Preparation and application of hACE2 recombinant protein

Abstract

The invention discloses an hACE2 recombinant antigen, and a preparation method of the hACE2 recombinant antigen comprises the following steps: (1) according to a GenBank database, obtaining a gene sequence of human angiotensin transferase 2 as shown in SEQ ID NO.1, and performing codon optimization on the gene sequence to obtain a gene sequence as shown in SEQ ID NO.2; (2) inserting the gene sequence SEQ ID NO.2 obtained in the step (1) into a plasmid vector pCDNA3.1 to form a recombinant expression vector; (3) transfecting the recombinant expression vector in the step (2) into Hek293 suspension cells to form recombinant cells; (4) culturing the recombinant cells, and expressing a human angiotensin transferase 2 recombinant antigen; and (5) separating and purifying the human angiotensin transferase 2 recombinant antigen. The recombinant protein obtained by the invention can be expressed at a higher level, the production cost and the time cost are reduced, and the recombinant protein has obvious advantages in the aspect of antigen specificity; the sensitivity of the antigen is detected by using a competition method by taking the antigen as a coating antigen.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture recombinant proteins, and in particular to the preparation and application of hACE2 recombinant protein. Background Art

[0002] At present, there are two sources of protein: one is from natural protein, and the other is to form recombinant protein by plasmid transfection so that the engineered cells carry the target gene fragment to express the target protein. The activity and purity of recombinant protein are higher than those of natural protein, and it is safer and has been widely used.

[0003] Studies have confirmed that the new coronavirus SARS-CoV-2 enters host cells through the same receptor angiotensin converting enzyme-2 (ACE2) as SARS-CoV. Moreover, studies have continuously confirmed that ACE2 is closely related to the occurrence of tissue damage and complications in the later stages of the new coronavirus infection. As an important potential therapeutic target for the treatment of the new coronavirus, angiotensin converting enzyme-2 is of great significance for studying the pathogenesis of the new coronavirus and developing drugs to treat the new coronavirus.

[0004] Therefore, how to provide a humanized ACE2 recombinant antigen and apply this recombinant antigen to enzyme-linked immunosorbent assay, colloidal gold and other platforms to detect coronavirus, and improve the sensitivity and specificity of coronavirus detection are technical problems that technicians in this field urgently need to solve. Summary of the invention

[0005] The present invention aims to solve the above problems. In a first aspect, a hACE2 recombinant antigen is provided. The preparation method of the hACE2 recombinant antigen is as follows: (1) The gene sequence of human angiotensin converting enzyme 2 was obtained from the GenBank database as shown in SEQ ID NO.1, and codons were optimized to obtain the gene sequence as shown in SEQ ID NO.2; (2) inserting the gene sequence SEQ ID NO.2 obtained in step (1) into the plasmid vector pCDNA3.1 to form a recombinant expression vector; (3) Transfecting the recombinant expression vector in step (2) into Hek293 suspension cells to form recombinant cells; (4) culturing the recombinant cells to express human angiotensin-converting enzyme 2 recombinant antigen; (5) Isolate and purify the recombinant human angiotensin-converting enzyme 2 antigen.

[0006] Further preferably, in step (2), the gene sequence SEQ ID NO.2 obtained in step (1) is inserted between the BamHI and EcoRI restriction sites of the plasmid vector pCDNA3.1 to form a recombinant expression vector.

[0007] Based on the above technical solution, the culture conditions of the CO2 shaker incubator for culturing the recombinant cells in step (4) of the preparation method are as follows: CO2 concentration 5%-8%, temperature 35-37°C, humidity 70%, and rotation speed 100-120r / min.

[0008] Based on the above technical solution, in step (3), 0.6-1 ug of plasmid DNA is added to 1 ml of cell solution during transfection, and the transfection reagent used is linear polyethyleneimine hydrochloride PEI MAX, with a concentration of 1-2 ug / ul, and 3-4.5 ul is added for every 1 ml of cell solution transfected.

