A recombinant poly-IgA protein, its preparation method and uses

Abstract

The present invention discloses a recombinant immunoglobulin A complex, its preparation method and uses. The recombinant poly-IgA protein is named rPolyIgA and is composed of a recombinant human IgA amino acid fragment, biotin and streptavidin. The amino acid sequence of the recombinant human IgA amino acid fragment is as shown in SEQ ID No.1. The preparation method of the recombinant poly-IgA protein includes obtaining the recombinant human IgA amino acid fragment; connecting biotin to obtain an intermediate product; and mixing with streptavidin and heating to obtain the recombinant poly-IgA protein. During the process of preparing the rPolyIgA, an expression plasmid and an expression cell are also included. The prepared rPolyIgA can be applied to calibration products for IgA immunoassay and the research and development of IgA immunological drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a recombinant poly-IgA protein, a preparation method thereof, and uses thereof. Background Art

[0002] Currently, the calibrator for IgA immunoassay that can be traced to international standards is Human IgA Standard (IgA standard), which is mainly used to calibrate the content of IgA in samples. The limitations of using IgA standard as a calibrator for IgA immune complexes are as follows: First, the main protein component of IgA standard is monomeric IgA protein, and the concentration of IgA immune complexes in it is extremely trace; Second, when performing detections related to IgA immune complexes, the target protein is IgA immune complexes, rather than monomeric IgA protein. Especially when performing detections for IgA nephropathy, accurate risk prompts can only be obtained by detecting IgA immune complexes. Therefore, the most ideal calibrator for IgA immune complex detection is IgA immune complexes, rather than IgA standard. However, the existing method for obtaining IgA immune complexes is to directly purify and extract from human peripheral blood, which is expensive, has high technical requirements, and does not meet the basic requirement that in vitro diagnostic kits should not contain human samples, so it is difficult to be actually applied. Therefore, it is very important to construct a calibrator for IgA immune complex detection. Summary of the Invention

[0003] The purpose of the present invention is to provide a recombinant poly-IgA protein, a preparation method thereof, and uses thereof, so as to solve one or more technical problems existing in the prior art, and at least provide a beneficial choice or create conditions.

[0004] The first aspect of the present invention provides a recombinant poly-IgA protein.

[0005] The second aspect of the present invention provides a preparation method for the above-mentioned recombinant poly-IgA protein.

[0006] The third aspect of the present invention provides an expression plasmid.

[0007] The fourth aspect of the present invention provides an expression cell.

[0008] The fifth aspect of the present invention provides the application of the above-mentioned recombinant poly-IgA protein as a calibrator for IgA immunoassay.

[0009] The sixth aspect of the present invention provides the application of the above-mentioned recombinant poly-IgA protein in the research and development of IgA immune drugs.

[0010] A recombinant poly-IgA protein described in the first aspect of the present invention is named rPolyIgA. The rPolyIgA is composed of a recombinant human IgA amino acid fragment, biotin, and streptavidin. The amino acid sequence of the recombinant human IgA amino acid fragment is as shown in SEQ ID No.1. The rPolyIgA is linked with biotin on the basis of the recombinant human IgA amino acid fragment and polymerized through streptavidin, belonging to an IgA immune complex, which can be distinguished from the existing IgA standard product. In the detection of IgA nephropathy risk, the content of circulating IgA immune complex needs to be detected. Therefore, the rPolyIgA is more suitable as a calibrator for detecting circulating IgA immune complex than the IgA standard product mainly composed of monomeric IgA protein.

[0011] In some embodiments of the first aspect of the present invention, the nucleotide sequence encoding the recombinant human IgA amino acid fragment is as shown in SEQ ID No.2. The nucleotide sequence encodes an information peptide from the 7th to 66th positions at the 5' end, a 6×His tag from the 70th to 87th positions, and an AviTag from the 109th to 153rd positions. TM , the 154th to 852nd positions encode the Fc segment of IgA. The 5' end is the BamHI cleavage site GGATCC, and the 3' end is the stop codon TAG and the ApaI cleavage site GGGCCC. The molecular probe rCD89 for detecting circulating IgA immune complex, also known as the IgA Fc segment receptor. The rCD89 can specifically bind to the Fc segment of IgA on the complex, but hardly binds to monomeric IgA protein. Therefore, the recombinant human IgA amino acid fragment only needs to contain the Fc segment sequence of IgA, without adding the Fab segment or other segments.

