CN116789806A - Recombinant poly IgA protein and preparation method and application thereof - Google Patents

Abstract

The invention discloses a recombinant immunoglobulin A compound and a preparation method and application thereof. The recombinant poly IgA protein is named rPolyIgA and is formed by combining recombinant human IgA amino acid fragments, biotin and streptavidin, and the amino acid sequence of the recombinant human IgA amino acid fragments is shown as SEQ ID No. 1. The preparation method of the recombinant poly IgA protein comprises the steps of obtaining the recombinant human IgA amino acid fragment; connecting biotin to obtain an intermediate product; mixing with streptavidin, and heating to obtain the recombinant poly IgA protein. The rPolyIgA is prepared by a process comprising an expression plasmid and an expression cell. The prepared rPolyIgA can be applied to calibration products for IgA immunodetection and research and development of IgA immune medicines.

Description

Recombinant poly IgA protein and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a recombinant multimeric IgA protein, a preparation method and application thereof.

Background

Currently, the standard of traceable international standard related to IgA immunodetection is Human IgA Standard (IgA standard), and is mainly used for calibrating the content of IgA in a sample. Limitations of using IgA standards as an IgA immune complex calibrator are: 1. the main protein component of the IgA standard substance is monomeric IgA protein, wherein the concentration of the IgA immune complex is very small; 2. in carrying out IgA immune complex related assays, the protein of interest is an IgA immune complex, rather than a monomeric IgA protein. In particular, in IgA nephropathy detection, it is necessary to detect IgA immune complexes to obtain accurate risk indication. Therefore, the most ideal calibrator for IgA immune complex detection is IgA immune complex, rather than IgA standard. However, the existing method for obtaining IgA immune complex is directly purified and extracted from human peripheral blood, which has high price and high technical requirement, and does not meet the basic requirement that an in-vitro diagnostic kit should not contain human samples, so that the method is difficult to be practically applied. Therefore, it is important to construct a calibrator for IgA immune complex detection.

Disclosure of Invention

The invention aims to provide a recombinant multimeric IgA protein, a preparation method and application thereof, which are used for solving one or more technical problems in the prior art and at least providing a beneficial selection or creation condition.

In a first aspect the invention provides a recombinant multimeric IgA protein.

The second aspect of the present invention provides a method for producing the recombinant multimeric IgA protein described above.

In a third aspect, the invention provides an expression plasmid.

In a fourth aspect, the invention provides an expression cell.

In a fifth aspect, the invention provides the use of a recombinant multimeric IgA protein as described above as a calibrator for IgA immunodetection.

The sixth aspect of the invention provides the use of the recombinant multimeric IgA protein described above in IgA immune drug development.

The recombinant poly IgA protein is named rPolyIgA, the rPolyIgA is formed by combining recombinant human IgA amino acid fragments, biotin and streptavidin, and the amino acid sequence of the recombinant human IgA amino acid fragments is shown as SEQ ID No. 1. The rPolyIgA is connected with biotin on the basis of recombinant human IgA amino acid fragments, and multimerization is realized through streptavidin, and belongs to an IgA immune complex which can be distinguished from the existing IgA standard. In IgA nephropathy risk detection, the content of the circulating IgA immune complex is required. Thus, the rPolyIgA is more suitable as a calibrator for detecting circulating IgA immune complexes than IgA standards whose main component is monomeric IgA protein.

In some embodiments of the first aspect of the invention, the nucleotide sequence encoding the recombinant human IgA amino acid fragment is set forth in SEQ ID No. 2. The nucleotide sequence codes for information peptide from 7 th to 66 th of 5' end, the 70 th to 87 th codes for 6 XHis tag, and the 109 th to 153 th codes for AviTag ^TM The 154 th to 852 th Fc segment of the coded IgA has BamHI enzyme cutting site GGATCC at the 5 'end and stop codon TAG and ApaI enzyme cutting site GGGCCC at the 3' end. The molecular probe rCD89 of the circulating IgA immune complex, also known as IgA Fc segment receptor, is detected. The rCD89 is capable of specifically binding the Fc segment of IgA on the complex, but hardly binds to monomeric IgA protein, so the recombinant human IgA amino acid fragment need only comprise the Fc segment sequence of IgA, without the addition of Fab segments or other fragments.

In some embodiments of the first aspect of the invention, the rPolyIgA is made from 1 of the streptavidin combined with 4 biotin-linked recombinant human IgA amino acid fragments.