[0009] Based on the above technical solution, the Hek293 suspension cell culture medium includes amino acids, inorganic salts, vitamins, trace elements, carbohydrates and organic molecules.

[0010] Furthermore, the amino acids in the culture medium include: glycine 20-60 mg / L, L-histidine 30-80 mg / L, L-isoleucine 80-180 mg / L, L-leucine 160-200 mg / L, L-lysine 250-300 mg / L, L-methionine 50-100 mg / L, L-phenylalanine 80-150 mg / L, L-threonine 100-200 mg / L, L-tryptophan 35-75 mg / L, L-valine 100-200 mg / L, L- Alanine 6-25mg / L, L-glutamine 1000-1500mg / L, L-arginine 200-600mg / L, L-asparagine 150-300mg / L, L-aspartic acid 20-60mg / L, L-cysteine ​​25-75mg / L, L-cystine 40-100mg / L, L-glutamate 10-30mg / L, L-proline 55-165mg / L, L-serine 50-150mg / L, L-tyrosine 50-100mg / L; The inorganic salts in the culture medium include: calcium chloride 100-500 mg / L, magnesium sulfate 50-150 mg / L, potassium chloride 300-500 mg / L, sodium bicarbonate 2000-5000 mg / L, disodium hydrogen phosphate 150-300 mg / L, zinc sulfate 1-2 mg / L; The vitamins in the culture medium include: vitamin B1 2-10 mg / L, vitamin B2 0.1-1 mg / L, vitamin B12 0.5-4 mg / L, vitamin H 0.1-1 mg / L, folic acid 2-5 mg / L, riboflavin 0.1-0.5 mg / L, biotin 0.1-0.5 mg / L, lipoic acid 0.1-1 mg / L, inositol 2-10 mg / L, vitamin B6 2-10 mg / L, vitamin C 2.5-10 mg / L, vitamin E 1-10 mg / L, D-calcium pantothenate 1-10 mg / L, nicotinamide 1-10 mg / L, choline chloride 2-6 mg / L; The trace elements in the culture medium include: CuSO4·5H2O 0.1-0.5mg / L, AlCl3 0.1-1mg / L, CoCl2·6H2O 0.2-1mg / L, Na2SeO3 0.1-0.5mg / L, NiCl2 0.1-1mg / L, MnCl2 0.5-5mg / L, ZnSO4·7H2O 0.5-2mg / L, FeSO4·7H2O 0.2-1mg / L; The carbohydrates and organic molecules in the culture medium include: 2000-5000 mg / L glucose and 50-200 mg / L dextran.

[0011] In the second aspect, based on the same invention, the present invention also provides a recombinant expression vector obtained by recombining the plasmid vector pCDNA3.1 and the human angiotensin converting enzyme 2 codon-optimized gene sequence SEQ ID NO.2.

[0012] In a third aspect, the present invention further provides a recombinant cell, wherein the recombinant cell is formed by transfecting the recombinant expression vector described in the second aspect into Hek293 suspension cells.

[0013] Furthermore, the hACE2 recombinant antigen is an extracellular expression product of Hek293 cells, which means that hACE2 is secreted into the culture medium outside the Hek293 cells and does not need to be extracted from the inside of the cells, which plays a beneficial and critical role in the subsequent purification and application of hACE2.

[0014] In a fourth aspect, the present invention also provides the use of a hACE2 recombinant antigen as described in the first aspect, a recombinant expression vector as described in the second aspect, and a recombinant cell as described in the third aspect in detecting coronavirus.

