Mutated human erythropoietin expressed based on insect cell expression system and its use

Abstract

This invention relates to the field of biotechnology, specifically disclosing a mutant human erythropoietin expressed using an insect cell expression system and its application in the detection of erythropoietin receptor agonists. The invention utilizes an artificially designed mutant erythropoietin INS1 expressed via an insect cell expression system. This mutant has a lower molecular weight than endogenous human EPO, does not disrupt the protein's tertiary structure, is easy to purify, and can be recognized by existing commercially available erythropoietin antibodies. Simultaneously, a mutant erythropoietin INS2 based on the EPO gene polymorphism c.577del is constructed. This mutant has a lower molecular weight than the endogenous mutant erythropoietin encoded by the human EPO gene polymorphism c.577del and can be recognized by existing antibodies against mutant erythropoietin encoded by the EPO gene polymorphism c.577del. Using both mutant erythropoietins INS1 and INS2 as internal standards for monitoring the detection of erythropoietin receptor agonists can improve the reliability of the detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a mutant human erythropoietin expressed based on an insect cell expression system and its application in doping detection. Background Technology

[0002] Erythropoietin (EPO) is a glycoprotein secreted by peritubular interstitial cells of the kidney. It is encoded by the EPO gene located on chromosome 7, which generates a 193-amino acid peptide chain. After removing the N-terminal 27-amino acid signal peptide and the C-terminal 193 arginine residue, the mature EPO protein is formed with a 165-amino acid backbone, modified by three N-glycosyl groups (asparagine residues at positions 24, 38, and 83) and one O-glycosyl group (serine residue at position 126). The peptide chain contains two pairs of disulfide bonds (Cys7-Cys161, Cys29-Cys33). As an important hormone in the human body, EPO binds to its receptor, leading to receptor dimerization. This activates multiple intracellular signaling pathways, promoting the growth of erythroid progenitor cells and erythroblasts while inhibiting their apoptosis, thereby stimulating erythrocyte production.

[0003] With the development of DNA recombination technology since the 1980s, recombinant human erythropoietin (rEPO), as one of the early recombinant protein drugs, was approved for production and clinical use in 1989, mainly for the treatment of renal anemia and anemia caused by chemotherapy for non-myeloid malignant tumors. In recent years, in order to prolong the half-life for convenient clinical use and improve efficacy, a variety of rEPO derivatives have been developed, mainly including dEPO, Continuous Erythropoietin Receptor Activator (CERA), and recombinant fusion protein comprising EPO linked to the human immunoglobulin Fc domain (EPO-Fc). All of these substances are collectively referred to as erythropoietin receptor agonists (ERAs). Among them, dEPO introduces two additional N-glycosyl groups by replacing amino acids in the natural EPO molecule. The introduction of these additional glycosyl groups increases the maximum residue content of sialic acid, thereby prolonging the serum half-life and enhancing in vivo biological activity. CERA is produced by coupling rEPO with 30 kDa polyethylene glycol and is a third-generation EPO drug with a methoxylated polyethylene glycol polymer chain, exhibiting a longer half-life. EPO-Fc, also developed to slow down EPO metabolism in vivo or improve its therapeutic properties, is a fusion protein formed by fusing rEPO with the Fc domain of human IgG.

[0004] Red blood cells, as carriers of oxygen to various tissues and organs in the human body, play a vital physiological role. ERAs (erythrocyte reductase inhibitors) increase the body's oxygen-carrying capacity by promoting red blood cell production. This effect has led to the widespread abuse of ERAs in various endurance sports that require aerobic capacity, aiming to improve the body's aerobic performance. The abuse of ERAs not only violates the principles of sportsmanship and fair competition but also causes serious harm to athletes' health, thus earning them a place on WADA's prohibited doping list (Class S2).