The preparation method of the second aspect of the invention comprises the following steps:

1) Obtaining the recombinant human IgA amino acid fragment;

2) Connecting biotin to the N end of the recombinant human IgA amino acid fragment by using biotin ligase to obtain an intermediate product;

3) And mixing the intermediate product with streptavidin, and heating to obtain the recombinant multimeric IgA protein.

In some embodiments of the second aspect of the invention, the recombinant human IgA amino acid fragment may be obtained by:

a) Obtaining a nucleotide fragment with a sequence shown as SEQ ID No.2 through means of synthesis, amplification and the like;

b) Inserting the nucleotide fragment obtained in the step a) into an expression plasmid through a BamHI enzyme cutting site and an ApaI enzyme cutting site;

c) The expression plasmid obtained in the step b) can be transformed into cloning bacteria for expansion culture, and then the expression plasmid obtained after the expansion culture is extracted and transfected into expression cells;

d) Inducing the expression cell obtained in the step c) to express, and purifying to obtain the recombinant human IgA amino acid fragment.

In some embodiments of the second aspect of the invention, the expression plasmid is a mammalian cell expression vector plasmid, preferably pcDNA3.1/Hygro (+) (gold srey).

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

The expression plasmid provided by the third aspect of the invention comprises a nucleotide sequence shown as SEQ ID No. 2.

The expression cell provided in the fourth aspect of the present invention comprises the nucleotide sequence shown as SEQ ID No.2 or the expression plasmid provided in the third aspect of the present invention.

The nucleotide sequence shown as SEQ ID No.2 is optimized to conform to a cell expression system of a human source, so that the structure of the recombinant human IgA amino acid fragment obtained by expression is closest to the conformation of the recombinant human IgA amino acid fragment in a human body, and the accuracy of the rPolyIgA serving as a calibrator for detecting the circulating IgA immune complex is improved.

Compared with the prior art, the invention has the beneficial effects that: by comparing the statistical analysis of the detection values in Healthy (HC) and IgA nephropathy (IgAN) populations using rPolyIgA and IgA standards, respectively, as calibrators, the upper limit of the healthy range of the IgA immune complex measured by rPolyIgA as the calibrator was 88.3U/mL, while the upper limit of the healthy range measured by IgA standard as the calibrator was 138.24U/mL, indicating that the healthy range measured by rPolyIgA calibrator was more compact, thereby reducing false positives beyond the normal range. In addition, the experiment proves that rPolyIgA is used for distinguishing healthy people from IgA nephropathy people, and the result is more accurate.

Drawings

FIG. 1 is a sequence diagram of pcDNA3.1/Hygro (+) plasmid used in example 1;

FIG. 2 is a map of the expression plasmid synthesized in example 1;

FIG. 3 is a flow chart showing the plasmid preparation in example 1;

FIG. 4 is an agarose gel electrophoresis of the plasmid extracted in 1-1 of example 1;

FIG. 5 is a transfection flow diagram of example 1-2;

FIG. 6 is a graph of protein purification in examples 1-3;

FIG. 7 is an agarose gel electrophoresis of the protein samples of examples 1-4;

FIG. 8 is an agarose gel electrophoresis of the protein samples of examples 1-6;

FIG. 9 is a schematic structural diagram of the rPolyIgA;

FIG. 10 is a graph of a fitted standard for verifying rPolyIgA as a calibrator for IgA immunodetection in example 2.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with specific embodiments, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Preparation of the reagent:

a binding buffer (PB buffer at a concentration of 20mM, naCl solution at a concentration of 0.5M, pH adjusted to 7.4), a washing buffer (PB buffer at a concentration of 20mM, naCl solution at a concentration of 0.5M, imidazole solution at a concentration of 20mM, pH adjusted to 7.4) and an elution buffer (PB buffer at a concentration of 20mM, naCl solution at a concentration of 0.5M, imidazole solution at a concentration of 500mM, pH adjusted to 7.4) for purification were prepared.

Example 1 preparation of rPolyIgA.