[0015] The present invention has the following beneficial effects: The present invention is based on human angiotensin-converting enzyme 2 (hACE2), and through codon optimization, it is constructed into an expression vector for eukaryotic cell expression. Because it has the function of post-translational modification of protein, and after the transfection method and reagent dosage are optimized, as well as the content of the culture medium and feed medium components are adjusted and optimized, the recombinant protein can be expressed at a higher level, and the production and time costs are reduced. In addition, the test found that the protein is extracellularly expressed, and after protein purification, a recombinant human angiotensin-converting enzyme 2 protein with high purity and high biological activity is obtained, which has obvious advantages in antigen specificity; the human angiotensin-converting enzyme 2 recombinant antigen protein is used as a coating antigen to detect its sensitivity using a competitive method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0017] Figure 1 : The recombinant expression vector of the present invention; Figure 2 : HEK293 suspension cell growth picture of the present invention; Figure 3 : The cell transfection effect diagram of the present invention (plasmid with green fluorescent label); Figure 4 : Electrophoresis diagram of the recombinant protein human angiotensin converting enzyme 2 purified by the present invention. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0019] Example 1: Recombinant plasmid construction (1) With reference to the gene sequence analysis of the GenBank database, the sequence reference NM_001386260.1 was used to optimize the nucleotides by using the Jcat online codon optimization software. The sequence before codon optimization is shown in SEQ ID NO.1, and the sequence after codon optimization is shown in SEQ ID NO.2. Human angiotensin converting enzyme 2 (hACE2) was obtained, and its GC content accounted for 55%; the corresponding gene sequence is as follows (i.e., the sequence after codon optimization is SEQ ID NO.2): Sequence before codon optimization SEQ ID NO.1: Codon optimized sequence SEQ ID NO.2: (2) Using Snap Gene design software, the gene sequence was inserted between the BamHI and EcoRI restriction sites of the plasmid vector pCDNA3.1 and sent to Qingdao Branch of Beijing Qingke Biotechnology Co., Ltd. for gene synthesis.

[0020] (3) Expand the culture of the synthesized E. coli cells and extract the recombinant plasmid for subsequent cell expression.

