Core streptavidin and preparation method and application thereof

Abstract

The application provides a core streptavidin and a preparation method and application thereof, the amino acid sequence of the core streptavidin is shown as SEQ ID NO:1, and / or the core streptavidin can be obtained by first expressing streptavidin by using a recombinant bacterium and then performing enzymatic cutting on the streptavidin by using a fixed enzyme technology.The core streptavidin provided by the application has high activity and good activity stability when combined with biotin, the preparation method of the core streptavidin is simple, and the core streptavidin prepared by the preparation method has high CSA purity.The core streptavidin and the preparation method thereof can be applied in enzyme-linked immunosorbent assay, immunohistochemistry, time-resolved immunofluorescence technology, chemiluminescence, quantitative PCR, single-stranded DNA preparation, biomolecule purification, monoclonal antibody preparation, and the like, and are especially applied in preparation of a hepatitis C virus antibody detection kit.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically a core streptavidin, its preparation method, and its application. Background Technology

[0002] Streptavidin (SA) is a homotetramer of approximately 80 kDa secreted by the fungus *Streptomyces avidinii*. It is one of the most stable proteins known in nature, maintaining its biological activity even under conditions of high temperature, extreme pH, denaturants, and enzymatic degradation. Due to its unique properties, streptavidin can specifically bind to biotin, forming the biotin-streptavidin system. The dissociation constant Kd of this system is 10⁻⁶. -15 mol / L represents the strongest known non-covalent binding force, far exceeding the antigen-antibody binding capacity (10 mol / L). -5 ~10 -11 The concentration of biotin (mol / L) is at least 10,000 times higher. This system exhibits high specificity and wide applicability because biotin readily binds to active biomolecules. Through the specific binding of streptavidin to biotin, signal amplification is achieved without affecting the activity of the biomolecules, making it particularly prevalent in the biosciences. Therefore, obtaining highly active and pure SA is a prerequisite for its important applications.

[0003] Streptavidin's full-length coding region consists of 183 amino acids (aa), including a 24-aa signal peptide, with a mature molecule of 159 aa. Intact streptavidin has poor solubility and readily aggregates. Early researchers obtained naturally occurring SA proteins that were shorter than the full-length protein. For example, when Stapley EO et al. first discovered SA in 1963, the purified SA had a molecular weight of approximately 60 kDa, with each single chain having a molecular weight of approximately 15 kDa. In the 1980s, researchers found that the natural SA monomeric peptide chain consisted of 169 amino acids, with a single chain molecular weight of 16.5 kDa and a tetramer molecular weight of 66.0 kDa. Studies have confirmed that this is because the full-length protein is easily cleaved by extracellular proteases during the exocrine fermentation of *Streptomyces avidinii*, resulting in incomplete SA proteins. This incomplete SA is formed by removing the amino acid fragments at both ends of the full-length protein. Later studies showed that the sequences at the amino acid ends of SA are hydrophobic, which is key to reducing the solubility of the full-length protein and also weakens the affinity of SA for biotin. When the protein is secreted outside the bacterial cell, these two sequences are removed by protease cleavage, restoring the solubility of SA. This greatly reduces the cytotoxicity caused by excessive biotin binding within the cell, thus protecting the cell. Core Streptavidin (CSA) is the core segment of streptavidin that exerts its biological activity. CSA is characterized by good solubility, high biological activity, stability, acid resistance, and resistance to urea and guanidine hydrochloride. It is currently the main form of streptavidin used in China.

[0004] Given the understanding of CSA, researchers have proposed several methods for expressing CSA using host cells. For example, in 1998, Anna Gallizia et al. successfully expressed CSA protein with a T7 tag using the expression vector pET11a and the E. coli expression host BL21(DE3), achieving an expression level of 70 mg / L. In 2005, Xipeng Liu et al. expressed SA and CSA with signal peptides using the expression vector pET28a and the expression host BL21(DE3), respectively, with expression levels around 80 mg / L. However, studies have shown that direct expression of CSA in host cells results in low yields. Furthermore, direct expression of recombinant core streptavidin in E. coli prokaryotic cells, being a bacterial protein, is too fast to fold correctly and is mostly expressed as inclusion bodies, resulting in relatively weak biotin-binding activity after refolding. To overcome the problems associated with direct expression of CSA, researchers have recently attempted to achieve exocrine expression of SA in *E. coli*. For example, in 2008, Gerhard Miksch et al. constructed a special expression vector using the guide sequence of the phoA gene, the corresponding strong promoter, and the kil gene, achieving high-volume exocrine expression of SA in *E. coli* BL21(DE3). Through optimization of expression conditions, the yield of exocrine SA reached 1715 nM (109.76 mg / L). Direct expression of SA effectively solves the problem of low CSA yield. However, obtaining CSA still requires enzymatic digestion. Current techniques primarily involve enzymatic digestion of SA to obtain CSA. However, enzymatic digestion with proteases presents the following problems: ① Proteases are highly stable, and incomplete protease removal can affect the protein stability of CSA; ② Incomplete removal of the digested polypeptide fragments or tag regions can affect the biotin-binding activity per unit mass of CSA.