[0012] In some embodiments of the first aspect of the present invention, the rPolyIgA is formed by combining 1 streptavidin with 4 recombinant human IgA amino acid fragments linked with biotin.

[0013] The preparation method described in the second aspect of the present invention includes the following steps:

[0014] 1) Obtain the recombinant human IgA amino acid fragment;

[0015] 2) Use biotin ligase to link biotin to the N-terminus of the recombinant human IgA amino acid fragment to obtain an intermediate product;

[0016] 3) Mix the intermediate product with streptavidin and heat to obtain the recombinant poly-IgA protein.

[0017] In some embodiments of the second aspect of the present invention, the recombinant human IgA amino acid fragment can be obtained by the following steps:

[0018] a) Obtain a nucleotide fragment with the sequence shown in SEQ ID No.2 by means of synthesis, amplification, etc.;

[0019] b) Insert the nucleotide fragment obtained in step a) into an expression plasmid through the BamHI restriction site and the ApaI restriction site;

[0020] c) The expression plasmid obtained in step b) can be transformed into a cloning bacterium for enlarged culture, and then the expression plasmid after enlarged culture is extracted and transfected into an expression cell;

[0021] d) Induce the expression cell obtained in step c) to express, and obtain the recombinant human IgA amino acid fragment after purification.

[0022] In some embodiments of the second aspect of the present invention, the expression plasmid is a mammalian cell expression vector plasmid, preferably pcDNA3.1 / Hygro(+)(GenScript).

[0023] In some embodiments of the second aspect of the present invention, the expression cell is a HEK-293 cell.

[0024] The expression plasmid provided in the third aspect of the present invention contains the nucleotide sequence shown in SEQ ID No.2.

[0025] The expression cell provided in the fourth aspect of the present invention contains the nucleotide sequence shown in SEQ ID No.2 or the expression plasmid provided in the third aspect of the present invention.

[0026] The nucleotide sequence shown in SEQ ID No.2 has been optimized to conform to the human cell expression system, so that the structure of the recombinant human IgA amino acid fragment obtained by expression is closest to its conformation in the human body, which helps to improve the accuracy of the rPolyIgA as a calibrator for detecting circulating IgA immune complexes.

[0027] Compared with the prior art, the beneficial effects of the present invention are: by using rPolyIgA and IgA standard as calibrators respectively, and conducting statistical analysis and comparison of the detected values in healthy individuals (HC) and IgA nephropathy (IgAN) patients, the upper limit of the healthy range of IgA immune complexes measured with rPolyIgA as a calibrator is 88.3 U / mL, while the upper limit of the healthy range measured with IgA standard as a calibrator is 138.24 U / mL, indicating that the healthy range measured with the rPolyIgA calibrator is more compact, thus reducing false positives beyond the normal range. In addition, it has been experimentally proven that rPolyIgA has a greater separation in distinguishing healthy populations from IgA nephropathy patients, and the results are more accurate. Description of the Drawings

[0028] Figure 1It is the sequence map of the pcDNA3.1 / Hygro(+) plasmid used in Example 1;

[0029] Figure 2 It is the map of the expression plasmid synthesized in Example 1;

[0030] Figure 3 It is the flow chart of large-scale plasmid extraction in Example 1;

[0031] Figure 4 It is the agarose gel electrophoresis map of the plasmid extracted in 1-1 of Example 1;

[0032] Figure 5 It is the transfection flow chart in 1-2 of Example 1;

[0033] Figure 6 It is the protein purification curve graph in 1-3 of Example 1;