1-1. Entrusting the gene fragment with the nucleotide sequence shown as SEQ ID No.2 by the Kirschner Biotech Co., ltd. And entrusting the construction of the gene fragment into corresponding vectors, transforming the vector into competent cells, and finally delivering the vector in the form of high-copy glycerol bacteria (with correct sequencing). The nucleotide sequence codes for information peptide from 7 th to 66 th of 5' end, the 70 th to 87 th codes for 6 XHis tag, and the 109 th to 153 th codes for AviTag ^TM The 154 th to 852 th parts encode Fc segment of human immunoglobulin A, the 5 'end is BamHI enzyme cutting site GGATCC, and the 3' end is stop codon TAG and ApaI enzyme cutting site GGGCCC. The gene fragment was inserted into a plasmid vector pcDNA3.1/Hygro (+) (the sequence diagram is shown in FIG. 1), the cloning site is located between the cleavage sites BamHI and ApaI, and the synthetic expression plasmid diagram is shown in FIG. 2. Inoculating 100 μl of glycerol bacteria into a 15mL centrifuge tube, adding 4mL of LB culture medium containing ampicillin, sealing the sealing film, fixing in a constant temperature shaking table, setting the temperature at 37deg.C, and culturing at 220rpm for 8 hr to activate glycerol bacteria; then, the whole 4mL of the bacterial liquid was added to a conical flask containing 250mL of the culture medium, and the culture was continued for 15 hours to allow the strain to fully expand, and the OD was measured ₆₀₀ About 0.8 shows that the clonal strain is in the logarithmic growth phase and has the optimal activity; and (3) using sterilized 50% glycerol to protect bacterial liquid, adding 600 mu L of bacterial liquid and 400 mu L of glycerol into each EP tube, fully and uniformly mixing, sealing by using a sealing film, and placing in a refrigerator at the temperature of-80 ℃ for preservation for later use, wherein each amplification strain only needs to be inoculated according to the proportion of 1:500. The remaining bacterial liquid was centrifuged at 6000rpm for 10 minutes, and then plasmid extraction was performed using a large extraction kit (AxyPrep endotoxin-free plasmid bulk kit), the procedure being shown in FIG. 3 provided by the kit. Agarose gel electrophoresis was performed on the extracted plasmid (see FIG. 4), and the plasmid obtained by the large extraction was not different from the small sample plasmid provided by Kirschner Biotech Co., ltd. And finally, detecting the concentration by using a Qubit fluorometer, and storing at the temperature of-20 ℃ for later use.

1-2 resuscitate suspended HEK-293 cells (from the Rayleigh Biotechnology of Pearl sea ), shake flask the cells into CO ₂ Shake suspension culture is carried out in a carbon dioxide incubator shaker with the concentration of 5 percent and the rotating speed of 120rpm and the temperature of 37 ℃; after 3 days of culture, the cells were counted, and the cell density and the viability were recorded when the cell density reached 3 to 6X 10 ⁶ The cells were directly diluted with fresh culture medium at 0.3X10 of subculture per mL ⁶ And each mL. Subculturing in large scale until the cell mass is sufficient for transfection and the cells are in the growth exponential phase (density of about 2-4X 10 ⁶ Number of cells per mL), cell viability was greater than 98%. After the cells reached transfection conditions, the cells were diluted to a density of 2.0X10 with fresh medium ⁶ The diluted cells were placed in 5% CO per mL ₂ Transfection was initiated after 10 minutes of incubation in a constant temperature shaker at 37℃and 120 rpm. Two sterile centrifuge tubes were prepared, and KPM (purchased from Pinctada martensii Rayleigh) 5% by volume of the total volume of the cell culture fluid to be transfected and 1. Mu.g/mL (plasmid/cell culture fluid volume) of sterile plasmid DNA were added to one of the tubes and gently swirled and mixed; the other separation tube is added with KPM with the same volume and TA-293 transfection reagent (purchased from the pearl sea Rui) with the volume of 5 mu L/mL (transfection reagent/cell culture solution volume), and the mixture is gently beaten and mixed; transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and lightly blowing and uniformly mixing; the plasmid-vector complex was prepared by standing at room temperature for 10 minutes. Extracting the expression cells from the constant temperature shaker, adding the prepared plasmid-vector complex while shaking, and returning to CO ₂ Shake culture in a constant temperature shaker, transfection procedure such as SOP (FIG. 5) from the Rayleigh biosystems, arisaema, pearl sea . And (5) finishing transfection to obtain the expression cell.