[0005] The deletion of adenosine at position c.577 in the EPO gene coding sequence results in the substitution of arginine (Arg) for aspartic acid (Asp). Since Arg at position 193 is the last amino acid in the wild-type EPO (WT-EPO) peptide chain, theoretically, this frameshift mutation would not alter the preceding amino acid sequence of the mutant EPO (Erythropoietin_p.Arg193AspfsTer28, VAR-EPO), but would invalidate the original stop codon at position 194. This would extend the peptide chain by 26 amino acids compared to WT-EPO before terminating translation, resulting in a molecular weight increase of approximately 3 kDa. Both WT-EPO and VAR-EPO are endogenously secreted and are referred to as endogenous EPO. WT-EPO is a glycoprotein composed of 165 amino acids, modified with glycosyl groups. Asparaginate (Asn) at positions 24, 38, and 83 modifies the N-glycosyl groups linked by N-glycosidic bonds, while serine (Ser) at position 126 modifies the O-glycosyl groups linked by O-glycosidic bonds. Although rEPO and WT-EPO have the same amino acid sequence, the composition and structure of the glycosyl groups vary depending on the species, cell line, and manufacturing process. Therefore, rEPO and WT-EPO differ in isoelectric point and molecular weight, with rEPO having a molecular weight approximately 3 kDa higher than WT-EPO. Currently, the ERA detection method used in the global doping control field distinguishes between WT-EPO and rEPO based on this molecular weight characteristic, thus achieving the purpose of detecting rEPO. The bands of VAR-EPO appear very close to those of rEPO during detection and interfere with each other. This makes it impossible to distinguish between rEPO and VAR-EPO using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE).

[0006] Currently, in the detection of ERAs (Extracorporeal Methionine Acids), a "no band" result may occur, meaning that the endogenous band is not detected. This phenomenon mainly occurs in urine samples, and occasionally in blood samples. The main reasons for the "no band" result include: (1) the concentration of endogenous EPO in the urine sample is too low, below the detection limit; (2) the sample is contaminated with proteases, leading to protein degradation in the sample; (3) factors such as sample spoilage may produce proteases, causing protein degradation; (4) improper experimental operation or quality problems of reagents and consumables may lead to the endogenous EPO not being detected.

[0007] In response to the various situations described above, doping control laboratories will take different measures. For example, if endogenous EPO is not detected due to equipment or antibody issues, the sample needs to be retested; if exogenous proteases are suspected in the sample, additional testing is required. However, since routine ERA testing currently does not include internal standards, the specific cause of "no band" samples cannot be determined. Therefore, adding internal standards to the sample during testing and monitoring the entire testing process is an effective way to address this issue.

[0008] The discovery of the EPO gene polymorphism c.577del in 2021 has deepened the understanding of endogenous EPO in the field of doping control, and has also had a significant impact on the current testing requirements and related judgments for ERAs. The bidirectional immunopurification method is an effective detection method developed by Chinese scientists for the EPO gene polymorphism c.577del. This method distinguishes between WT-EPO, VAR-EPO, and rEPO by sequentially enriching and separating different substances. Compared to traditional ERA doping control methods, this method replaces the original single-step immunoaffinity purification with a two-step immunoaffinity purification: reverse immunopurification (RI) and normal immunopurification (NI). The reverse immunopurification step uses a monoclonal antibody that specifically recognizes trace amounts of VAR-EPO to enrich VAR-EPO in the sample. Normal immunopurification follows the reverse immunopurification step, using EPO antibodies to enrich WT-EPO and rEPO that remain in the sample. After immunopurification, the enriched substances from both reverse and forward immunopurification were detected using SAR-PAGE combined with Western blotting. If the sample is an rEPO-negative sample from an EPO gene polymorphism c.577del carrier population, reverse immunopurification will detect the VAR-EPO target band, while forward immunopurification will not detect a tail or band above WT-EPO. If the sample is an rEPO-positive sample from a non-EPO gene polymorphism c.577del carrier population, reverse immunopurification will not detect the VAR-EPO target band, while forward immunopurification will detect a tail or band above WT-EPO, thus indicating rEPO positivity.