[0021] Example 2: Recombinant Antigen Expression and Purification 2.1 Inducible expression A. HEK293 suspension cells (purchased from Hangzhou Peiding Biotechnology Co., Ltd.) were revived. The cell cryovials were taken out from liquid nitrogen and rotated in a 37°C water bath for 1-2 minutes to thaw the cells quickly. The thawed cells were centrifuged at 1000rpm for 10 minutes in a centrifuge. The cryovials were opened in a clean bench and the supernatant was discarded. 1ml of HEK293 suspension cell culture medium was added to the cryovials and mixed evenly. The resuspended cell solution was transferred to a shaking flask containing 30ml of HEK293 suspension cell culture medium. After calculating the cell viability, the cells were placed in a CO2 shaking incubator (5% CO2, 75% humidity, and 100rpm / min speed). B. Cell counting: add 20ul HEK293 cell suspension culture medium to the cell counting plate, cover with a coverslip, and count under a microscope; C. Calculation of cell viability: add a small amount of trypan blue dye solution to the cell culture medium, observe the number of live and dead cells under a microscope, and calculate the cell viability; D. After 2-4 days of cell culture, count the cells and calculate the viability. When the cell concentration reaches 2-3*10 6 / ml, dilute to 2-3*10 5 1000 cells / ml for subculture, and then cultured in a CO2 shaker incubator (the incubator conditions are the same as step A); Cell transfection, when the cell density grows to 3*10 6 When the cell volume reaches the required transfection volume and the cell viability reaches 90%, use HEK293 suspension cell culture medium to dilute the cells to 1-2*10 one day before transfection. 6 / ml, culture overnight, and the cell viability was detected on the next day to reach 90%, and the cell number was calculated and diluted to 3-5*10 using HEK293 suspension cell culture medium. 6Each 100ml cell solution was used as the transfection solution. 0.4-1ug of plasmid DNA was added to each transfection of 100ml of cell solution, and 40ul of HEK293 suspension cell culture medium was used to dilute the plasmid DNA and mix well. After mixing well, 2-8ul of linear polyethyleneimine hydrochloride PEIMAX with a concentration of 1ug / ul was added and shaken to mix well. The plasmid vector mixture was incubated at room temperature for 15min to form the plasmid vector mixture. The HEK293 suspension cell culture medium was composed of the following components and concentrations: 20mg / L glycine, 40mg / L L-histidine, 100mg / L L-isoleucine, 160mg / L L-leucine, L-Lysine 260mg / L, L-Methionine 50mg / L, L-Phenylalanine 80mg / L, L-Threonine 110mg / L, L-Tryptophan 40mg / L, L-Valine 110mg / L, L-Alanine 12mg / L, L-Glutamine 1000mg / L, L-Arginine 300mg / L, L-asparagine 160mg / L, L-aspartic acid 30mg / L, L-cysteine ​​35mg / L, L-cystine 45mg / L, L-glutamic acid 15mg / L, L-proline 85mg / L, L-serine 70mg / L, L-tyrosine 50mg / L, calcium chloride 150mg / L, magnesium sulfate 75mg / L, potassium chloride 300mg / L, sodium bicarbonate 2000mg / L, disodium hydrogen phosphate 180mg / L, zinc sulfate 1mg / L, Vitamin B1 5mg / L, Vitamin B2 0.2mg / L, Vitamin B12 0.5mg / L, Vitamin H 0.2mg / L, Folic acid 2mg / L, Riboflavin 0.2mg / L, Biotin 0.1mg / L, Lipoic acid 0.3mg / L, Inositol 2mg / L, Vitamin B6 3mg / L, Vitamin C 3mg / L, Vitamin E 3mg / L, D-calcium pantothenate 3mg / L, Nicotinamide 2mg / L, Choline chloride 2mg / L, CuSO4 .5H2O 0.1mg / L, AlCl3 0.4mg / L, CoCl2 .6H2 O 0.2mg / L, Na2 SeO3 0.3mg / L, NiCl2 0.2mg / L, MnCl2 2mg / L, ZnSO4 .7H2 O 0.5mg / L, FeSO4 .7H2 O 0.25mg / L, glucose 3000mg / L, dextran 100mg / L E. Add the mixture to the cells to be transfected, mix gently, and place in a CO2 shaker incubator for culture (the incubator conditions are the same as step A); F. After 24 hours, feed medium ① (glucose 60g / L, glutamic acid 0.7g / L, L-glutamine 1g / L, glycine 0.6g / L, aspartic acid 0.6g / L, cysteine ​​0.6mg / L, arginine hydrochloride 1g / L, folic acid 0.1mg / L, inositol 0.2mg / L, biotin 10mg / L, riboflavin 15mg / L, calcium chloride 300mg / L, potassium chloride 85mg / L, magnesium sulfate 80mg / L, manganese chloride 10mg / L, zinc chloride 6mg / L) was added at a ratio of 10%; after 72 hours, feed medium ② (glucose 150g / L) was added at a ratio of 2%, and feed medium ② was added at 2% every 24 hours until the cell viability reached 20% after 10 days, and cell fluid was collected; G. After collecting the cell culture medium, centrifuge it at 9000 rpm for 35 minutes, discard the precipitate and take the supernatant for collection and purification of human angiotensin converting enzyme 2 recombinant protein.

[0022] 2.2 Protein purification: 2.2.1 Column A purification Take AT Protein A Diamond protein chromatography column (purchased from Boglon (Shanghai) Biotechnology Co., Ltd.), balance the column with 10 column volumes of ultrapure water, and then balance the column with 10 column volumes of equilibration buffer (1×PBS (pH7.4)) and add the above-mentioned human angiotensin converting enzyme 2 recombinant protein cell supernatant intermediate. After the loading is completed, wash with 5 column volumes of 1×PBS (PH7.4), then elute and collect the protein with 50mM glycine (PH3.0). Treat the column with 5 column volumes of 0.2M NaOH, and then wash with 10 column volumes of water for later use.

