New use of cytochrome C in preparing ELISA kit

Abstract

The application discloses a new use of cytochrome C in preparing an ELISA kit and relates to the field of biotechnology. The application uses cytochrome C (Cytochrome c, CC) and streptavidin (Streptavidin, SA) to construct a fusion protein, and the fusion protein has a good detection effect in an ELISA experiment. The fusion protein can simplify a production process, reduce production cost and has a good market conversion prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel use of cytochrome C in the preparation of ELISA kits. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) is currently the most widely used immunoenzymatic technique for the qualitative and quantitative detection of antibodies (antigens). Antigens or antibodies immobilized on the surface of a carrier do not become inactive and retain their immunoreactivity. Enzyme-labeled antibodies or antigens possess both immunological and enzymatic catalytic activity. In actual detection, the antigen or antibody is first immobilized on a solid-phase carrier. Then, the analyte is added, allowing it to bind to the immobilized carrier, forming a solid-phase antigen-antibody complex. Next, enzyme-labeled antibodies or antigens are added, binding to the solid-phase complex to form an enzyme-labeled complex. Finally, a substrate solution is added, triggering an enzyme-catalyzed colorimetric reaction. Qualitative or quantitative analysis is then performed based on the color intensity.

[0003] Double-antibody sandwich ELISA kits are currently the most commonly used type on the market. Their principle is as follows: Figure 1 As shown, its components include a fixed primary antibody, a biotin-labeled secondary antibody, horseradish peroxidase (HRP)-labeled streptavidin (HRP-SA), a chromogenic solution, and a stop solution. HRP-SA is typically composed of SA in the covalently coupled form of HRP. SA can bind biotin, and the coupled HRP polymer provides a high level of enzyme activity, thus facilitating detection using an appropriate substrate system.

[0004] The preparation process of HRP-SA is as follows: Figure 2 As shown, the process typically involves four steps: 1. Extracting and purifying SA from Streptomyces avicenniatum; 2. Extracting and purifying HRP from horseradish; 3. Reacting SA and HRP to form a complex using chemical cross-linking agents such as sodium iodate or glutaraldehyde; 4. Purifying to remove free HRP and SA, yielding a high-purity HRP-SA complex. In this production process, extracting the corresponding proteins from Streptomyces and horseradish separately requires significant economic resources. The subsequent cross-linking reaction and purification processes are cumbersome, reducing production efficiency. Therefore, the entire HRP-SA preparation process incurs substantial costs in terms of both cost and labor. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a novel use of cytochrome c in the preparation of ELISA kits. This invention connects cytochrome c (CC) and streptavidin (SA) via a short polypeptide segment to form a fusion protein, such as... Figure 3 As shown, this fusion protein possesses both a CC domain, exhibiting high peroxidase activity and the ability to catalyze the oxidation of substrates such as luminol to produce a colorimetric reaction; and an SA domain, demonstrating a strong binding affinity for biotin. The preparation of an ELISA kit using the recombinant CC-SA fusion protein of this invention simplifies the preparation process, reduces production costs, improves production efficiency, and offers high sensitivity, demonstrating promising market application prospects.

[0006] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows:

[0007] A novel use of cytochrome C in the preparation of ELISA kits.

[0008] A recombinant CC-SA fusion protein, wherein the recombinant CC-SA fusion protein is formed by the linkage of cytochrome C and streptavidin via a polypeptide, and its amino acid sequence is shown in SEQ ID NO.1.

[0009] A gene encoding a recombinant CC-SA fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] A method for preparing a recombinant CC-SA fusion protein includes the following steps:

[0011] S1. Based on the amino acid sequence of the recombinant CC-SA fusion protein, synthesize the encoding gene of the recombinant CC-SA fusion protein using gene amplification technology;

[0012] S2. Insert the coding gene of the recombinant CC-SA fusion protein into the expression vector for recombination to obtain the recombinant plasmid;

[0013] S3. The recombinant plasmid was transferred into competent cells, and expression and culture were induced by isopropyl-β-D-thiogalactoside IPTG. After purification, the recombinant CC-SA fusion protein was obtained.