[0009] Using specific mutant erythropoietin antibodies is currently the most effective method for doping detection. This method distinguishes between VAR-EPO and rEPO by detecting the presence of mutant erythropoietin in the sample. However, if VAR-EPO is missed in the sample using this method, it could lead to a false positive for rEPO, with serious consequences. Therefore, it is essential to include an internal standard that can be recognized by VAR-EPO antibodies to monitor the testing process.

[0010] In existing research, internal standards are mainly prepared by artificially modifying antigen proteins. For example, Reihlen et al. conjugated NHS-PEG with the primary amine group of rEPO to obtain PEG-EPO conjugates of different molecular weights and screened candidate internal standards that did not interfere with ERA detection; however, these PEG-EPO conjugates could not be recognized by mutant EPO antibodies, and therefore could not monitor the VAR-EPO detection process. The Beijing Anti-Doping Laboratory, on the other hand, used NHS-PEG to modify VAR-EPO to obtain VAR-PEG conjugates. The screened internal standards can be used for both ERA detection and monitoring of the VAR-EPO detection process.

[0011] However, the internal standards synthesized by the above methods are all high-molecular-weight modified proteins, which can interfere with ERA detection if they degrade. Therefore, designing and synthesizing low-molecular-weight internal standards has important and wide-ranging application value. Summary of the Invention

[0012] To address the shortcomings of existing synthetic internal standards, which are high-molecular-weight proteins and thus have limitations in detecting erythropoietin receptor agonists (ERAs) due to their degradation interfering with ERA detection, this invention provides a mutant human erythropoietin expressed using an insect cell expression system and its application in doping detection.

[0013] This invention provides a recombinant human erythropoietin based on a wild-type EPO gene mutation, wherein the recombinant human erythropoietin is an INS1 protein, and its amino acid sequence is shown in SEQ ID NO.2.

[0014] The above-mentioned recombinant human erythropoietin based on wild-type EPO gene mutation encodes the INS1 protein with the nucleic acid sequence shown in SEQ ID NO.1.

[0015] The above-mentioned application of recombinant human erythropoietin based on wild-type EPO gene mutation in the detection of erythropoietin receptor agonist stimulants.

[0016] The present invention also provides another recombinant erythropoietin variant based on the EPO gene polymorphism c.577del, wherein the recombinant erythropoietin variant is the INS2 protein, the amino acid sequence of which is shown in SEQ ID NO.4.

[0017] The aforementioned recombinant erythropoietin variant based on the EPO gene polymorphism c.577del, encoding the INS2 protein, has the nucleic acid sequence shown in SEQ ID NO.3.

[0018] The above-mentioned application of the recombinant erythropoietin variant based on the EPO gene polymorphism c.577del in the detection of erythropoietin receptor agonist-like stimulants. Compared with existing technologies, the beneficial effects of this invention are as follows: The artificially designed mutant erythropoietin INS1 expressed through an insect cell expression system has a lower molecular weight than endogenous human EPO, making it easier to purify without damaging the protein's tertiary structure. It can be recognized by existing commercially available erythropoietin antibodies, and can be used as an internal standard for monitoring erythropoietin receptor agonist doping detection, effectively assessing the reliability of the detection. The mutant erythropoietin INS2 constructed in this invention, based on the EPO gene polymorphism c.577del, has a lower molecular weight than the endogenous mutant erythropoietin encoded by the human EPO gene polymorphism c.577del. It can be recognized by existing mutant erythropoietin antibodies encoded by the EPO gene polymorphism c.577del, and can be used as an internal standard for monitoring erythropoietin receptor agonist doping detection. It can detect whether the sample contains VAR-EPO encoded by the EPO gene polymorphism c.577del, improving the reliability of the detection. Attached Figure Description