[0005] In summary, there is an urgent need to develop a method for producing highly active streptavidin at low cost and high efficiency. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a core streptavidin, its preparation method, and its application. This core streptavidin exhibits high activity and good activity stability when bound to biotin. The preparation method of the core streptavidin is simple, and the CSA purity of the prepared core streptavidin is high.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a core streptavidin, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] The sequence of SEQ ID NO:1 is as follows:

[0010] AEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAA.

[0011] In some embodiments of the present invention, the core streptavidin is obtained by first expressing streptavidin using recombinant bacteria, and then enzymatically digesting streptavidin using immobilization enzyme technology.

[0012] In some embodiments of the present invention, the expression of streptavidin using recombinant bacteria includes constructing the amino acid sequence of streptavidin into the pET28a expression vector using genetic engineering techniques, then transforming the expression vector into Rosseta competent cells of Escherichia coli, culturing the cells, collecting the recombinant bacterial cells, and isolating streptavidin from the recombinant bacterial cells.

[0013] In some embodiments of the present invention, the streptavidin amino acid sequence constructed into the pET28a expression vector contains the amino acid sequence of SEQ ID NO:1; preferably, the streptavidin amino acid sequence is selected from any one or more of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. More preferably, the streptavidin amino acid sequence is selected from any one or two of SEQ ID NO:3 and SEQ ID NO:4. Most preferably, the streptavidin amino acid sequence is selected from SEQ ID NO:3.

[0014] The sequence of SEQ ID NO:2 is as follows (the 135aa streptavidin amino acid sequence):

[0015] DPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKP.

[0016] The sequence of SEQ ID NO:3 is as follows (the 138aa streptavidin amino acid sequence):

[0017] DPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAA.

[0018] The sequence of SEQ ID NO:4 is as follows (the 147aa streptavidin amino acid sequence):

[0019] DPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAG.

[0020] The sequence of SEQ ID NO:5 is as follows (the 159aa streptavidin amino acid sequence):

[0021] DPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ.

[0022] In some embodiments of the present invention, the sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 can be obtained by truncating the sequence of SEQ ID NO:5.

[0023] In some embodiments of the present invention, culturing the cells includes: culturing to OD. 600 When the concentration is 0.6–1.2, add the inducing agent IPTG (isopropylβ-D-Thiogalactoside) to a final concentration of 1 mM, and induce at 36–38 °C for 4–6 h.

[0024] In some embodiments of the present invention, the isolation of streptavidin from recombinant bacterial cells includes extraction, chromatography, and dialysis steps; preferably, the recombinant bacterial cells are added to a disruption buffer and homogenized under high pressure to disrupt the cells and release streptavidin, and then subjected to NI column affinity chromatography and dialysis in sequence to obtain purified streptavidin.

[0025] In some embodiments of the present invention, the enzymatic digestion of streptavidin using immobilized enzyme technology includes coupling a protease with a filler to form a protease conjugate, then performing an enzymatic digestion reaction with streptavidin at 16–25°C, and then removing the protease conjugate.

[0026] In some embodiments of the present invention, the protease is selected from one or more of proteinase K, pepsin, papain, trypsin, subtilisin, and plasmin. Preferably, the protease is selected from any one or two of proteinase K and trypsin; most preferably, the protease is selected from proteinase K.

[0027] In some embodiments of the present invention, the protease is tagged with histidine.

[0028] In some embodiments of the present invention, the ratio of the protease to the filler is 5-10 mg of protease per 1 mL of filler.

[0029] In some embodiments of the present invention, the filler is a hydrogen bromide activated and / or NHS activated agarose matrix.

[0030] In some embodiments of the present invention, during the enzymatic digestion reaction, the ratio of protease conjugate to streptavidin is 22-25 mg streptavidin per 1 mL of protease conjugate, and the digestion time is 50-70 min.

[0031] Secondly, this invention provides the application of the core streptavidin described in the first aspect in enzyme-linked immunosorbent assays, immunohistochemistry, time-resolved immunofluorescence, chemiluminescence, quantitative PCR, single-stranded DNA preparation, biomolecule purification, and monoclonal antibody preparation. In particular, it is used in the preparation of hepatitis C virus antibody detection kits.

[0032] Thirdly, the present invention provides a method for preparing core streptavidin, the method comprising the following steps:

[0033] (1) Using recombinant bacteria to express streptavidin;

[0034] (2) Streptavidin was digested using immobilized enzyme technology.

[0035] According to the preparation method of the present invention, the core streptavidin comprises the core streptavidin with the amino acid sequence shown in SEQ ID NO:1.

[0036] In some embodiments of the present invention, step (1) includes using genetic engineering technology to construct the amino acid sequence of streptavidin into the pET28a expression vector, then transforming the expression vector into Rosseta competent cells of Escherichia coli, culturing the cells, collecting recombinant cells, and isolating streptavidin from the recombinant cells.

[0037] In some embodiments of the present invention, the streptavidin amino acid sequence constructed into the pET28a expression vector contains the amino acid sequence in SEQ ID NO:1.

[0038] In some embodiments of the present invention, the streptavidin amino acid sequence constructed into the pET28a expression vector is selected from any one or more of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. More preferably, the streptavidin amino acid sequence is selected from any one or two of SEQ ID NO:3 and SEQ ID NO:4. Most preferably, the streptavidin amino acid sequence is selected from SEQ ID NO:3.