[0014] Furthermore, the expression vector is selected from pET-32a(+) vector, and the competent cells are selected from BL21(DE3) competent cells.

[0015] Application of a recombinant CC-SA fusion protein in the preparation of an ELISA kit. Compared with the prior art, the advantages and beneficial effects of this invention are as follows:

[0016] 1. This invention proposes a new use of cytochrome c in the preparation of ELISA kits. A fusion protein is constructed by using cytochrome c (CC) and streptavidin (SA). This fusion protein has the functions of catalyzing the oxidation of recombinant cytochrome c and binding biotin to SA without interference. It can fully exert its effects in ELISA experiments, and there is no possibility of the two domains being broken, so the structure is stable.

[0017] 2. The recombinant CC-SA fusion protein of the present invention is expressed through competent cells, requiring only simple culture and extraction. The economic and labor costs are far lower than those of existing products on the market, and it has a very good market prospect.

[0018] 3. The ELISA kit prepared using the recombinant CC-SA fusion protein of the present invention not only has comparable detection effect compared with the traditional ELISA kit prepared by HRP-SA, but also eliminates the need for redundant cross-linking and other preparation processes, greatly simplifying the preparation process, significantly reducing production costs, and improving production efficiency, thus showing great market application prospects. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the detection principle of a double-antibody sandwich ELISA kit.

[0020] Figure 2 A comparison of the preparation flowcharts for HRP-SA and CC-SA.

[0021] Figure 3 This is a schematic diagram of the structural construction of the recombinant CC-SA fusion protein in Example 1.

[0022] Figure 4 This is a plasmid diagram of the recombinant CC-SA fusion protein from Example 1.

[0023] Figure 5 This is an SDS-PAGE gel electrophoresis image of the recombinant CC-SA prepared in Example 1.

[0024] Figure 6 The image shows the effect of an ELISA kit prepared using the recombinant CC-SA fusion protein from Example 1 on detecting human angiotensin-converting enzyme (ACE).

[0025] Figure 7 The image shows the effect of the ELISA kit prepared with the recombinant CC-SA fusion protein of Example 1 on detecting human complement system 3a protein standard (C3a).

[0026] Figure 8 The image shows the effect of an ELISA kit prepared using the recombinant CC-SA fusion protein from Example 1 to detect C3a protein in the culture supernatant of human cervical cancer cells (HeLa). Detailed Implementation

[0027] To facilitate understanding and implementation of the present invention by those skilled in the art, the present invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described herein are for illustration and explanation only and are not intended to limit the present invention. Unless otherwise specified, the reagents and biological materials used in the embodiments of the present invention were commercially available. Methods without specific experimental conditions are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or as recommended by the equipment manufacturer.

[0028] Example 1

[0029] This embodiment compares the preparation method using recombinant CC-SA fusion protein, as follows:

[0030] 1. Construction of recombinant CC-SA fusion protein particle:

[0031] Recombinant CC-SA fusion protein particle design, such as Figure 4 As shown, the amino acid sequences of the target proteins CC and SA were obtained by searching the NCBI database. The CC-SA gene was synthesized by Aoke (Wuhan) Biotechnology Co., Ltd., and restriction endonucleases HindIII and NdeI were introduced at both ends for digestion.

[0032] First, the target gene and the empty vector pET-21(a) plasmid were digested with restriction endonucleases HindIII and NdeI at 37℃ for 2 h. The digestion conditions are shown in Table 1.

[0033] Table 1 Restriction endonuclease digestion system

[0034]

[0035] The fragments were then separated by agarose gel electrophoresis, and the target fragment was recovered using a gel extraction kit. The two fragments were then mixed, and T4 ligase was added for ligation for 10 hours to obtain the recombinant CC-SA plasmid. The enzyme ligation system is shown in Table 2.