[0019] Figure 1 This is a sequencing peak diagram of the mutant erythropoietin INS1 of this invention; Figure 2 This is the purification result of the mutant erythropoietin INS1 of this invention; in the figure, M is the protein molecular weight marker; Lord is the sample loaded onto the chromatography column; FT is the sample flowed through the chromatography column; E is the imidazole elution buffer, and the imidazole concentration of the elution buffer is E2>E1; Figure 3 This is a sequencing peak diagram of the mutant erythropoietin INS2 of this invention; Figure 4 This is the purification result of the mutant erythropoietin INS2 of this invention; M in the figure is the protein molecular weight marker; Lord is the sample loaded onto the chromatography column; FT is the sample flow-through of the chromatography column; Ni ion affinity chromatography elution buffer imidazole concentration E2>E1; ion exchange chromatography elution buffer NaCl concentration E5>E4>E3>E2>E1; Figure 5 This invention provides the detection results of endogenous EPO or other ERAs by using mutant erythropoietin INS1 as an internal standard. Figure 6 The results of VAR-EPO detection using mutant erythropoietin INS2 as an internal standard are presented in this invention. Detailed Implementation

[0020] Baculoviruses are a common tool for recombinant protein expression. A common approach involves infecting insect cells with baculoviruses and using the cells' own components for viral amplification and target protein expression. Based on this, the Baculovirus Expression Vector System (BEVS) has been widely used in scientific research and industrial production. Compared to other commonly used recombinant protein expression systems, the baculovirus expression system offers advantages such as high levels of exogenous gene expression, ease of expression of heterologous multimeric proteins, and high safety. Insect cells possess the protein processing mechanisms of eukaryotic cells, capable of complex post-translational modifications such as glycosylation, phosphorylation, and acylation, producing active proteins in their native structural and functional states. Furthermore, insect cells can grow in suspension, facilitating large-scale culture and promoting the expression of recombinant proteins on a large scale. This invention utilizes the Invitrogen Bac-to-Bac® system to recombinantly express human erythropoietin and uses it as an internal standard to monitor the stimulant detection of erythropoietin receptor agonists. The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments (Note: The vectors and cells involved in the present invention are commercial products, and the primers are from Beijing Yiqiao Shenzhou Technology Co., Ltd.).

[0021] The nucleic acid sequence of the artificially designed low molecular weight recombinant human erythropoietin variant INS1 provided by the present invention is shown in SEQ ID NO.1, and the amino acid sequence of INS1 is shown in SEQ ID NO.2.

[0022] The nucleic acid sequence of the low molecular weight recombinant human erythropoietin variant INS2 based on the EPO gene polymorphism c.577del provided by the present invention is shown in SEQ ID NO.3, and the amino acid sequence of INS2 is shown in SEQ ID NO.4.

[0023] Example 1: Preparation of low molecular weight rEPOINS1 derived from wild-type EPO gene mutation expressed by an insect cell expression system S1. Construction of expression vector (1) Gene fragment of INS1 was obtained by gene synthesis and used as the target gene; (2) The target gene and vector are ligated and transformed into competent cells; (3) Design primer pairs and sequence the constructed expression vector to obtain plasmids containing the correct sequences; the plasmid peak diagram is shown below. Figure 1 As shown. The INS1 gene sequence is: (SEQ ID NO.1).

[0024] (4) Transfect the plasmid containing the target protein into competent cells for amplification to obtain a sufficient amount of expression plasmid.

[0025] S2. Constructing recombinant rod-shaped plasmids The obtained expression plasmid was transformed into H10Bac competent cells. White spots were selected from blue-white colonies, and the plasmid was extracted and confirmed by PCR to be correct, thus obtaining the recombinant rod plasmid. S3, Rescuing Recombinant Baculovirus The correctly identified recombinant baculovirus was transferred into Sf9 cells and cultured at 27°C for 3 days. The cell culture supernatant was collected, and the P1 generation recombinant baculovirus was transferred into new SF9 cells at a volume ratio of 2%. The cells were cultured again to obtain high-titer P2 generation recombinant virus, and the virus titer was measured.

[0026] S4. Protein Expression and Purification P2 generation recombinant baculovirus was used to inoculate large quantities of cultured Hi-5 host cells at an MOI of 1 / 200, with each cell containing 100 mL of the virus. The infected cells were then cultured at 27°C for 2–4 days to express the target protein.