[0039] In some embodiments of the present invention, culturing the cells includes: culturing to OD. 600 When the concentration is 0.6–1.2, add the inducing agent IPTG (isopropylβ-D-Thiogalactoside) to a final concentration of 1 mM, and induce at 36–38 °C for 4–6 h.

[0040] In some embodiments of the present invention, step (1) further includes adding the collected recombinant bacterial cells to a disruption buffer for high-pressure homogenization to disrupt the cells and release streptavidin, and then performing NI column affinity chromatography and dialysis sequentially to obtain purified streptavidin.

[0041] In some embodiments of the present invention, the composition of the disruption buffer includes: 18-22 mM PB (phosphate buffer), 0.45-0.55 M NaCl, and the pH of the disruption buffer is 6.2-6.8.

[0042] In some embodiments of the present invention, the ratio of the amount of lysis buffer to recombinant bacterial cells is 1 mg of recombinant bacterial cells per 15-25 mL of lysis buffer.

[0043] In some embodiments of the present invention, the temperature of the high-pressure homogenizer is -2 to 2°C, and the homogenizing pressure is 550 to 700 Barg.

[0044] In some embodiments of the present invention, when performing NI column affinity chromatography, the loading buffer system is 18–22 mM PB + 0.45–0.55 M NaCl, pH = 6.2–6.8; the washing buffer system is 18–22 mM PB + 0.45–0.55 M NaCl + 28–32 mM imidazole, pH = 6.2–6.8; and the elution buffer system is 18–22 mM PB + 0.45–0.55 M NaCl + 250–350 mM imidazole, pH = 6.2–6.8.

[0045] In some embodiments of the present invention, the dialysis is performed using 18-22 mM PBS with pH = 6.2-6.8.

[0046] In some embodiments of the present invention, step (2) includes coupling the protease with the filler to form a protease conjugate, then performing an enzymatic digestion reaction with streptavidin at 16–25°C, and then removing the protease conjugate.

[0047] In some embodiments of the present invention, the protease is selected from one or more of proteinase K, pepsin, papain, trypsin, subtilisin, and plasmin. Preferably, the protease is selected from any one or two of proteinase K and trypsin; most preferably, the protease is selected from proteinase K.

[0048] In some embodiments of the present invention, the protease is tagged with histidine.

[0049] In some embodiments of the present invention, the ratio of the protease to the filler is 5-10 mg of protease per 1 mL of filler.

[0050] In some embodiments of the present invention, the filler is a hydrogen bromide activated and / or NHS activated agarose matrix.

[0051] In some embodiments of the present invention, during the enzymatic digestion reaction, the ratio of protease conjugate to streptavidin is 22-25 mg streptavidin per 1 mL of protease conjugate, and the digestion time is 50-70 min.

[0052] In some embodiments of the present invention, step (2) further includes performing NI affinity chromatography and benzalkonium chloride affinity chromatography sequentially on the product obtained after removing the protease conjugate following the enzymatic digestion reaction to remove residual protease and histidine tags as well as the cleaved polypeptide fragments, and then collecting the flow-through of the chromatography for dialysis.

[0053] In some embodiments of the present invention, the loading buffer system for NI affinity chromatography and benzalkonium affinity chromatography is 18-22 mM PBS with pH = 6.2-6.8.

[0054] In some embodiments of the present invention, the collected chromatographic flow is dialyzed using a dialysis bag with a molecular weight cutoff of 12-14 kDa. Preferably, the dialysate is a mixed solution formed by adding one or more of sucrose, trehalose, lactose, ethylene glycol, glycerol, and sorbitol to a phosphate buffer system; more preferably, the amounts of sucrose, trehalose, lactose, ethylene glycol, glycerol, and sorbitol added to the phosphate buffer system are such that the mass concentrations of sucrose, trehalose, lactose, ethylene glycol, glycerol, and sorbitol in the phosphate buffer system are 2-4 wt%, 2-4 wt%, 2-4 wt%, 0.2-0.8 wt%, 4-6 wt%, and 1-3 wt%, respectively; for example, a phosphate buffer system solution containing 2-4 wt% sucrose or a phosphate buffer system solution containing 2-4 wt% sucrose and 2-4 wt% trehalose is used as the dialysate.

[0055] In some embodiments of the present invention, the phosphate buffer system in the dialysis solution has a PB concentration of 18–22 mM and a pH value of 6.2–6.8.

[0056] In some embodiments of the present invention, the preparation method further includes dissolving the prepared core streptavidin in a protective agent and freeze-drying it, or dissolving the prepared core streptavidin in a protective agent and then storing it directly in liquid form at 2-8°C. The protective agent is selected from a mixed solution formed by adding one or more of sucrose, trehalose, lactose, ethylene glycol, glycerol, and sorbitol to a phosphate buffer system; the amounts of sucrose, trehalose, lactose, ethylene glycol, glycerol, and sorbitol added to the phosphate buffer system are such that the mass concentrations of sucrose, trehalose, lactose, ethylene glycol, glycerol, and sorbitol in the phosphate buffer system are 2-4 wt%, 2-4 wt%, 2-4 wt%, 0.2-0.8 wt%, 4-6 wt%, and 1-3 wt%, respectively. Preferably, the protective agent is selected from a mixed solution formed by adding one or more of sucrose, trehalose, and ethylene glycol to a phosphate buffer system.