[0036] Table 2 Enzyme ligation system

[0037]

[0038] 2. Expression of recombinant CC-SA fusion protein:

[0039] Add 1-2 μL of the recombinant CC-SA fusion protein plasmid to the prepared BL21(DE3) competent cells, freeze for 30 min, then heat shock in a 42℃ water bath for 90 s, and then freeze again on ice for 1 min. Add 500 μL of liquid LB medium, then incubate on a shaker at 220 rpm and 37℃ for 30 min. After incubation, evenly spread the BL21(DE3) competent cells onto solid agar plates containing ampicillin (Amp), and then incubate at 37℃ for 8-10 h. After single colonies grow to visible size on solid agar plates, pick a single colony and transfer it to 5 mL of LB liquid medium containing Amp resistance. Place the plate on a shaker and incubate at 220 rpm and 37°C for 8-10 hours. Transfer the bacterial culture to 1 L of LB liquid medium containing Amp resistance and incubate again at 220 rpm and 37°C for 8-10 hours. When the OD600 of the bacterial culture reaches 0.8-1.0, add IPTG to a final concentration of 0.1 mM and continue incubating at 60 rpm and 30°C for 18-24 hours. Since CC-SA is red, when it is overexpressed in BL21(DE3) competent cells, the cells will turn red. When the cells are clearly reddened, it indicates successful expression and the cells can be harvested. Harvesting conditions: centrifuge at 6000 rpm for 15 minutes at 4°C.

[0040] 3. Extraction of recombinant CC-SA fusion protein:

[0041] The centrifuged bacterial cells were resuspended in 50 mL of Lysis Buffer (50 mM Tris, 5 mM EDTA), followed by the addition of lysozyme (1.5 mg / mL wet cells) and 5 μL of DNase, and digested on a shaker for 1 h. The cells were then lysed using a high-pressure autoclave. The lysed cells were centrifuged at 4 °C and 20,000 rpm for 30 min, and the supernatant was collected. Ammonium sulfate (150 g / L) was added for salting out for 3-6 h. The salted-out sample was then centrifuged at 4 °C and 20,000 rpm for 30 min, and the supernatant was collected and sealed in a 0.22 μm dialysis bag. The bag was then dialyzed in dialysis buffer (5 L 20 mM PB Na) for 3-6 h. The dialyzed sample was then filtered through a 0.22 μm filter membrane, and the resulting filtrate was the crude extract of the target protein.

[0042] 4. Purification of recombinant CC-SA fusion protein:

[0043] The first step of protein purification used a Hitrap SP HP cation exchange column. Elution was performed using a gradient buffer containing 20 mM PB Na and 1 M NaCl (pH 7.0) to obtain a crude extract of the target protein (the target protein is red; collect the red sample). After elution, the extract was concentrated to 5 mL using a 30 kDa molecular weight cutoff tube. This purified protein was used for the second purification step. The second purification step used a HiLoad Superdex 200 pg preparative size exclusion column. Elution was performed using a 20 mM PB Na and 150 mM NaCl (pH 7.0) buffer to obtain a purified protein extract. The purified protein was then washed three times with ddH2O using a 30 kDa molecular weight cutoff tube to remove salts and lyophilized at -80°C. Finally, the purified target protein was analyzed and identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 5 As shown, the band on the gel image is approximately 30 kDa, consistent with its theoretical molecular weight, indicating successful protein expression.

[0044] Example 2

[0045] This example compares the detection of rabbit angiotensin-converting enzyme (ACE) using a recombinant CC-SA fusion protein and an ELISA kit prepared using HRP-SA (Shanghai Beyotime Biotechnology Co., Ltd.) from a commercial kit. The specific methods are as follows:

[0046] 1. Dilute the ACE standard 30-fold with PBS buffer to obtain the ACE working solution. Add 100 μL of the ACE working solution to each of the three wells of an ELISA plate. Pour the ACE working solution into the bottom of the wells, avoiding contact with the well walls as much as possible. Gently shake to mix, and finally seal the wells with a clear sealing film. Set up three groups of standard wells as described above: one control group and two experimental groups, with three standard wells in each group.