[0023] 2.2.2 Protein dialysis Transfer the collected eluted protein to a dialysis bag with 2L 1×PBS (pH7.4) as the dialysis fluid, dialyze overnight at 4°C. On the second day, change the dialysis fluid to 2L 1×PBS (pH7.4) for 8h, then change to 2L 1×PBS (pH7.4) for dialyzing overnight at 4°C. After the dialysis, take out the protein sample from the dialysis bag and place it at -20°C for standby use. (The dialysis fluid needs to be prepared in advance and pre-cooled at 4°C; the entire dialysis process is performed at 4°C) In addition, the transfection conditions of Example 2 were optimized, and the yield of purified hACE2 protein was gradually increased: Optimization 1: The cell density during transfection is 3*10 6Each 100 ml of cell solution was transfected with 0.4 ug of plasmid DNA, and the plasmid DNA was diluted with 40 ul of HEK293 suspension cell culture medium and mixed. After mixing, 2 ul of linear polyethyleneimine hydrochloride PEI MAX at a concentration of 1 ug / ul was added, and the expression was cultured for 5 days before purification.

[0024] Optimization 2: The cell density during transfection is 3*10 6 Each 100 ml of cell solution was transfected with 0.4 ug of plasmid DNA, and the plasmid DNA was diluted with 40 ul of HEK293 suspension cell culture medium and mixed. After mixing, 1 ug / ul of linear polyethyleneimine hydrochloride PEI MAX: 4 ul was added, and the expression was cultured for 5 days before purification.

[0025] Optimization 3: The cell density during transfection is 4*10 6 Each 100 ml of cell solution was transfected with 0.6 ug of plasmid DNA, and the plasmid DNA was diluted with 40 ul of HEK293 suspension cell culture medium and mixed. After mixing, 3 ul of linear polyethyleneimine hydrochloride PEI MAX at a concentration of 1 ug / ul was added, and the expression was cultured for 4 days before purification.

[0026] Optimization 4: The cell density during transfection is 4*10 6 Each time 100 ml of cell solution was transfected, 6 ug of plasmid DNA was added, and 40 ul of HEK293 suspension cell culture medium was used to dilute the plasmid DNA and mix well. After mixing well, 4 ul of linear polyethyleneimine hydrochloride PEI MAX at a concentration of 1 ug / ul was added, and the expression was cultured for 7 days before purification.

[0027] The yield of purified hACE2 protein after expression under each optimized condition is shown in Table 1 below: Table 1

[0028] Example 3: Competitive detection of hACE2 recombinant protein S-RBD (the full name of Spike Receptor-Binding Domain) (Chinese name: Spike protein receptor binding domain, which is a part of the spike protein on the surface of the new coronavirus and is responsible for binding to the ACE2 receptor of the host cell) was used as the test sample. The method was a competitive method. S-RBD and self-produced S-RBD-HRP (S-RBD protein labeled with horseradish peroxidase) competed for the coated ACE2 at the same time. It can be seen from the data in Table 2 that, overall, the hACE recombinant protein has a higher binding ability with S-RBD.

[0029] Table 2

[0030] Clinical samples were used as test samples, the methodology was competitive method, and the sample addition amount was: 50μl clinical samples (neutralizing antibodies) + 50μl 2.5μg / ml S-RBD-HRP. Neutralizing antibodies in clinical samples and coated hACE2 competed for S-RBD-HRP at the same time. The lower the value, the stronger the binding ability between S-RBD-HRP and neutralizing antibodies in clinical samples, and the weaker the binding ability between coated hACE2 and S-RBD-HRP. It can be seen from the data in Table 3 that hACE titer and linearity are good.

[0031] Table 3

[0032] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A hACE2 recombinant antigen, characterized in that: The preparation method of the hACE2 recombinant antigen is as follows: (1) The gene sequence of human angiotensin converting enzyme 2 was obtained from the GenBank database as shown in SEQ ID NO.1, and codons were optimized to obtain the gene sequence as shown in SEQ ID NO.2; (2) inserting the gene sequence SEQ ID NO.2 obtained in step (1) into the plasmid vector pCDNA3.1 to form a recombinant expression vector; (3) Transfecting the recombinant expression vector in step (2) into Hek293 suspension cells to form recombinant cells; (4) culturing the recombinant cells to express human angiotensin-converting enzyme 2 recombinant antigen; (5) Isolate and purify recombinant human angiotensin-converting enzyme 2 antigen.