[0057] In some embodiments of the present invention, the concentration of PB in the phosphate buffer system in the protective agent is 18-22 mM and the pH value is 6.2-6.8.

[0058] Fourthly, the present invention provides a core streptavidin prepared by the core streptavidin preparation method described in the third aspect.

[0059] Fifthly, this invention provides an application of core streptavidin prepared by the method described in the third aspect in enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, time-resolved immunofluorescence, chemiluminescence, quantitative PCR, single-stranded DNA preparation, biomolecule purification, and monoclonal antibody preparation. In particular, it is used in the preparation of hepatitis C virus antibody detection kits.

[0060] In a sixth aspect, the present invention provides a method for preparing a hepatitis C virus antibody detection kit containing the core streptavidin described in the first aspect or the core streptavidin prepared by the preparation method described in the third aspect, comprising: using the core streptavidin described in the first aspect or the core streptavidin prepared by the preparation method described in the third aspect to replace conventional core streptavidin and preparing the hepatitis C virus antibody detection kit by conventional methods.

[0061] The beneficial effects of this invention are as follows:

[0062] This invention provides a core streptavidin with the sequence SEQ ID NO:1. This core streptavidin exhibits high activity and good activity stability when binding to biotin, maintaining high biotin-binding activity even after long-term storage. Furthermore, this invention provides a method for preparing the core streptavidin. The method involves first expressing streptavidin using recombinant bacteria, then digesting the streptavidin using immobilization enzyme technology. The resulting core streptavidin contains the amino acid sequence shown in SEQ ID NO:1. The obtained core streptavidin has high CSA purity and strong biotin-binding ability, and the preparation method is simple. The core streptavidin of this invention, or the core streptavidin prepared by the method described herein, shows promising applications in enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, time-resolved immunofluorescence (TIR), chemiluminescence, quantitative PCR, single-stranded DNA preparation, biomolecule purification, and monoclonal antibody preparation. Attached Figure Description

[0063] Figure 1 This is an SDS-PAGE identification result of purified streptavidin isolated from recombinant bacterial cells obtained by expressing streptavidin with sequences SEQ ID NO:2 and SEQ ID NO:3. In the figure, A represents the tetrameric form of streptavidin, and B represents the monomeric form of streptavidin.

[0064] Figure 2This is an SDS-PAGE identification result of purified streptavidin isolated from recombinant bacterial cells obtained by expressing streptavidin with sequences SEQ ID NO:4 and SEQ ID NO:5. In the figure, B represents the monomeric form of streptavidin.

[0065] Figure 3 The image shows the SDS-PAGE identification results of core streptavidin obtained by enzymatic digestion with different proteases. In the image, 1 indicates the use of proteinase K, 2 indicates the use of papain, 3 indicates the use of pepsin, and 4 indicates the use of trypsin.

[0066] Figure 4 This is an SDS-PAGE image of the core streptavidin obtained by digestion with proteinase K at different digestion times.

[0067] Figure 5 This is an SDS-PAGE identification result of streptavidin at different stages (or different treatments) during the preparation of core streptavidin. In the figure, 1 represents streptavidin after enzyme digestion to remove the protease and the cleaved polypeptide fragments; 2 represents streptavidin after enzyme digestion to remove the cleaved polypeptide fragments but not the protease; 3 represents streptavidin after enzyme digestion to remove the protease and the cleaved polypeptide fragments; and 4 represents streptavidin before enzyme digestion.

[0068] Figure 6 These are images of the core streptavidin prepared in Example 1 of this invention, after being freeze-dried for a period of time using a phosphate buffer system containing a protective agent as a freeze-drying protectant, then reconstituted, and the reconstituted liquid was placed at 2-8°C for 9 months.

[0069] Figure 7 This is an SDS-PAGE identification result diagram of the core streptavidin obtained in Example 1 of the present invention and streptavidin purchased from Thermo, Invitrogen, and Agilent; in the figure, 1 represents Agilent SA10, 2 represents Agilent SA26, 3 represents Thermo SA, 4 represents Thermo CSA, 5 represents Invitrogen CSA, and 6 represents the core streptavidin obtained in Example 1 of the present invention.

[0070] Figure 8 This is the size exclusion chromatogram of the core streptavidin obtained in Example 1 of the present invention.

[0071] Figure 9 This is a size exclusion chromatogram of CSA purchased from Thermo.

[0072] Figure 10This is a size exclusion chromatogram of CSA purchased from Invitrogen.

[0073] Figure 11 This is a size exclusion chromatogram of Agilent SA10 purchased from Agilent.

[0074] Figure 12 This is a size exclusion chromatogram of Agilent SA26 purchased from Agilent. Detailed Implementation

[0075] The following examples further illustrate the technology of the present invention. These examples are illustrative and exemplary of the present invention and do not limit the scope of the invention in any way.

[0076] Example 1

[0077] A method for preparing core streptavidin includes the following steps:

[0078] (1) The amino acid sequence of streptavidin SEQ ID NO:3 was constructed into the pET28a expression vector using genetic engineering technology. The expression vector was then transformed into Rosseta competent cells of Escherichia coli and the Rosseta competent cells were cultured.