[0034] Figure 7 It is the agarose gel electrophoresis map of the protein sample in 1-4 of Example 1;

[0035] Figure 8 It is the agarose gel electrophoresis map of the protein sample in 1-6 of Example 1;

[0036] Figure 9 It is the schematic structural diagram of the said rPolyIgA;

[0037] Figure 10 It is the fitting standard curve graph for verifying rPolyIgA as a calibration product for IgA immunoassay in Example 2. Detailed implementation manners

[0038] Next, in combination with specific examples, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] Preparation of reagents:

[0040] Prepare the binding buffer for purification (PB buffer with a concentration of 20 mM, NaCl solution with a concentration of 0.5 M, adjust the pH to 7.4), the washing buffer (PB buffer with a concentration of 20 mM, NaCl solution with a concentration of 0.5 M, imidazole solution with a concentration of 20 mM, adjust the pH to 7.4), and the elution buffer (PB buffer with a concentration of 20 mM, NaCl solution with a concentration of 0.5 M, imidazole solution with a concentration of 500 mM, adjust the pH to 7.4).

[0041] Example 1, Preparation of rPolyIgA.

[0042] 1-1. Entrust GenScript Biotech Corporation to synthesize the gene fragment with the nucleotide sequence shown in SEQ ID No. 2, construct it into the corresponding vector, transform it into competent cells, and finally deliver it in the form of high-copy glycerol bacteria (correctly sequenced). The nucleotide sequence encodes an information peptide from the 7th to 66th positions at the 5' end, a 6×His tag from the 70th to 87th positions, and an AviTag from the 109th to 153rd positions. TM , the 154th to 852nd positions encode the Fc segment of human immunoglobulin A. The 5' end has a BamHI restriction site GGATCC, and the 3' end has a stop codon TAG and an ApaI restriction site GGGCCC. The gene fragment is inserted into the plasmid vector pcDNA3.1 / Hygro(+) (GenScript, sequence map as Figure 1 shown), and the cloning site is between the restriction sites BamHI and ApaI. The synthetic expression plasmid map is as Figure 2 shown. Take 100 μL of glycerol bacteria and inoculate it into a 15 mL centrifuge tube, add 4 mL of LB medium containing ampicillin, seal it with a sealing film and fix it in a constant temperature shaker. The shaker is set at a temperature of 37°C and a rotation speed of 220 rpm, and cultured for 8 hours to activate the glycerol bacteria; then add all 4 mL of the bacterial solution to a conical flask containing 250 mL of medium and continue to culture for 15 hours to fully amplify the strain. When the OD 600 is about 0.8, it indicates that the cloned bacteria are in the logarithmic growth phase and have the best viability; use sterilized 50% glycerol to preserve the bacterial solution. Add 600 μL of the bacterial solution and 400 μL of glycerol to each EP tube, mix well, seal it with a sealing film, and store it in a -80°C refrigerator for later use. For each subsequent amplification of the bacteria, only inoculate according to a ratio of 1:500. Centrifuge the remaining bacterial solution at 6000 rpm for 10 minutes, and then use a large-scale plasmid extraction kit (AxyPrep endotoxin-free large-scale plasmid extraction kit) for plasmid extraction. The process is as shown in the Figure 3 provided by the kit. Identify the extracted plasmid by agarose gel electrophoresis (see Figure 4 ), and there is no difference between the plasmid obtained by large-scale extraction and the small-scale plasmid provided by GenScript Biotech Corporation. Finally, use a Qubit fluorometer to detect the concentration and store it at -20°C for later use.

