New application of cytochrome C in preparation of ELISA kit

Abstract

The invention discloses a novel application of cytochrome C in preparation of an ELISA kit, and relates to the technical field of biology. According to the present invention, the cytochrome C (Cytochrome c, CC) and the streptavidin (SA) are adopted to construct the fusion protein, and the good detection effect is provided in the ELISA experiment. By utilizing the fusion protein, the production process can be simplified, the production cost can be reduced, and the fusion protein has a very good market transformation prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a new use of cytochrome C in preparing an ELISA kit. Background Art

[0002] Enzyme-linked immunosorbent assay (ELISA) is currently the most widely used immunoenzyme technology for qualitative and quantitative detection of antibodies (antigens). The antigens or antibodies fixed on the surface of the carrier will not be inactivated and will retain their immunoreactivity. The enzyme-labeled antibody or antigen has both immunological activity and enzyme catalytic activity. In actual detection, the antigen or antibody is first fixed on a solid phase carrier, and then the test object is added to allow the test substance to bind to the solid-phase substance to form a solid-phase "antigen-antibody" complex. Then, the enzyme-labeled antibody or antigen is added to bind to the solid-phase complex to form an enzyme-labeled complex. Finally, the substrate solution is added to cause an enzyme-catalyzed substrate color development reaction, and qualitative or quantitative analysis is performed based on the color depth.

[0003] The double antibody sandwich ELISA kit is the most commonly used one on the market. Figure 1 As shown, its composition includes fixed primary antibody, biotin-labeled secondary antibody, horseradish peroxidase (HRP)-labeled streptavidin (HRP-SA), color development solution, stop solution, etc. Among them, HRP-SA is usually composed of SA covalently coupled to polymerized HRP, SA can bind to biotin, and the coupled HRP polymer can provide a higher level of enzyme activity, so that it is convenient to use an appropriate substrate system for detection.

[0004] The preparation process of HRP-SA is as follows Figure 2 As shown, it usually includes 4 steps: 1. Extracting SA from Streptomyces avicllrdi and purifying it; 2. Extracting HRP from horseradish and purifying it; 3. Reacting SA and HRP to form a complex through chemical cross-linking reagents such as sodium iodate or glutaraldehyde; 4. Removing free HRP and SA through purification to obtain a high-purity HRP-SA complex. In this production process, the corresponding proteins are extracted from Streptomyces and horseradish respectively, which requires a large economic cost. The subsequent cross-linking reaction and purification and other production processes are cumbersome, which reduces the production efficiency. Therefore, the preparation process cost and labor cost of the entire HRP-SA are extremely high. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a new use of cytochrome c in preparing an ELISA kit. In the present invention, cytochrome c (CC) and streptavidin (SA) are connected via a short polypeptide to form a fusion protein, such as Figure 3 As shown. The fusion protein has both a CC domain, which has high peroxidase activity and can catalyze substrates such as luminol to produce a color reaction by oxidation; and an SA domain, which has a strong binding ability with biotin. The use of the recombinant CC-SA fusion protein of the present invention to prepare an ELISA kit simplifies the preparation process, reduces production costs, improves production efficiency, and has high sensitivity, and has a good market application prospect.

[0006] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0007] New use of cytochrome C in preparing ELISA kits.

[0008] A recombinant CC-SA fusion protein, wherein the recombinant CC-SA fusion protein is formed by connecting cytochrome C and streptavidin through 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 the gene encoding is shown in SEQ ID NO.2.

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

[0011] S1. According to the amino acid sequence of the recombinant CC-SA fusion protein, the coding gene of the recombinant CC-SA fusion protein is synthesized by gene amplification technology;

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

[0013] S3. The recombinant plasmid is transferred into competent cells, and isopropyl-β-D-thiogalactoside IPTG is used to induce expression and culture, and then purified to obtain the recombinant CC-SA fusion protein.

[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 the present invention are:

[0016] 1. The present invention proposes a new use of cytochrome C in the preparation of ELISA kits, and uses cytochrome C (Cytochrome c, CC) and streptavidin (Streptavidin, SA) to construct a fusion protein. The fusion protein has the functions of recombinant cytochrome C catalytic oxidation and SA binding to biotin, and does not interfere with each other. It can fully exert its efficacy in ELISA experiments, and the two domains are unlikely to be disconnected, and the structure is stable.

