. The amino acid sequence is reverse translated into a nucleotide sequence, and the codon is optimized according to the codon preference of Escherichia coli, as shown in SEQ ID NO: 4 to SEQ ID NO: 6. The whole gene synthesis method is used for synthesis, and it is directly synthesized into the pET-30a vector to obtain a recombinant plasmid.

[0081] 2. Inducible expression

[0082] 1) The recombinant plasmids were transferred into BL21(DE3) competent cells to construct prokaryotic expression engineering strains, which were named: BL21-pET30a-SA01, BL21-pET30a-SA02, BL21-pET30a-SA03;

[0083] 2) Pick a single colony with uniform morphology and size and inoculate it into 20 ml LB (containing Kan+) medium for overnight culture;

[0084] 3) The seed solution was then inoculated into 1 L LB liquid medium and cultured at 37°C for 2-4 h. 600 When the concentration reached 0.8-1, IPTG was added to induce the cells at a final concentration of 0.2 mM. The cells were cultured for 4-6 h and the precipitate was collected by centrifugation.

[0085] 3. Inclusion body transformation, renaturation purification and purification

[0086] 1) Resuspend the bacterial pellet in 50 mM Tris + 5 mM EDTA pH 8.0 buffer at a ratio of 1:20 and stir until there are no obvious bacterial clumps;

[0087] 2) Ultrasonic disruption was performed in an ice bath, 300 W for 3 s, 3 s rest, 10 min / time, 2 times in total, and then centrifuged at 4°C for 20 min to collect the precipitate;

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

[0089] 4) Dissolve the precipitate with 6 M guanidine hydrochloride, stir at low temperature (4°C or ice bath) until there are no obvious bacterial clumps and 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 renaturation, slowly stir at 2-8℃ for 3 h, and centrifuge at 4℃ to collect the supernatant;

[0091] 6) Add 3 M NaCl to the refolded supernatant, pass through the hydrophobic column Butyl S, balance the column with the starting buffer (20 mM PBS + 3M NaCl pH 7.4), elute with the buffer (20 mM PBS pH 7.4), and collect the protein at the elution peak. Continue to pass through the affinity column Ni, balance the column with the starting buffer (20 mM PBS pH 7.4), elute with the buffer (20 mM PBS + 1 M imidazole pH7.4), and collect the protein at the elution peak. Replace the eluted protein with the buffer to 20 mM PB pH 7.4, pass through the ion exchange column DEAE, balance the column with the 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 as shown in Figure 1 As shown, HPLC analysis Figure 2 The collected protein was replaced with 50 mM Tricine pH 9.0 buffer by ultrafiltration and the protein was concentrated to 20 mg / ml for storage.

[0092] 4. Marking

[0093] 1) Weigh horseradish peroxidase and dissolve it in ultrapure water, then add freshly prepared periodic acid solution and mix for 60 min at 2~8℃ in the dark to activate horseradish peroxidase. Add ethylene glycol to the activated horseradish peroxidase solution and mix well. Place it at 2~8℃ in the dark for 30 min to terminate the activation reaction. The activated horseradish peroxidase is dialyzed into 10 mM acetic acid pH 4.4 buffer for 3~4 times.

[0094] 2) The streptavidin antigen was dialyzed into 0.01 M CB pH 9.6 buffer in advance, and 4 times the mass of horseradish peroxidase was added. The mixture was placed in a 2-8°C water bath for 22-26 h. The next day, freshly prepared NaBH4 was added and allowed to stand for another 2 h at 2-8°C.

[0095] 3) Place the above liquid into a dialysis bag and place it in 0.067 M PBS pH 6.8 buffer, and dialyze 3 to 5 times at 4°C. Dissolve the dialysis bag and 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 particle chemiluminescence method) uses a biotin-avidin system. This kit uses the double antigen sandwich method and double antibody sandwich method for detection. In order to evaluate the titer and background of the enzyme-labeled avidin, the p24 antigen positive control, negative control and negative sample were tested respectively. The titer of the enzyme-labeled avidin was adjusted to make the p24 antigen positive signal value between 11 million and 13 million.

[0098] Table 1 shows the titers of enzyme-labeled antigens of different constructions.

[0099] Table 1: Titer and background determination

[0100]

[0101] From Table 1, we can know that if the concentration of enzyme-labeled avidin is increased, the signal value of the antigen positive control will change as shown in Table 2:

[0102] Table 2

[0103]

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

[0105] In summary, the results showed that the titer of horseradish peroxidase-streptavidin prepared without fusion of histidine tags was significantly low (Table 1), and the increase in the antigen positive control was not obvious with the gradient increase of dilution ratio (Table 2). However, the titer of horseradish peroxidase-streptavidin prepared with fusion of histidine tags was higher (Table 1), especially the titer of horseradish peroxidase-streptavidin prepared with fusion of 6 histidine tags increased significantly (Table 2).

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

[0107] Table 3: Titer and background determination 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, and the titer deviations of different batches were within 33%. The background of the five batches of enzyme-labeled antigens was low, avoiding interference with the test samples.

[0110] Comparative Example

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

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

[0113]

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as 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.

2. The streptavidin according to claim 1, characterized in that The flexible linker has (GGGGS) n sequence.

3. The streptavidin according to claim 1 or 2, characterized in that The histidine tag consists of 3 or 6 histidines.

4. The streptavidin according to claim 1, characterized in that have: (1) an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3; or (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (1) or (2); The plurality is 2 to 5.

5. A nucleic acid molecule encoding the streptavidin according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that have: (4) a nucleotide sequence as shown in any one of SEQ ID NOs: 4 to 6; or (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases to the nucleotide sequence shown in (4), and having the same or similar function as (4); or (6) A nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (4) or (5); The number is 2 to 15.

7. A method for preparing the streptavidin according to any one of claims 1 to 4, characterized in that: include: Expressing the nucleic acid molecule according to claim 5 or 6, harvesting the protein, purifying the protein, and obtaining the streptavidin; The purification comprises: (i) Purification by denaturation and renaturation of inclusion bodies; and / or (ii) at least one of hydrophobic chromatography, affinity chromatography or ion exchange chromatography.

8. Streptavidin labeled with horseradish peroxidase, characterized in that The streptavidin is the streptavidin according to any one of claims 1 to 4.

9. The method for preparing streptavidin according to claim 8, characterized in that: The method comprises coupling horseradish peroxidase with the streptavidin according to any one of claims 1 to 4.

10. Use of the streptavidin according to any one of claims 1, 2, 3, 4 or 8 in the preparation of a detection kit.

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