[0043] 1-2. Resuscitate and suspend HEK-293 cells (from Zhuhai Kairui Biotechnology Company), place the cell flask in a carbon dioxide incubator shaker with a CO2 concentration of 5%, a rotation speed of 120 rpm, and a temperature of 37°C for shaking and suspension culture; count the cells 3 days after culture, record the cell density and viability. When the cell density reaches 3 - 6×10 6cells / mL, directly dilute the cells with fresh culture medium for subculture, and the subculture density is 0.3×10 6 cells / mL. Subculture and amplify until the cell quantity is sufficient for transfection, and the cells are in the growth exponential phase (density is about 2 - 4×10 6 cells / mL), and the cell viability is greater than 98%. When the cells reach the transfection conditions, dilute the cells with fresh culture medium to a density of 2.0×10 6 cells / mL, place the diluted cells in a 5% CO2 constant temperature shaker, and start transfection after culturing at 37°C and 120 rpm for 10 minutes. Prepare two sterile centrifuge tubes. In one of them, add 5% of the total volume of the cell culture medium to be transfected with KPM (purchased from Zhuhai Kairui) and 1 μg / mL (plasmid / volume of cell culture medium) of sterile plasmid DNA, and gently pipette and mix well; in the other centrifuge tube, add the same volume of KPM and 5 μL / mL (transfection reagent / volume of cell culture medium) of TA-293 transfection reagent (purchased from Zhuhai Kairui), and gently pipette and mix well; transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently pipette and mix well; let it stand at room temperature for 10 minutes to prepare the plasmid-vector complex. Take out the expressing cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and put it back into the CO2 constant temperature shaker for shaking culture. The transfection process is as per the SOP of Zhuhai Kairui Biotech Co., Ltd.( Figure 5 ). The transfected cells are obtained upon completion of transfection.

[0044] 1-3. After 24 hours of transfection, the protein expression enhancer (KE-293) required for 0.6% HEK-293 cells and 2% transient transfection nutrient additive (KT-Feed 50×) can be added; continue to culture for 6 days after transfection. Take out the cell flask from the incubator, detect and record the cell density and viability, then transfer the cell culture medium to the collection bottle, centrifuge at 10,000 rpm for 10 minutes, and pour the centrifuged supernatant into a 0.2 μm filtration device for suction filtration. Thus, the supernatant containing the recombinant human IgA amino acid fragment is obtained. Use an ultrafiltration membrane package (Sartorius Vivaflow200) with a molecular weight cut-off of 10 kD to replace the buffer of the culture supernatant, replace the medium in it with binding buffer, and then concentrate it to 100 mL; wash the pipeline of the AKTA protein purifier with pure water, and connect the Ni-TED 6FF pre-packed chromatography column (purchased from Sangon Biotech) for purifying His-tagged proteins on the instrument; wash the nickel column with 5-10 column volumes of pure water at a flow rate of 5 mL / min to remove the ethanol used for storing the column. Balance the medium with 5-10 column volumes of binding buffer at a flow rate of 5 mL / min to ensure that the components and pH of the solution in the medium are the same as those of the culture supernatant to be purified; load the supernatant at a flow rate of 5 mL / min, and at this time, it can be seen that the UV line first gradually rises and then levels off; wash the impurities with 10-20 column volumes of washing solution at a flow rate of 5 mL / min to wash the miscellaneous proteins non-specifically adsorbed on the nickel column, and collect the washing solution for subsequent analysis. During the washing process, it can be seen that the UV line gradually goes down and then levels off, or there are small protruding peaks during the descending process; elute with 5-10 column volumes of eluent at a low flow rate, and during the elution process, it can be seen that the UV line shows a protruding peak (as Figure 6 shown), collect the eluent when this elution peak appears; after purification, wash the resin with 5-10 column volumes of pure water at 0.5 mL / min to remove the eluent in the medium; then wash the medium with 5-10 column volumes of 20% ethanol at 0.5 mL / min, and finally remove the column from the instrument and store it at 4-8 °C. The collected eluent is processed using a desalting column (Zeba TM Desalting Spin Column, molecular weight cut-off 7000) to remove imidazole and replace the buffer with PBS buffer solution with a pH of 7.4, that is, the recombinant human IgA amino acid fragment is obtained.