1-3. 24 hours after transfection, 0.6% of the protein expression enhancer required for HEK-293 cells (KE-293) and 2% of transient transfection nutrition additive (KT-Feed 50X) may be added; culturing for 6 days, taking out cell shake flask from incubator, detecting density and activity of recorded cells, transferring cell culture solution to collecting bottle, centrifuging at 10000rpm for 10 min, and filtering with 0.2 μm filter to obtain the final productSupernatant of the recombinant human IgA amino acid fragment. Using ultrafiltration membrane bag (Sartorius Vivaflow 200) with cut-off molecular weight of 10kD to replace culture supernatant with buffer solution, replacing culture medium therein with binding buffer solution, and concentrating to 100mL; washing the pipeline of AKTA protein purifier with pure water, and connecting Ni-TED 6FF pre-packed chromatographic column (purchased from biological company) for purifying His tag protein; the nickel column was washed with 5 to 10 column volumes of pure water at a flow rate of 5mL/min to remove ethanol used to store the column. Balancing the medium with 5-10 times of column volume of binding buffer solution at a flow rate of 5mL/min to ensure that the components and pH of the solution in the medium are consistent with those of the culture supernatant to be purified; loading the supernatant at a flow rate of 5mL/min, wherein the UV rays are gradually increased and then leveled; washing impurities with 10-20 times of column volume of impurity washing liquid at a flow rate of 5mL/min, washing impurity proteins non-specifically adsorbed on the nickel column, and collecting the impurity washing liquid for subsequent analysis, wherein the UV rays are gradually downward and leveled in the impurity washing process or have raised small peaks in the descending process; eluting with 5-10 times of column volume eluent at low flow rate, wherein a convex peak appears in UV rays during elution (as shown in figure 6), and collecting the eluent when the eluting peak appears; after the purification is finished, cleaning the resinoid with purified water with 5-10 times of column volume at 0.5mL/min, and removing eluent in the medium; and then 5-10 times of column volume of 20% ethanol is used for cleaning the cleaning medium at the speed of 0.5mL/min, and finally the column is detached from the instrument and stored at the temperature of 4-8 ℃. The collected eluate was purified using a desalting column (Zeba ^TM Desalting the centrifugal column, intercepting the molecular weight 7000), and removing the imidazo replacement buffer solution to be PBS buffer solution with pH of 7.4 to obtain the recombinant human IgA amino acid fragment.

1-4. Using a 12% SDS-PAGE preform, 5. Mu.g of the recombinant human IgA amino acid fragment and non-reducing Loading buffer (Loading buffer) were added to the first well (left), label (Marker) was added to the second well (right) and 5. Mu.g of the recombinant human IgA amino acid fragment and reducing Loading buffer were added to the third well (right). The voltage was 120V and electrophoresis was performed for 1 hour. The gel diagram (shown in FIG. 7) shows that the first porin sample has a molecular weight of about 70kD and is CH of two recombinant human IgA amino acid fragments ₃ -CH ₃ One formed by disulfide bondsIndividual recombinant human IgA Fc; after the reducing agent is added into the third hole, disulfide bonds are opened, and the recombined human IgA amino acid segment is reduced into a recombined CH ₂ -CH ₃ The molecular weight is about 35kD, which is in accordance with the designed molecular weight. The concentration of the purified protein was measured using a Qubit fluorometer and stored in a-80 ℃ refrigerator for further use.

1-5 the purified recombinant human IgA amino acid fragment has AviTag ^TM Can specifically bind to biotin. The recombinant human IgA amino acid fragment was biotinylated using the birA Enzyme kit, i.e.biotin (Biotin) was attached to the N-terminus of the fragment. The components are added into an EP pipe according to the proportion shown in the table 1, the EP pipe is blown and evenly mixed by a liquid shifter, the EP pipe is placed into a water bath kettle at 30 ℃ for heating for 30 minutes after being sealed, the reaction liquid in the EP pipe is taken out and evenly mixed again, and the EP pipe is placed into the water bath kettle at 30 ℃ for heating for 30 minutes; the concentration of the intermediate was measured by removing unbound biotin from the reaction solution using a desalting column.

TABLE 1 biotinylation

1-6. Since one streptavidin molecule can bind 4 biotins, it has a strong affinity. Taking 57.1nmol of said intermediate requires the addition of a quarter amount of streptavidin, i.e. 14.28nmol. The streptavidin is added for 3 times, after each addition, the EP is fixed on a rotary mixer for uniformly mixing for 5 minutes at a low rotating speed, the mixture is put into a water bath kettle at 37 ℃ for heating for 20 minutes, and after the streptavidin is combined, the recombinant protein is finally combined into a polymer rPolyIgA containing recombinant human IgA amino acid fragments and the streptavidin. The SDS-page electrophoresis gel diagram (see FIG. 8) shows that rPolyIgA has a molecular weight of about 180kD (left) in the non-reduced state and about 35kD (right) in the reduced state, with a Marker in the middle. From these data, it is assumed that streptavidin is attached to rIgA by biotin as shown in FIG. 9.

Example 2, ELISA assay.