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

[0018] 3. The ELISA kit prepared by using the recombinant CC-SA fusion protein of the present invention not only has equivalent detection effect compared with the traditional ELISA kit prepared by HRP-SA, but also does not require redundant preparation processes such as cross-linking, which greatly simplifies the preparation process, greatly reduces production costs, improves production efficiency, and has a good market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2 Comparison of the preparation flow charts of HRP-SA and CC-SA.

[0021] Figure 3 Schematic diagram of the structural construction of the recombinant CC-SA fusion protein of Example 1.

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

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

[0024] Figure 6 This is a diagram showing the effect of detecting human angiotensin converting enzyme (ACE) using an ELISA kit prepared with the recombinant CC-SA fusion protein of Example 1.

[0025] Figure 7 This is a diagram showing the effect of using the ELISA kit prepared with the recombinant CC-SA fusion protein of Example 1 to detect human complement system 3a protein standard (Complement system 3a, C3a).

[0026] Figure 8 This is a diagram showing the effect of using the ELISA kit prepared with the recombinant CC-SA fusion protein of Example 1 to detect C3a protein in the culture supernatant of human cervical cancer cells (Hela). DETAILED DESCRIPTION

[0027] In order to facilitate the understanding and implementation of the present invention by those of ordinary skill in the art, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention. Unless otherwise specified, the reagents and biological raw materials in the embodiments of the present invention are purchased commercially. Among them, the methods for which specific experimental conditions are not indicated are usually carried out according to conventional conditions such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the equipment manufacturer.

[0028] Example 1

[0029] This example compares the preparation of recombinant CC-SA fusion protein, and the specific method is as follows:

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

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

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

[0033] Table 1 Restriction endonuclease digestion system

[0034]

[0035] Then, the fragments were separated by agarose gel electrophoresis, and the target fragment was recovered by gel recovery kit, and then the two were mixed and ligated with T4 ligase for 10 hours to finally 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] 1-2 μL of the recombinant CC-SA fusion protein plasmid was added to the prepared BL21 (DE3) competent cells, ice-shocked for 30 minutes, taken out and placed in a 42°C water bath for heat shock for 90 seconds, and then placed back on ice for ice shock for 1 minute, followed by adding 500 μL of liquid LB medium, and then placed on a shaker, and cultured at 220 rpm and 37°C for 30 minutes. After the culture, the BL21 (DE3) competent cells were evenly spread on a solid plate culture medium containing ampicillin (Ampicillin, Amp), and then placed in an incubator and continued to be cultured at 37°C for 8-10 hours. After a single colony visible to the naked eye grows on the solid plate culture medium, pick up a single colony and place it in 5 mL of LB liquid culture medium containing Amp resistance, place it on a shaker, and culture it at a speed of 220 rpm and 37°C for 8-10 hours. Transfer the bacterial solution to 1L of LB liquid culture medium containing Amp resistance, then place it on a shaker, and culture it at a speed of 220 rpm and 37°C for 8-10 hours. When the OD600 of the bacterial solution reaches 0.8-1.0, add IPTG with a final concentration of 0.1 mM to the bacterial solution, and continue to culture it at a speed of 60 rpm and 30°C for 18-24 hours. Since CC-SA is red, when it is overexpressed in BL21 (DE3) competent cells, the bacteria will turn red. When the bacteria can be observed to turn red with the naked eye, it means that the expression is successful and the bacteria can be harvested. The conditions for harvesting bacteria are: centrifugation at 6000 rpm for 15 minutes at 4°C.

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

[0041] After centrifugation, the bacteria were resuspended with 50mL Lysis Buffer (50mM Tris, 5mM EDTA), and then lysozyme (1.5mg / mL wet bacteria) and 5μL DNA enzyme were added, and digested on a flip oscillator for 1h. Then, the bacteria were broken with a high-pressure lysing instrument; the broken bacteria were centrifuged at 4°C, 20000rpm for 30min, the supernatant was collected, and ammonium sulfate (150g / L) was added for salting out for 3-6h, and the sample after salting out was collected and centrifuged at 4°C and 20000rpm for 30min, the supernatant was collected, and packaged in a 0.22μm dialysis bag, placed in a dialysis solution (5L 20mM PB Na) for 3-6h, and the dialyzed sample was filtered through a 0.22μm filter membrane, and the filtrate obtained was the crude extract of the target protein.