2. The hACE2 recombinant antigen according to claim 1, characterized in that: In the step (2), the gene sequence SEQ ID NO.2 obtained in the step (1) is inserted between the BamHI and EcoRI restriction sites of the plasmid vector pCDNA3.1 to form a recombinant expression vector.

3. The hACE2 recombinant antigen according to claim 1, characterized in that: The culture conditions of the CO2 shaker incubator for culturing the recombinant cells in step (4) of the preparation method are as follows: CO2 concentration 5%-8%, temperature 35-37°C, humidity 70%, and rotation speed 100-120 r / min.

4. The hACE2 recombinant antigen according to claim 1, characterized in that: In the step (3), 0.6-1 ug of plasmid DNA is added to 1 ml of cell solution during transfection, and the transfection reagent used is linear polyethyleneimine hydrochloride PEI MAX, with a concentration of 1-2 ug / ul. 3-4.5 ul is added for every 1 ml of cell solution transfected.

5. The hACE2 recombinant antigen according to claim 1, characterized in that: The Hek293 suspension cell culture medium comprises amino acids, inorganic salts, vitamins, trace elements, carbohydrates and organic molecules.

6. The hACE2 recombinant antigen according to claim 5, characterized in that: The amino acids in the culture medium include: 20-60 mg / L glycine, 30-80 mg / L L-histidine, 80-180 mg / L L-isoleucine, 160-200 mg / L L-leucine, 250-300 mg / L L-lysine, 50-100 mg / L L-methionine, 80-150 mg / L L-phenylalanine, 100-200 mg / L L-threonine, 35-75 mg / L L-tryptophan, 100-200 mg / L L-valine, and L-alanine. 6~25mg / L, L-glutamine 1000~1500mg / L, L-arginine 200~600mg / L, L-asparagine 150~300mg / L, L-aspartic acid 20~60mg / L, L-cysteine ​​25~75mg / L, L-cystine 40~100mg / L, L-glutamate 10~30mg / L, L-proline 55~165mg / L, L-serine 50~150mg / L, L-tyrosine 50~100mg / L; The inorganic salts in the culture medium include: calcium chloride 100-500 mg / L, magnesium sulfate 50-150 mg / L, potassium chloride 300-500 mg / L, sodium bicarbonate 2000-5000 mg / L, disodium hydrogen phosphate 150-300 mg / L, zinc sulfate 1-2 mg / L; The vitamins in the culture medium include: vitamin B1 2-10 mg / L, vitamin B2 0.1-1 mg / L, vitamin B12 0.5-4 mg / L, vitamin H 0.1-1 mg / L, folic acid 2-5 mg / L, riboflavin 0.1-0.5 mg / L, biotin 0.1-0.5 mg / L, lipoic acid 0.1-1 mg / L, inositol 2-10 mg / L, vitamin B6 2-10 mg / L, vitamin C 2.5-10 mg / L, vitamin E 1-10 mg / L, D-calcium pantothenate 1-10 mg / L, nicotinamide 1-10 mg / L, choline chloride 2-6 mg / L; The trace elements in the culture medium include: CuSO4·5H2O 0.1-0.5mg / L, AlCl3 0.1-1mg / L, CoCl2·6H2O 0.2-1mg / L, Na2 SeO3 0.1-0.5mg / L, NiCl2 0.1-1mg / L, MnCl2 0.5-5mg / L, ZnSO4·7H2O0.5-2mg / L, FeSO4·7H2O 0.2-1mg / L; The carbohydrates and organic molecules in the culture medium include: 2000-5000 mg / L glucose and 50-200 mg / L dextran.

7. A recombinant expression vector, characterized in that: The gene is obtained by recombining the plasmid vector pCDNA3.1 and the gene sequence SEQ ID NO.2 after codon optimization of human angiotensin converting enzyme 2.

8. A recombinant cell, characterized in that The recombinant cells are formed by transfecting the recombinant expression vector as claimed in claim 7 into Hek293 suspension cells.

9. Use of a hACE2 recombinant antigen as claimed in claim 1, a recombinant expression vector as claimed in claim 7, and a recombinant cell as claimed in claim 8 in detecting coronavirus.

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