[0079] (2) When the OD of Rosseta cells were cultured to competent cells 600 When the concentration of IPTG was 0.9, IPTG was added to the culture medium until the final concentration of IPTG was 1 mM. The expression of streptavidin amino acid sequence SEQ ID NO:3 in competent cells Rosseta was induced at 37℃. The recombinant cells in the culture medium were collected after 5 h.

[0080] (3) The collected recombinant cells were added to the disruption buffer and homogenized under high pressure to break the cells and release streptavidin. The ratio of disruption buffer to recombinant cells was 1 mg of recombinant cells per 20 mL of disruption buffer. The disruption buffer consisted of 22 mM PB and 0.5 M NaCl, and the pH of the disruption buffer was 6.5. The temperature of the high-pressure homogenization was 0 °C and the homogenization pressure was 620 Barg.

[0081] (4) The slurry obtained after high-pressure homogenization was sequentially subjected to NI column affinity chromatography and dialysis to separate purified streptavidin. For NI column affinity chromatography, the loading buffer system was 20 mM PB + 0.5 M NaCl, pH = 6.5; the washing buffer system was 20 mM PB + 0.5 M NaCl + 30 mM imidazole, pH = 6.5; and the elution buffer system was 20 mM PB + 0.5 M NaCl + 300 mM imidazole, pH = 6.5. Dialysis was performed using 20 mM PBS at pH = 6.5.

[0082] (5) The histidine-tagged proteinase K and the hydrogen bromide activated agarose matrix were mixed at a ratio of 8 mg proteinase K per 1 mL of filler and coupled to form a proteinase conjugate. Then, the proteinase conjugate was mixed with the isolated and purified streptavidin at a ratio of 24 mg streptavidin per 1 mL of proteinase conjugate and placed at 20 °C for enzymatic digestion. After 60 min, the proteinase conjugate was removed.

[0083] (6) The enzyme digestion products obtained after removing the protease conjugate were subjected to NI affinity chromatography and benzomidine affinity chromatography in sequence to remove residual protease, histidine tag and cleaved polypeptide fragments, and the flow-through of the chromatography was collected. The loading buffer system for both NI affinity chromatography and benzomidine affinity chromatography was 20 mM PBS at pH 6.5.

[0084] (7) The collected chromatographic flow was dialyzed using a dialysis bag with a molecular weight cutoff of 13 kDa to obtain purified core streptavidin. The dialysate was a phosphate buffer system containing 3 wt% trehalose, wherein the concentration of PB in the phosphate buffer system was 20 mM and the pH value was 6.4.

[0085] Example 2

[0086] A method for preparing core streptavidin is the same as that in Example 1, except that: step (1) involves constructing the streptavidin amino acid sequence SEQ ID NO:2 into the pET28a expression vector, and step (2) involves inducing the expression of the streptavidin amino acid sequence SEQ ID NO:2 in competent cells Rosseta.

[0087] Example 3

[0088] A method for preparing core streptavidin is the same as that in Example 1, except that: step (1) involves constructing the streptavidin amino acid sequence SEQ ID NO:4 into the pET28a expression vector, and step (2) involves inducing the expression of the streptavidin amino acid sequence SEQ ID NO:4 in competent cells Rosseta.

[0089] Example 4

[0090] A method for preparing core streptavidin is the same as that in Example 1, except that: step (1) involves constructing the streptavidin amino acid sequence SEQ ID NO:5 into the pET28a expression vector, and step (2) involves inducing the expression of the streptavidin amino acid sequence SEQ ID NO:5 in competent cells Rosseta.

[0091] Example 5

[0092] A method for preparing core streptavidin is the same as that in Example 1, except that step (5) involves mixing papain with histidine tag with agarose matrix activated by hydrogen bromide and coupling the two to form a protease conjugate.

[0093] Example 6

[0094] A method for preparing core streptavidin is the same as that in Example 1, except that step (5) involves mixing pepsin with histidine tag with agarose matrix activated by hydrogen bromide and coupling the two to form a protease conjugate.

[0095] Example 7

[0096] A method for preparing core streptavidin is the same as that in Example 1, except that step (5) involves mixing a histidine-tagged trypsin with a hydrogen bromide-activated agarose matrix and coupling the two to form a protease conjugate.

[0097] Amino acid sequence analysis was performed on the core streptavidins prepared in Examples 1-7. The results showed that the core streptavidins all included the core streptavidin with the amino acid sequence shown in SEQ ID NO:1.

[0098] Application Examples

[0099] A method for preparing a hepatitis C virus antibody detection kit, comprising using the core streptavidin prepared in Examples 1-7 as the streptavidin component to prepare a hepatitis C virus antibody detection kit containing streptavidin according to conventional hepatitis C virus antibody detection kit production methods.

[0100] Methodological examination and effectiveness verification:

[0101] 1. Comparison of expression results using different streptavidin amino acid sequences as raw materials:

[0102] (1) The SDS-PAGE identification results of purified streptavidin (i.e., the purified streptavidin obtained in Examples 1-4) isolated from recombinant bacteria expressed using streptavidin with sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 are shown in the figure below. Figure 1-2 As shown in the figure, when streptavidin with sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 is used as a raw material for expression, monomeric forms are visible in the purified streptavidin obtained. When streptavidin with sequence SEQ ID NO:3 is used as a raw material for expression, a clear tetramer form is visible in the purified streptavidin obtained, and heating cannot significantly destroy it.