[0045] 1-4. Use a 12% SDS-PAGE precast gel. Add 5 μg of the recombinant human IgA amino acid fragment and non-reducing loading buffer to the first well (left), add a marker to the second well (middle), and add 5 μg of the recombinant human IgA amino acid fragment and reducing loading buffer to the third well (right). The voltage is 120 V, and electrophoresis is carried out for 1 hour. The gel pattern (as Figure 7As shown, the molecular weight of the first porin sample is about 70 kD, which is a recombinant human IgA Fc formed by the CH3-CH3 of two recombinant human IgA amino acid fragments through disulfide bonds. After adding a reducing agent to the third well, the disulfide bonds were opened, and the recombinant human IgA amino acid fragments were reduced to a recombinant CH2-CH3 with a molecular weight of about 35 kD, which is in line with the designed molecular weight. The concentration of the purified protein was detected using a Qubit fluorometer and stored at -80 °C in the refrigerator for future use.

[0046] 1-5. The above purified recombinant human IgA amino acid fragments have AviTag TM , which can specifically bind to biotin. The recombinant human IgA amino acid fragments were biotinylated using a BirA Enzyme kit, that is, biotin (Biotin) was linked to the N-terminus of the fragments. Each component was added to an EP tube according to the ratio shown in Table 1, and the mixture was pipetted and mixed well. After sealing the EP tube, it was placed in a 30 °C water bath and heated for 30 minutes. The EP tube was taken out and the reaction solution in the tube was mixed again, and then continued to be placed in a 30 °C water bath and heated for 30 minutes. A desalting column was used to remove the unlinked biotin in the reaction solution, and the concentration of the intermediate product was detected.

[0047] Table 1. Biotinylation

[0048]

[0049] 1-6. Since one streptavidin molecule can bind 4 biotins and has a strong affinity. Taking 57.1 nmol of the intermediate product, one-quarter of the amount of streptavidin in terms of substance amount needs to be added, that is, 14.28 nmol. Streptavidin was added in 3 portions. After each addition, the EP was first fixed on a rotary mixer and mixed at a low speed for 5 minutes, and then placed in a 37 °C water bath and heated for 20 minutes. After binding streptavidin, the recombinant protein was finally combined into a polymer containing recombinant human IgA amino acid fragments and streptavidin - rPolyIgA. The SDS-page electrophoresis gel image (see Figure 8 ) shows that the molecular weight of rPolyIgA is about 180 kD in the non-reduced state (left) and about 35 kD in the reduced state (right), and the middle is the marker (Marker). Based on these data, it is presumed that the way streptavidin links rIgA through biotin is as Figure 9 shown.

[0050] Example 2, ELISA detection.

[0051] Dilute the molecular probe rCD89 to 5 μg / mL using PBS buffer solution with a pH of 7.4. Add 100 μL to each well on the enzyme-linked immunosorbent assay (ELISA) plate and coat overnight at 4 °C. Wash the ELISA plate 3 times with PBST. Add 2.0% BSA solution and incubate at room temperature for 1 hour to block the ELISA plate. Then wash 3 times. Dilute rPolyIgA to 500 ng / mL using the sample diluent, and then perform two-fold serial dilution for a total of 7 concentrations, namely 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 ng / mL, denoted as STD1 - STD7 respectively. Add the 7 groups of samples to the ELISA plate, with two wells for each group, 100 μL per well, and incubate at room temperature for 1 hour. Wash 3 times. Add the goat anti-human IgA enzyme-labeled secondary antibody diluted 10,000-fold and incubate at room temperature for 30 minutes. Wash 3 times. Add 100 μL of TMB chromogenic solution to each well, keep in the dark and stand for 10 minutes. Add 50 μL of 2 M H2SO4 to terminate the color development. Read the absorbance at 450 / 620 nm using an ELISA reader. The results are shown in Table 2.