[0027] The recombinant protein INS1 was obtained after purification using a Ni ion affinity ladder, and the results are as follows: Figure 2 As shown.

[0028] Ni ion affinity chromatography buffer: Loading buffer= 20 mM PB, pH=7.0, NaCl, glycerol, imidazole; Washing buffer=20 mM PB, pH=7.0, NaCl, glycerol, imidazole.

[0029] The amino acid sequence of the target protein INS1 was obtained as follows: APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACHHHHHHH (SEQ ID NO. 2).

[0030] The basic physicochemical properties of the target protein are: amino acid length 167, molecular weight 18.62 kDa, and isoelectric point 8.21 (the physicochemical properties of the protein are calculated based on the mature protein (excluding the signal peptide)).

[0031] Example 2: Preparation of low molecular weight VAR-EPO based on the EPO gene polymorphism c.577del expressed by an insect expression system S1. Construction of expression vector (1) Gene fragment of INS2 was obtained by gene synthesis and used as the target gene; (2) The target gene and vector are ligated and transformed into competent cells; (3) Design primer pairs and sequence the constructed expression vector to obtain plasmids containing the correct sequences; the plasmid peak diagram is shown below. Figure 3 As shown. The INS2 gene sequence is: (SEQ ID NO.3).

[0032] (4) Transfect the plasmid containing the target protein into competent cells for amplification to obtain a sufficient amount of expression plasmid.

[0033] S2. Constructing recombinant rod-shaped plasmids The obtained expression plasmid was transformed into H10Bac competent cells. White spots were selected from blue-white colonies, and the plasmid was extracted and confirmed by PCR to be correct, thus obtaining the recombinant rod plasmid. S3, Rescuing Recombinant Baculovirus The correctly identified recombinant baculovirus was transferred into Sf9 cells and cultured at 27°C for 3 days. The cell culture supernatant was collected, and the P1 generation recombinant baculovirus was transferred into new SF9 cells at a volume ratio of 2%. The cells were cultured again to obtain high-titer P2 generation recombinant virus, and the virus titer was measured.

[0034] S4. Protein Expression and Purification P2 generation recombinant baculovirus was used to inoculate large quantities of cultured Hi-5 host cells at an MOI of 1 / 200, with each cell containing 100 mL of the virus. The infected cells were then cultured at 27°C for 2–4 days to express the target protein.

[0035] The recombinant protein INS2 was obtained after purification by Ni ion affinity layer and exchange layer, as shown in the following results. Figure 4 As shown.

[0036] Ni ion affinity chromatography buffer: Loading buffer= 20 mM PB, pH=7.0, NaCl, glycerol, imidazole; Washing buffer=20 mM PB, pH=7.0, NaCl, glycerol, imidazole.

[0037] Ni ion exchange chromatography buffer: Loading buffer= 20 mM PB, pH=7.0, NaCl, glycerol; Washing buffer= 20 mM PB, pH=7.0, NaCl, glycerol.

[0038] The amino acid sequence of the target protein INS2 was obtained as follows: APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDDDQVCPPGHIHHLPHQHCLCHTLPRHS (SEQ ID NO.4).

[0039] The basic physicochemical properties of the target protein are: amino acid length 192, molecular weight 21.32 kDa, and isoelectric point 7.75 (the physicochemical properties of the protein are calculated based on the mature protein (excluding the signal peptide)).

[0040] Example 3: Mutant erythropoietin INS1 as an internal standard for the detection of erythropoietin receptor agonists. (1) Preparation of internal standard solution Take 10 µL of 1 µg / mL mutant erythropoietin INS1 protein stock solution and dilute it in 490 µL of 0.05% casein PBS solution to prepare a 20 ng / mL mutant erythropoietin INS1 protein working solution. Take 25 µL of mutant erythropoietin INS1 protein working solution and dilute it in 975 µL of 0.05% casein PBS solution to prepare a 0.5 ng / mL INS1 internal standard solution.