PBS buffer with pH 7.4The molecular probe rCD89 was diluted to 5. Mu.g/mL, 100. Mu.L of each well was added to the plate, the plate was coated overnight at 4℃and washed 3 times with PBST, 2.0% BSA solution was added, the plate was left at room temperature for 1 hour, the plate was blocked, washed 3 times, and rPolyIgA was diluted to 500ng/mL with sample diluent, followed by two-fold gradient dilution, and total dilution was performed for 7 concentrations, i.e., 500, 250, 125, 62.5, 31.25, 15.625, 7.8125ng/mL, and designated STD1-STD7, respectively. Adding 7 groups of samples into an ELISA plate, adding two holes in each group, incubating for 1 hour at room temperature for 3 times with 100 mu L of each hole, adding 10000 times of diluted goat anti-human IgA ELISA secondary antibody, incubating for 30 minutes at room temperature, washing 3 times, adding 100 mu L of TMB color development liquid in each hole, standing for 10 minutes in a dark place, and adding 50 mu L of H with the concentration of 2M ₂ SO ₄ The color development was terminated and a 450/620nm dual wavelength reading was performed using a microplate reader, the results of which are shown in Table 2.

TABLE 2 OD values corresponding to rPolyIgA dilution gradient

Concentration (ng/ml)	OD 450	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

The Logistic four-parameter fitting standard curve is shown in fig. 10, standard equation:

y＝(A-D)/[1+(x/C)^B]+D；

A＝7.81487，

B＝-4.29002，

C＝3.16153，

D＝0.08058，

r ² ＝0.99978；

substituting the measured value of the sample to be measured into the equation can calculate the concentration of the IgA immune complex in the sample.

Example 3, comparison test of rPolyIgA with IgA standards.

By comparing the statistical analysis of the detection values in healthy people (HC) with IgA nephropathy (IgAN) population by using rPolyIgA and Human IgA Standard (IgA standards) as calibrators, respectively, the upper limit of the healthy people range of the IgA immune complex measured by rPolyIgA as the calibrator is 88.3U/mL, and the upper limit of the healthy people range measured by IgA standard as the calibrator is 138.24U/mL, which indicates that the healthy people range measured by rPolyIgA calibrator is tighter, thereby reducing false positives beyond the normal range.

On the other hand, in the case of the IgA nephropathy sample, the IgA immune complex index measured by rPolyIgA as a calibrator and the index measured by IgA standard as a calibrator were not significantly reduced. The comparative experiments used 64 healthy persons and 39 IgA nephropathy peripheral blood, respectively, with rPolyIgA and IgA standards as calibrator, and then T-test (T-test) was used to compare the statistical differences between healthy persons and IgA nephropathy as shown in Table 3.

TABLE 3 comparison of healthy population with IgA nephropathy detection value T-test results

As can be seen from Table 3, the p-value for rPolyIgA is 1.11E-06, while the p-value for IgA standard is 0.04, indicating that rPolyIgA is more accurate for distinguishing healthy from IgA nephropathy.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (10)

1. The recombinant poly IgA protein is characterized by being formed by combining a recombinant human IgA amino acid fragment, biotin and streptavidin, wherein the amino acid sequence of the recombinant human IgA amino acid fragment is shown as SEQ ID No. 1.

2. The recombinant multimeric IgA protein of claim 1 wherein the nucleotide sequence encoding said recombinant human IgA amino acid fragment is set forth in SEQ ID No. 2.

3. A method of preparing a recombinant multimeric IgA protein according to any one of claims 1 to 2 comprising the steps of:

1) Obtaining the recombinant human IgA amino acid fragment;

2) Connecting biotin to the N end of the recombinant human IgA amino acid fragment by using biotin ligase to obtain an intermediate product;

3) And mixing the intermediate product with streptavidin, and heating to obtain the recombinant multimeric IgA protein.

4. The method of claim 3, wherein the recombinant human IgA amino acid fragment is obtained by:

a) Obtaining a nucleotide fragment with a sequence shown as 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 the step c) to express, and purifying to obtain the recombinant human IgA amino acid fragment.

5. The method of claim 4, wherein the expression plasmid is a mammalian cell expression vector plasmid.

6. The method of claim 4, wherein the expression cell is a HEK-293 cell.

7. An expression plasmid comprising the nucleotide sequence set forth in SEQ ID No. 2.

8. An expression cell comprising the nucleotide sequence set forth in SEQ ID No.2 or the expression plasmid of claim 7.

9. Use of a recombinant multimeric IgA protein according to any one of claims 1 to 2 as a calibrator for IgA immunodetection.

10. Use of a recombinant multimeric IgA protein according to any one of claims 1 to 2 in the development of IgA immune-based medicaments.