[0103] (2) The core streptavidin (i.e. the core streptavidin obtained in Examples 1-4) was finally prepared by expression using streptavidin with the sequence SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 as raw materials. The activity of the core streptavidin is shown in Table 1 below. As can be seen from Table 1, the core streptavidin prepared by expression using streptavidin with the sequence SEQ ID NO:3 as raw material has the best activity, followed by the core streptavidin prepared by expression using streptavidin with the sequence SEQ ID NO:4 as raw material.

[0104] Table 1. Comparison of the activity of core streptavidin prepared from different raw materials.

[0105]

[0106] The core streptavidin activity in Table 1 was calculated using the following formula (the streptavidin activity data mentioned in this invention are all streptavidin-biotin binding activity detected by the HBAB method, and all given streptavidin (including core streptavidin) activity values ​​were calculated using the following formula):

[0107]

[0108] Where: M: molar mass of d-Biotin (244 g / mole);

[0109] V: Test volume, in liters (0.001L);

[0110] v: Volume of SA (0.05 mL);

[0111] C: Concentration of SA in the sample;

[0112] ε: Molar extinction coefficient of HABA-SA at 500 nm (34500M) -1 ).

[0113] 2. Comparison of enzymatic digestion results using different proteases:

[0114] (1) After isolating and purifying streptavidin from recombinant bacterial cells expressed using streptavidin with the sequence SEQ ID NO:3 as raw material, protease conjugates were prepared using histidine-tagged proteinase K, papain, pepsin, and trypsin, respectively, and then the purified streptavidin was digested with enzymes. The SDS-PAGE identification results of the prepared core streptavidin (i.e., the core streptavidin obtained in Examples 1 and 5-7) are shown in the figure below. Figure 3 As shown in the figure, the core streptavidin obtained by enzymatic digestion of purified streptavidin using the protease conjugates prepared with these four proteases all exhibited obvious tetramer forms. Among them, the core streptavidin obtained by enzymatic digestion of purified streptavidin using the protease conjugate prepared with proteinase K had a clearer background and a lighter background color.

[0115] (2) After purifying streptavidin from recombinant bacterial cells expressed using streptavidin with the sequence SEQ ID NO:3 as raw material, the activities of core streptavidin (i.e., the core streptavidin obtained in Examples 1 and 5-7) prepared by enzymatic digestion of purified streptavidin using proteinase K with histidine tag, papain, pepsin and trypsin respectively are shown in Table 2 below. As can be seen from Table 2, the core streptavidin prepared by enzymatic digestion of purified streptavidin using proteinase K with histidine tag has the highest activity, followed by trypsin.

[0116] Table 2. Comparison of the activities of core streptavidin obtained by enzymatic digestion with different proteases.

[0117]

[0118] 3. Comparison of results with different enzyme digestion times:

[0119] After purifying streptavidin from recombinant bacterial cells expressed using streptavidin (SEQ ID NO:3) as a raw material, a protein conjugate was prepared using a histidine-tagged proteinase K to digest the purified streptavidin. The protein conjugate was removed after digestion for 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively. The SDS-PAGE identification results of the prepared core streptavidin (preparation method as described in Example 1) are shown in the figure below. Figure 4 As shown in the figure, when the enzyme digestion time is 60 min, both monomeric and tetrameric forms of core streptavidin can be clearly seen.

[0120] 4. Comparison of SDS-PAGE identification results of streptavidin at different stages (or different treatments) during the preparation of core streptavidin:

[0121] The SDS-PAGE identification results of streptavidin before enzyme digestion (such as the purified streptavidin obtained by separation in step (4) of Example 1), streptavidin without removal of protease and cleaved polypeptide fragments after enzyme digestion (such as the enzyme digestion product obtained by removing the protease conjugate in step (5) of Example 1), streptavidin with removal of cleaved polypeptide fragments but without removal of protease after enzyme digestion (such as streptavidin obtained by NI affinity chromatography but not yet by benzalkonium affinity chromatography in step (6) of Example 1), and streptavidin with removal of protease and cleaved polypeptide fragments after enzyme digestion (such as streptavidin obtained by NI affinity chromatography and benzalkonium affinity chromatography in step (6) of Example 1) are as follows: Figure 5 The results of the activity comparison are shown in Table 3; from Figure 5 As shown in Table 3, after enzymatic digestion, both the protease and the cleaved polypeptide fragments affect the activity of streptavidin. Moreover, the activity of the pure core streptavidin obtained after enzymatic digestion is higher than that of the streptavidin before enzymatic digestion.

[0122] Table 3 Comparison of the activities of different streptavidins

[0123]

[0124] 5. Investigation of the stability and storage methods of core streptavidin

[0125] (1) Study on preservation stability

[0126] The activity of the core streptavidin prepared in Example 1 of this invention and the streptavidin purchased from Thermo (Thermo SA) was first tested separately. Then, they were dispersed in a phosphate buffer system containing 3 wt% trehalose and stored at 2-8°C for 9 months. Their activity was tested again. The results are shown in Table 4 below. It can be seen from the results in Table 4 that the core streptavidin prepared in this invention can maintain long-term stable activity and is not easily inactivated when stored at low temperature under the protection of the protective agent.