[0041] (2) Sample ultrafiltration concentration Take 15 mL of the urine sample to be tested, and add 300 µL of protease inhibitor solution, 50 µL of INS1 internal standard solution, and 1.5 mL of Tris-HCl buffer (3.75 M, pH 7.4) sequentially. Mix thoroughly to adjust the pH of the sample, and centrifuge for 20 minutes at a centrifugal force of 4000 g. After centrifugation, pour the supernatant into a 15 mL 10 kD ultrafiltration centrifuge tube (Millipore) and concentrate it to 200 µL-500 µL. Replace the supernatant with PBS buffer, and transfer the recovered supernatant to a 0.5 mL 30 kD ultrafiltration centrifuge tube (Millipore) and concentrate it to approximately 20 µL.

[0042] (3) Immunopurification Transfer the concentrated sample from step (2) into an EPO antibody-coated ELISA plate (Stemcell) and incubate overnight at 4°C. After incubation, wash the ELISA plate, add 20 µL of SAR loading buffer, and incubate at 95°C for 5 minutes to elute the EPO protein. After incubation, cool the ELISA plate for sample loading.

[0043] (4) Western blot The elution buffer from step (3) was used for Western blotting of proteins. The method was as follows: SDS-PAGE gel electrophoresis was performed using a 10% Bis-Tris protein gel (Invitrogen) with an SDS-MOPS buffer system at a constant voltage of 200V for 75 minutes. Proteins were transferred to a 0.45µm PVDF membrane (Millipore) using Towbin transfer buffer and a semi-dry transfer method, at 1.0 mA / cm² for 60 minutes and 1.56 mA / cm² for 20 minutes. After transfer, the PVDF membrane was blocked in 5% LFM / PBS for 60 minutes. After blocking, the PVDF membrane was rinsed with PBS and then incubated overnight at 4°C with 20 mL of biotin-based primary antibody solution diluted in 1% LFM / PBS (0.5 µg / mL, BAM2871, R&D).

[0044] After primary antibody incubation, the PVDF membrane was rinsed three times with PBS, then incubated for 60 minutes at room temperature with 40 mL of streptavidin-labeled HRP solution (0.25 µg / mL, Streptavidin Protein HRP, Thermo) diluted with 1% LFM / PBS. After incubation, the PVDF membrane was rinsed five times with PBS, and then developed using HRP chemiluminescent substrate chromogenic reagent (SuperSignal West Femto). The detection results and interpretation are as follows. Figure 5 As shown in Table 1 below.

[0045]

[0046] When endogenous EPO is not detected, the presence or absence of the INS1 protein band is used to determine the reason for the lack of detection, thereby assessing the validity of the test. Strictly rule out false negatives caused by system failure, avoid misjudging the absence of EPO in the sample due to experimental operation problems, and ensure the reliability of the test results.

[0047] Example 4: Mutant erythropoietin INS2 was used as an internal standard for the detection of recombinant erythropoietin stimulants. The detection was performed using a two-way immunopurification method combined with Western blotting, including the following steps: (1) Preparation of internal standard solution Take 10 µL of 1 µg / mL mutant erythropoietin INS2 protein stock solution and dilute it in 490 µL of 0.05% casein PBS solution to prepare a 20 ng / mL mutant erythropoietin INS2 protein working solution. Take 25 µL of mutant erythropoietin INS2 protein working solution and dilute it in 975 µL of 0.05% casein PBS solution to prepare a 0.5 ng / mL INS2 internal standard solution.

[0048] (2) Sample ultrafiltration concentration Take 15 mL of the urine sample to be tested, and add 300 µL of protease inhibitor solution, 50 µL of INS2 internal standard solution, and 1.5 mL of Tris-HCl buffer (3.75 M, pH 7.4) sequentially. Mix thoroughly to adjust the pH of the sample, and centrifuge for 20 minutes at a centrifugal force of 4000 g. After centrifugation, pour the supernatant into a 15 mL 10 kD ultrafiltration centrifuge tube (Millipore) and concentrate it to 200 µL-500 µL. Replace the supernatant with PBS buffer and transfer the concentrated sample to a 1.5 mL low-adsorption centrifuge tube.