[0052] Table 2. OD values corresponding to the dilution gradient of rPolyIgA

[0053] Concentration (ng / ml) <![CDATA[OD 450 > <![CDATA[OD 620 > 500 2.749 2.739 250 1.952 1.969 125 1.25 1.288 62.5 0.749 0.79 31.25 0.436 0.457 15.625 0.265 0.266 7.8125 0.17 0.172 0 0.062 0.063

[0054] The Logistic four-parameter fitting standard curve is as Figure 10 shown, and the standard equation:

[0055] y = (A - D) / [1 + (x / C)^B] + D;

[0056] A = 7.81487,

[0057] B = -4.29002,

[0058] C = 3.16153,

[0059] D = 0.08058,

[0060] r 2 = 0.99978;

[0061] Substitute the measured value of the sample to be tested into the above equation to calculate the concentration of IgA immune complex in the sample.

[0062] Example 3. Comparative test of rPolyIgA and IgA standard.

[0063] By using rPolyIgA and Human IgA Standard as calibrators respectively, a statistical analysis and comparison of the detected values were carried out between healthy individuals (HC) and IgA nephropathy (IgAN) patients. The upper limit of the healthy range of IgA immune complexes measured with rPolyIgA as the calibrator was 88.3 U / mL, while the upper limit of the healthy range measured with IgA Standard as the calibrator was 138.24 U / mL, indicating that the healthy range measured with rPolyIgA as the calibrator was tighter, thus reducing false positives beyond the normal range.

[0064] On the other hand, for IgA nephropathy samples, there was no significant decrease in the IgA immune complex index measured with rPolyIgA as the calibrator compared to that measured with IgA Standard as the calibrator. In the comparative experiment, 64 healthy individuals and 39 peripheral blood samples of IgA nephropathy patients were used. Detection was carried out using rPolyIgA and IgA Standard as calibrators respectively, and then the statistical differences between the healthy population and IgA nephropathy were compared using the T-test (t-test). The results are shown in Table 3.

[0065] Table 3. Comparison of T-test results of detected values between healthy population and IgA nephropathy

[0066]

[0067] As can be seen from Table 3, the p-value corresponding to rPolyIgA was 1.11E-06, while the p-value corresponding to IgA Standard was 0.04, indicating that rPolyIgA had a greater separation in distinguishing between the healthy population and IgA nephropathy patients, and the results were more accurate.

[0068] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

Claims

1. A recombinant poly-IgA protein, characterized in that, It is composed of a recombinant human IgA amino acid fragment, biotin, and streptavidin. The amino acid sequence of the recombinant human IgA amino acid fragment is as shown in SEQ ID No.

1. During the synthesis process, the recombinant human IgA amino acid fragment first binds to the biotin to form an intermediate product, and then the intermediate product binds to streptavidin to form the recombinant poly-IgA protein. The molar ratio of streptavidin to the intermediate product is 1:

4.

2. The preparation method of the recombinant poly-IgA protein according to claim 1, characterized in that, It includes the steps of: 1) Obtaining the recombinant human IgA amino acid fragment; 2) Using a biotin ligase to link biotin to the N-terminus of the recombinant human IgA amino acid fragment to obtain an intermediate product; 3) Mixing the intermediate product with streptavidin and heating to obtain the recombinant poly-IgA protein.

3. The preparation method according to claim 2, characterized in that, The recombinant human IgA amino acid fragment is obtained by the following steps: a) Obtaining a nucleotide fragment with a sequence as shown in SEQ ID No.2; b) Inserting the nucleotide fragment obtained in step a) into an expression plasmid; c) Transfecting the expression plasmid obtained in step b) into an expression cell; d) Inducing the expression cell obtained in step c) to express, and obtaining the recombinant human IgA amino acid fragment after purification.

4. The preparation method according to claim 3, characterized in that, The expression plasmid is a mammalian cell expression vector plasmid.

5. The preparation method according to claim 3, characterized in that, The expression cell is a HEK-293 cell.

6. An expression plasmid, characterized in that, It contains a nucleotide sequence as shown in SEQ ID No.

2.

7. An expression cell, characterized in that, It contains a nucleotide sequence as shown in SEQ ID No.2 or the expression plasmid described in claim 6.

8. Use of the recombinant poly-IgA protein according to claim 1 in the preparation of a calibrator for IgA immunoassay.

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