[0047] 2. Incubate the microplates treated in step 1 at room temperature for 120 minutes. After incubation, wash the plates 5 times and finally place each microplate on thick absorbent paper to dry.

[0048] 3. Add biotin-labeled ACE antibody (100 μL / well) to each of the three sets of standard wells, and seal each standard well with a sealing film (transparent).

[0049] 4. Incubate the microplates treated in step 3 at room temperature for 60 minutes. After incubation, wash the plates 5 times and finally place each microplate on thick absorbent paper to dry.

[0050] 5. Dissolve HRP-SA and CC-SA separately in 20mM PBS buffer to prepare 0.1mM HRP-SA solution and HRP-SA solution respectively. Add 0.1mM HRP-SA solution and CC-SA solution (100μL / well) to the standard wells of the two experimental groups respectively, and seal the standard wells of the two experimental groups with white sealing film.

[0051] 6. Incubate the three standard wells of each group at room temperature in the dark for 20 min, 40 min and 60 min respectively. After incubation, wash the plate 5 times and finally place the microplate on thick absorbent paper to dry.

[0052] 7. Add TMB solution (100 μL / well) to each well of the standard sample treated in step 6, seal each well with white sealing film, and incubate at room temperature in the dark for 18 min.

[0053] 8. Add 50 μL of stop solution (2M H2SO4 solution) to each well of the standard sample treated in step 7, mix well, and immediately measure the absorbance of ACE in the well of the standard sample and read the A450 value. Based on the A450 values ​​of each group of standard wells measured after incubation for 20 min, 40 min, and 60 min, compare the stability of rabbit angiotensin-converting enzyme (ACE) in the two experimental groups.

[0054] 9. The A450 values ​​of ACE standards detected by the ELISA kit prepared with recombinant CC-SA fusion protein and the commercially available HRP-SA ELISA kit are as follows: Figure 6 As shown, within the same incubation time, the ELISA kit prepared with the recombinant CC-SA fusion protein of the present invention and the ELISA kit prepared with commercial HRP-SA achieved comparable testing results. In fact, within a shorter incubation time, the ELISA kit prepared with the recombinant CC-SA fusion protein of the present invention achieved better testing results.

[0055] Example 3

[0056] This embodiment utilizes an ELISA kit prepared from recombinant CC-SA fusion protein to detect human complement system 3a (C3a) standards. The specific method is as follows:

[0057] 1. Dilute the C3a standard 30 times with PBS diluent to obtain the C3a working solution. Add 100 μL of the C3a working solution to each well of the microplate, ensuring it doesn't touch the well walls. Gently shake to mix, and finally seal with a clear sealing film. Set up two groups of standard wells as described above: the first group is the experimental group, and the other is the control group.

[0058] 2. Incubate the microplates treated in step 1 at room temperature for 120 minutes. After incubation, wash the plates 5 times and finally place each microplate on thick absorbent paper to dry.

[0059] 3. Add biotin-labeled C3a antibody (100 μL / well) to each of the two sets of standard wells, and seal each standard well with a transparent sealing film.

[0060] 4. Incubate the microplates treated in step 3 at room temperature for 60 minutes. After incubation, wash the plates 5 times and finally place each microplate on thick absorbent paper to dry.

[0061] 5. Dissolve CC-SA in 20mM PBS to prepare a 0.1mM CC-SA solution. Add 0.1mM CC-SA solution (100μL / well) to the wells of the experimental group standard and seal the wells of the experimental group standard with white sealing film.

[0062] 6. Incubate the microplates treated in step 5 at room temperature in the dark for 20 minutes. After incubation, wash the plates 5 times and finally place each microplate on thick absorbent paper to dry.

[0063] 7. Add TMB solution (100 μL / well) to each of the two sets of standard wells, seal each well with white sealing film, and incubate at room temperature in the dark for 18 min.