[0127] Table 4 Results of the preservation stability study

[0128]

[0129] (2) Study on preservation and preservation agents

[0130] The core streptavidin prepared in Example 1 of this invention was lyophilized using a phosphate buffer system containing a preservative as the lyophilization protectant. The types and mass concentrations of the preservatives in each phosphate buffer system were 2 wt% sorbitol, 0.5 wt% ethylene glycol, 5 wt% glycerol, 3 wt% trehalose, 3 wt% sucrose, and 3 wt% lactose, respectively. After lyophilization for a period of time, the solution was reconstituted and then placed at 2-8°C for 9 months. Images of each solution are shown below. Figure 6 As shown in Table 5, the results of detecting the activity of core streptavidin are presented below. The results in Table 5, combined with... Figure 6 It can be seen that the core streptavidin prepared by this invention can maintain long-term activity stability under the protection of the protective agent. In particular, when the protective agent is a phosphate buffer system containing 0.5 wt% ethylene glycol, a phosphate buffer system containing 3 wt% trehalose, or a phosphate buffer system containing 3 wt% sucrose, the core streptavidin has high activity and good solubility.

[0131] Table 5 Results of the study on preservation and protective agents

[0132]

[0133] 6. Comparison of binding capacity between core streptavidin and protein biotin.

[0134] The results of comparing the core streptavidin prepared in Example 1 of this invention with streptavidin purchased from Thermo, Agilent, and Invitrogen are as follows: Figure 7 And as shown in Table 6. From Figure 7 It can be seen that the tetramer form of the core streptavidin of this invention is more pronounced, and the CSA content is higher. Table 6 shows that the core streptavidin of this invention exhibits significantly stronger activity in binding with biotin.

[0135] Table 6. Comparison of binding affinity between core streptavidin and proteokinin.

[0136] B (500nm absorption) 0.52354 0.51823 0.46742 0.46187 0.51522 Activity U / mg 16.93410 17.93661 14.99676 15.66369 16.49841 Streptavidin sample source Thermo CSA This invention Agilent SA10 Agilent SA26 Invitrogen CSA

[0137] In addition, the purity comparison results of the core streptavidin prepared in Example 1 of this invention with streptavidin purchased from Thermo, Agilent, and Invitrogen are as follows: Figure 8-12 As shown, Figure 8 The purity of CSA in the core streptavidin prepared in Example 1 of this invention is 100.00%. Figure 9 The purity of CSA in the streptavidin (Thermo CSA) purchased from Thermo was 94.75%. Figure 10The purity of CSA in the streptavidin (Invitrogen CSA) purchased from Invitrogen was 93.89%. Figure 11 The purity of CSA in the streptavidin (Agilent SA10) purchased from Agilent is 77.99%. Figure 12 The purity of CSA in the streptavidin (Agilent SA26) purchased from Agilent was 86.85%. Based on the purity comparison, the core streptavidin prepared in Example 1 of this invention has a higher purity of CSA. Furthermore, the comparison of binding activity results suggests that the binding activity of the core streptavidin to biotin may be related to the purity of CSA.

[0138] 7. Comparison of biotin binding capacity between core streptavidin-coated magnetic beads and biotin.

[0139] The magnetic beads coated with the core streptavidin prepared in Example 1 of this invention were compared with Dynal magnetic beads. The biotinylation ability of the two types of magnetic beads was determined by the biotinylated acridinium ester method and the fluorescent biotin titration method, respectively. The results are shown in Tables 7 and 8. As can be seen from Tables 7 and 8, the magnetic beads coated with the core streptavidin of this invention have a significantly stronger biotin-binding ability.

[0140] Table 7 shows the results determined using the biotinylated acridinium ester method.

[0141]

[0142] Table 8 shows the results determined by the fluorescent biotin titration method.

[0143] Magnetic bead concentration (mg / mL) 0.15 0.2 Magnetic bead volume (mL) 0.1 0.1 B4F concentration (nM) 97.2 137.8 Soln volume (mL) 0.2 0.2 Biotin binding site (pmol / mg) 1236 1378

[0144] 8. Evaluation of the effectiveness of core streptavidin in hepatitis C virus antibody detection kit

[0145] (1) Examination of long-term stability

[0146] Referring to the formulation of the Hepatitis C Virus Antibody Detection Kit (Direct Chemiluminescence Method) (National Medical Device Registration Certificate 20183400326) already sold by Mindray Bio-Medical Electronics Co., Ltd., streptavidin purchased from Thermo (Thermo SA) and the core streptavidin prepared in Example 1 of this invention were used as the streptavidin for preparing the kits. The prepared kits were stored at a constant temperature of 4°C. Three kits from each of the two types were taken at days 1, 7, 14, and 90 for Hepatitis C virus antibody detection. The results are shown in Table 9. As can be seen from Table 9, the kit prepared using the core streptavidin of this invention as the streptavidin for the kit has a higher signal retention rate, indicating that the core streptavidin of this invention has better long-term stability.