[0049] (3) Reverse immunopurification Add 1 µg of VAR-EPO monoclonal antibody (Sino-Pharmaceutical) to the concentrated sample, mix thoroughly, and incubate at room temperature for 1 hour. After incubation, add 775 µL of washed Anti-rabbit IgG magnetic beads (Dynabeads M-280 Sheep anti-Rabbit IgG, Invitrogen), mix thoroughly, and incubate overnight at 4°C. After incubation, collect the magnetic beads and supernatant separately.

[0050] After washing the enriched magnetic beads three times, add 25 µL of SAR loading buffer and incubate at 95°C for 5 minutes to elute the proteins.

[0051] (4) Forward immunopurification The supernatant recovered in step (3) was transferred to 50 µL of EPO antibody (AB-286-NA, R&D) Anti-rabbit IgG magnetic beads (Dynabeads M-280 Sheep anti-Rabbit IgG, Invitrogen) complex that had been incubated overnight at 4°C for 1 hour at room temperature. After incubation, the magnetic beads were collected. The enriched magnetic beads were washed 3 times, and then 25 µL of SAR loading buffer was added and incubated at 95°C for 5 minutes to elute the protein.

[0052] (5) Immunoblotting The magnetic bead elution buffers from steps (3) and (4) were then subjected to Western blotting. The procedure was as follows: SDS-PAGE gel electrophoresis was performed using a 10% Bis-Tris protein gel (Invitrogen) with an SDS-MOPS buffer system at a constant voltage of 200V for 75 minutes. Proteins were then transferred to a 0.45 µm PVDF membrane (Millipore) using Towbin transfer buffer and a semi-dry transfer method at 1.0 mA / cm². 2 Transfer at 60 minutes, connection at 1.56 mA / cm. 2 Transfer for 20 minutes. After transfer, block the PVDF membrane in 5% LFM / PBS for 60 minutes. After blocking, rinse the PVDF membrane with PBS, add 20 mL of biotin-based primary antibody solution diluted with 1% LFM / PBS (0.5 µg / mL, BAM2871, R&D), and incubate overnight at 4°C.

[0053] After primary antibody incubation, the PVDF membrane was rinsed three times with PBS, then incubated for 60 minutes at room temperature with 40 mL of streptavidin-labeled HRP solution (0.25 µg / mL, Streptavidin Protein HRP, Thermo) diluted with 1% LFM / PBS. After incubation, the PVDF membrane was rinsed five times with PBS, and then developed using HRP chemiluminescent substrate chromogenic reagent (SuperSignal West Femto). The results are as follows. Figure 6 As shown. The presence of VAR-EPO encoded by the EPO gene polymorphism c.577del can be detected by the bands in the magnetic bead elution buffer; the sample result is only acceptable if the quality control of the elution buffer in step (3) and the sample both detect the internal standard INS2.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Recombinant human erythropoietin based on mutation of wild type EPO gene, characterized in that: The recombinant human erythropoietin is INS1 protein, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The recombinant human erythropoietin based on wild-type EPO gene mutation as described in claim 1, characterized in that: The nucleic acid sequence encoding the INS1 protein is shown in SEQ ID NO.

1.

3. The application of recombinant human erythropoietin INS1 protein based on wild-type EPO gene mutation as described in claim 1 in the detection of erythropoietin receptor agonist stimulants.

4. A recombinant erythropoietin variant based on the EPO gene polymorphism c.577del, characterized in that: The recombinant erythropoietin variant is the INS2 protein, whose amino acid sequence is shown in SEQ ID NO.

4.

5. The recombinant erythropoietin variant based on the EPO genetic polymorphism c.577del according to claim 4, characterized in that: The nucleic acid sequence encoding the INS2 protein is shown in SEQ ID NO.

3.

6. The application of the recombinant erythropoietin variant INS2 protein based on the EPO gene polymorphism c.577del as described in claim 4 in the detection of erythropoietin receptor agonist stimulants.

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