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

[0043] The chromatographic column used in the first step of protein purification experiment is Hitrap SP HP cation exchange column, which is gradient eluted with 20mM PB Na, 1M NaCl (pH 7.0) buffer to obtain the crude extract of the target protein (the target protein is red, so just collect the red sample). After elution is completed, it is concentrated to 5mL using a concentrator with a molecular weight cutoff of 30kDa, and the obtained protein extract is used for the second step of purification; the chromatographic column used in the second step of purification is HiLoad Superdex 200pg preparative size exclusion column, which is eluted with 20mM PB Na, 150mM NaCl (pH 7.0) buffer to obtain the target protein extract; the purified protein is washed with ddH2O three times using a concentrator with a molecular weight cutoff of 30kDa to remove the salt inside, and then freeze-dried and stored at -80°C. Finally, the purified target protein is analyzed and identified by polyacrylamide gel electrophoresis (SDS-polyacrylamide gelelectrophoresis, SDS-PAGE), as shown in Figure 2. Figure 5 As shown, the band on the gel image is approximately 30 kD, which is consistent with its theoretical molecular weight, indicating that the protein is successfully expressed.

[0044] Example 2

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

[0046] 1. Dilute the ACE standard 30 times with PBS buffer to obtain the ACE working solution, and add the ACE working solution to the three wells of the ELISA plate, adding 100 μL of ACE working solution to each well. Add the ACE working solution to the bottom of the well of the ELISA plate, try not to touch the well wall, gently shake to mix, and finally seal the standard well with a transparent sealing film. According to the above method, set up 3 groups of standard wells, one of which is the control group, and the other two groups are experimental groups, each group has 3 standard wells.

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

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

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

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

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

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

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

[0054] 9. The A450 values ​​of ACE standards tested by the ELISA kit prepared with recombinant CC-SA fusion protein and the commercial 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 test results. Even within a shorter incubation time, the ELISA kit prepared with the recombinant CC-SA fusion protein of the present invention achieved better test results.

[0055] Example 3

[0056] In this example, an ELISA kit prepared by recombinant CC-SA fusion protein was used to detect human complement system 3a (C3a) standard. The specific method is as follows:

[0057] 1. Dilute the C3a standard 30 times with PBS diluent to obtain C3a working solution. Add the C3a working solution to the ELISA plate. Add 100 μL of C3a working solution to each well. Add the C3a working solution to the bottom of the ELISA plate well, try not to touch the well wall, gently shake to mix, and finally seal with a transparent sealing film. Set up 2 groups of standard wells according to the above method, the first group is the experimental group, and the other group is the control group.

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

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

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

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

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

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

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

[0066] Example 4

[0067] In this example, an ELISA kit based on recombinant CC-SA fusion protein was used to detect C3a protein in the supernatant of Hela cell culture. The specific method is as follows:

[0068] 1. Pipette the Hela cell culture supernatant and add it to the ELISA plate. Add 100μL Hela cell culture supernatant to each well. Add the Hela cell culture supernatant to the bottom of the ELISA plate well, try not to touch the well wall, gently shake to mix, and finally seal the well with a transparent sealing film. Set up 2 groups of actual sample wells according to the above method, one group is the experimental group, and the other group is the control group.

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

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

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

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

[0073] 6. Incubate the ELISA plate treated in step 5 at room temperature in the dark for 20 minutes. After the incubation, wash the plate 5 times and finally place each ELISA plate on thick absorbent paper and pat dry.

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

[0075] 8. Add 50 μL of stop solution (2M H2SO4 solution) to each of the two groups 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 by ELISA kit prepared with recombinant CC-SA fusion protein. Figure 8 As shown, compared with the control group, within the same incubation time, the ELISA kit prepared based on the recombinant CC-SA fusion protein of the present invention produced a significant A450 absorption value, indicating that the C3a protein in the Hela cell culture supernatant was successfully detected.

Claims

1. New use of cytochrome C in the preparation of ELISA kits.

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

1.

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

2.

4. A method for preparing the recombinant CC-SA fusion protein according to claim 2, characterized in that The steps include: S1. According to the amino acid sequence of the recombinant CC-SA fusion protein, the coding gene of the recombinant CC-SA fusion protein is synthesized by gene amplification technology; S2, inserting the coding gene of the recombinant CC-SA fusion protein into the expression vector for recombination to obtain a recombinant plasmid; S3. The recombinant plasmid is transferred into competent cells, and isopropyl-β-D-thiogalactoside IPTG is used to induce expression and culture, and then purified to obtain the recombinant CC-SA fusion protein.

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

6. Use of the recombinant CC-SA fusion protein according to claim 2 in preparing an ELISA kit.

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