[0147] Table 9 Results of Long-Term Stability Assessment

[0148]

[0149] (2) Stability assessment

[0150] Referring to the formulation of the Hepatitis C Virus Antibody Detection Kit (Direct Chemiluminescence Method) (National Medical Device Registration Certificate 20183400326) sold by Mindray Bio-Medical Electronics Co., Ltd., streptavidin (Thermo SA) purchased from Thermo and the core streptavidin prepared in Examples 1 and 3 of this invention were used as the streptavidin preparation kits. The prepared kits were temporarily stored at a constant temperature of 4°C. A batch of kits was taken from each kit to examine their signal retention rate after being placed in a water bath at 37°C and an air bath at 37°C for 7 days and 14 days, respectively. The results are shown in Table 10. The signal value of the kits was measured before the water bath or air bath as a control (i.e., the 4°C control in Table 10). As can be seen from Table 10, the kits have good signal retention rates in both the water bath and air bath at 37°C, that is, the core streptavidin in Examples 1 and 3 of this invention has good stability.

[0151] Table 10 Results of the stability test

[0152]

[0153] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications having the same function.

Claims

1. A method for preparing core streptavidin, characterized in that, The preparation method includes the following steps: (1) Using recombinant bacteria to express streptavidin; (2) Streptavidin was digested using immobilized enzyme technology; Step (1) includes using genetic engineering technology to construct the nucleic acid sequence of streptavidin into the pET28a expression vector, then transforming the expression vector into Rosseta competent cells of Escherichia coli, culturing the cells, collecting recombinant bacterial cells, and isolating streptavidin from the recombinant bacterial cells; the amino acid sequence corresponding to the nucleic acid sequence of streptavidin constructed into the pET28a expression vector is selected from any one of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; Step (2) includes coupling the protease with the filler to form a protease conjugate, then performing an enzymatic digestion reaction with streptavidin at 16-25°C, and then removing the protease conjugate; Step (2) also includes sequentially performing NI affinity chromatography and benzalkonium chloride affinity chromatography on the product obtained after removing the protease conjugate following the enzymatic digestion reaction, and then collecting the flow-through of the chromatography for dialysis; When the amino acid sequence corresponding to the nucleic acid sequence of streptavidin constructed into the pET28a expression vector is SEQ ID NO:3, the protease is selected from one or more of proteinase K, pepsin, papain, and trypsin; When the amino acid sequence corresponding to the nucleic acid sequence of streptavidin constructed into the pET28a expression vector is any one of SEQ ID NO:4 and SEQ ID NO:5, the protease is proteinase K.

2. The preparation method according to claim 1, characterized in that, The cell culture includes: culturing to OD. 600 When the concentration is 0.6 to 1.2, add IPTG as an inducer to a final concentration of 1 mM and induce at 36 to 38°C for 4 to 6 hours.

3. The preparation method according to claim 2, characterized in that, Step (1) further includes adding the collected recombinant cells to the disruption buffer for high-pressure homogenization to break the cells and release streptavidin, and then performing NI column affinity chromatography and dialysis in sequence to obtain purified streptavidin.

4. The preparation method according to claim 3, characterized in that, The composition of the lysis buffer includes: 18–22 mMPB, 0.2–0.8 M NaCl, and the pH of the lysis buffer is 6.2–6.8; And / or, the temperature of the high-pressure homogenizer is -2 to 2℃, and the homogenizing pressure is 550 to 700 Barg; And / or, in step (1), during NI column affinity chromatography, the loading buffer system is 18–22 mM PB + 0.2–0.8 M NaCl, pH = 6.2–6.8; the washing buffer system is 18–22 mM PB + 0.2–0.8 M NaCl + 28–32 mM imidazole, pH = 6.2–6.8; the elution buffer system is 18–22 mM PB + 0.2–0.8 M NaCl + 250–350 mM imidazole, pH = 6.2–6.8; And / or, the dialysis described in step (1) is performed using 18-22 mM PBS with pH 6.2-6.

8.

5. The preparation method according to claim 3, characterized in that, The ratio of lysis buffer to recombinant bacterial cells is 1 mg of recombinant bacterial cells per 15-25 mL of lysis buffer.

6. The preparation method according to any one of claims 1-5, characterized in that, The filler is a hydrogen bromide activated and / or NHS activated agarose matrix; And / or, during the enzymatic digestion reaction, the ratio of protease conjugate to streptavidin is 22-25 mg streptavidin per 1 mL of protease conjugate, and the digestion time is 50-70 min.

7. The preparation method according to claim 6, characterized in that, The protease is tagged with histidine.

8. The preparation method according to claim 7, characterized in that, The ratio of protease to filler material is 5-10 mg of protease per 1 mL of filler material.

9. The preparation method according to any one of claims 1-5 and 7-8, characterized in that, In step (2), the loading buffer system for NI affinity chromatography and benzalkonium chloride affinity chromatography is 18-22 mM PBS with pH = 6.2-6.8; And / or, in step (2), the collected chromatographic flow is dialyzed using a dialysis bag with a molecular weight cutoff of 12-14 kDa.

10. The preparation method according to claim 9, characterized in that, Dialysis fluid is a mixed solution formed by adding one or more of sucrose, trehalose, lactose, and ethylene glycol to a phosphate buffer system.

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