[0064] 8. Add 50 μL of stop solution (2M H2SO4 solution) to each of the two sets of standard wells, mix well, and immediately measure the absorbance of ACE in each standard well and read the A450 value.

[0065] The A450 value of standard C3a was detected using an ELISA kit prepared with recombinant CC-SA fusion protein. The results are as follows: Figure 7 As shown, compared with the control group, the ELISA kit prepared based on the recombinant CC-SA fusion protein of this invention produced a significant A450 value within the same incubation time, indicating that the standard C3a protein was successfully detected.

[0066] Example 4

[0067] This embodiment utilizes an ELISA kit based on recombinant CC-SA fusion protein to detect C3a protein in HeLa cell culture supernatant. The specific method is as follows:

[0068] 1. Pipe 100 μL of HeLa cell culture supernatant into each well of an ELISA plate. Add the supernatant to the bottom of each well, being careful not to let it touch the well walls. Gently shake to mix, and then seal the wells with a clear sealing film. Set up two sets of sample wells as described above: one experimental group and the other a control group.

[0069] 2. Incubate the microplate treated in step 1 at room temperature for 120 minutes. After incubation, wash the plate 5 times and finally place it on thick absorbent paper to dry.

[0070] 3. Add biotin-labeled C3a antibody (100 μL / well) to each of the two sets of actual sample wells, and seal each actual sample well with a transparent sealing film.

[0071] 4. Incubate the microplate treated in step 4 at room temperature for 60 minutes. After incubation, wash the plate 5 times and finally place it on thick absorbent paper to dry.

[0072] 5. Dissolve CC-SA in 20mM PBS to prepare a 0.1mM CC-SA solution. Add 0.1mM CC-SA solution (100μL / well) to the actual sample wells of the experimental group and seal the actual sample wells of the experimental group with white sealing film.

[0073] 6. After incubating the microplates treated in step 5 at room temperature in the dark for 20 minutes, wash the plates 5 times after incubation, and finally place each microplate on thick absorbent paper to dry.

[0074] 7. Add TMB solution (100 μL / well) to each of the two sets of actual sample wells, seal each actual sample well with white sealing film, and incubate at room temperature in the dark for 18 min.

[0075] 8. Add 50 μL of the stop solution (2M H2SO4 solution) to each of the two sets of actual samples, mix well, and immediately measure the absorbance and read the A450 value.

[0076] The A450 value of C3a protein in HeLa cell culture supernatant was detected using an ELISA kit prepared with recombinant CC-SA fusion protein. The results are as follows: Figure 8 As shown, compared with the control group, the ELISA kit prepared based on the recombinant CC-SA fusion protein of this invention produced a significant A450 absorbance value within the same incubation time, indicating that the C3a protein in the HeLa cell culture supernatant was successfully detected.

Claims

1. A recombinant CC-SA fusion protein, characterized in that: The recombinant CC-SA fusion protein is formed by linking CC and SA via a polypeptide, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The encoding gene of the recombinant CC-SA fusion protein according to claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A method for preparing the recombinant CC-SA fusion protein according to claim 1, characterized in that... Includes the following steps: S1. Based on the amino acid sequence of the recombinant CC-SA fusion protein, synthesize the encoding gene of the recombinant CC-SA fusion protein using gene amplification technology; S2. Insert the coding gene of the recombinant CC-SA fusion protein into the expression vector for recombination to obtain the recombinant plasmid; S3. The recombinant plasmid was transferred into competent cells, and expression and culture were induced by isopropyl-β-D-thiogalactoside IPTG. After purification, the recombinant CC-SA fusion protein was obtained.

4. The method for preparing the recombinant CC-SA fusion protein according to claim 3, characterized in that: The expression vector was selected from pET-32a(+) vector, and the competent cells were selected from BL21(DE3) competent cells.

5. The use of the recombinant CC-SA fusion protein according to claim 1 in the preparation of an ELISA kit.

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