Recombinant human-derived cd8 alpha protein and preparation method and application thereof

Abstract

The application discloses a recombinant human CD8 alpha protein and a preparation method and application thereof, and belongs to the technical field of biotechnology. Two signal peptides and three labels are designed for a human T cell surface glycoprotein CD8 alpha, and are combined for use. It is found that under the condition that the signal peptides B and C-human Fc label exist, efficient secretion expression of the target protein can be realized, the recombinant human CD8 alpha protein is successfully obtained through affinity purification, the SDS-PAGE electrophoresis strip of the target protein is clear and single, and the yield is high. The recombinant human CD8 alpha protein antigen prepared through the preparation method (production process) has good immunogenicity, and animals immunized with the recombinant human CD8 alpha protein antigen can generate specific polyclonal antibodies. The preparation method (production process) can be effectively applied to large-scale production of the recombinant human CD8 alpha protein antigen, and the recombinant human CD8 alpha protein antigen is used as an immunogen for immunization, antibody preparation and production, and is further used for diagnosis of a disease marker CD8 alpha of familial CD8 deficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a recombinant human CD8 alpha protein and a preparation method and application thereof. BACKGROUND

[0002] CD8 (cluster of differentiation 8) is a transmembrane glycoprotein present on the surface of some T cells, which can act as a co-receptor of T cell receptor (TCR). The protein has two subunits, namely alpha and beta. CD8 alpha plays a crucial role in immune response and performs multiple functions in response to external and internal attacks. In T cells, CD8 alpha mainly acts as a helper receptor of MHC class I molecules. Once the T cell receptor binds to its specific antigen, Lck phosphorylates various substrates, leading to the activation of transcription factors such as NFAT, NF-κB and AP-1, and ultimately leading to the production of lymphokines, movement, adhesion and activation of cytotoxic T lymphocytes (CTL). This mechanism enables CTL to recognize and eliminate infected cells and tumor cells. In NK cells, the presence of CD8 alpha homodimers on the cell surface provides a survival mechanism, allowing the binding and lysis of multiple target cells. CD8 alpha deficiency is the cause of familial CD8 deficiency, which is a new autosomal recessive immunodeficiency characterized by the absence of cytotoxic T cells with CD8 surface protein, which will lead to repeated bacterial infections. The development of antibodies targeting CD8 alpha can quickly screen against such conditions, which helps in the diagnosis of familial CD8 deficiency disease and provides a direction for subsequent new drug development.

[0003] Many important medically valuable proteins (such as antibodies) have no biological activity or very low activity in E. coli expression system, because many eukaryotic proteins cannot complete post-translational modifications such as glycosylation, amidation, correct connection of disulfide bonds and correct assembly of subunits in this system. In the mammalian cell expression system, these post-translational modification processes can proceed normally, producing molecules identical to natural proteins. Therefore, the mammalian cell expression system is gradually forming an industry.

[0004] In the actual production of human T cell surface glycoprotein CD8 alpha, there are often problems such as low secretion efficiency of recombinant protein and low yield, which cannot meet the subsequent experimental application. Screening of suitable signal peptides to improve the secretion efficiency of recombinant protein is a common strategy, and combined with efficient purification tags, the purity and yield of recombinant protein can be further improved on the basis of efficient secretion expression, and the production process constructed can be applied to large-scale production of recombinant protein, thereby promoting the application of the protein in the fields of medical treatment, life science, etc. However, in actual production, there are many types of signal peptides and purification tags, and their efficiencies are not the same for different recombinant proteins. Therefore, finding suitable signal peptides and purification tags has become the focus of research and development. SUMMARY

[0005] 1. PROBLEMS TO BE SOLVED

[0006] The present application is directed to the problem of low yield of recombinant human CD8 alpha protein expressed by mammalian cells, and provides a recombinant human CD8 alpha protein, a preparation method and application thereof. The recombinant human CD8 alpha protein comprises human CD8 alpha 22-182 aa, and a signal peptide and a purification tag screened by the present application, has good immunogenicity, and can be used as an antigen to prepare anti-CD8 alpha antibody. At the same time, the preparation method (production process) of the present application can significantly improve the secretion expression of recombinant human CD8 alpha protein by mammalian cells, significantly improve the yield of recombinant human CD8 alpha protein in mammalian cells, reduce the production cost of recombinant human CD8 alpha protein, and has wide industrial application prospect.

[0007] 2. TECHNICAL SCHEME

[0008] In order to solve the above problems, the technical scheme adopted by the present application is as follows:

[0009] The present application provides a recombinant human CD8 alpha protein, and the amino acid sequence of the recombinant human CD8 alpha protein is shown in any one of SEQ ID NO. 7-SEQ ID NO. 12.

[0010] Further, the amino acid sequence of the recombinant human CD8 alpha protein is shown in SEQ ID NO. 7.

[0011] Further, the amino acid sequence of the recombinant human CD8 alpha protein is shown in SEQ ID NO. 8.

[0012] Further, the amino acid sequence of the recombinant human CD8 alpha protein is shown in SEQ ID NO. 9.

[0013] Further, the amino acid sequence of the recombinant human CD8 alpha protein is shown in SEQ ID NO. 10.

[0014] Further, the amino acid sequence of the recombinant human CD8 alpha protein is shown in SEQ ID NO. 11.

[0015] Further, the amino acid sequence of the recombinant human CD8 alpha protein is shown in SEQ ID NO. 12.

[0016] The application also provides a polynucleotide encoding the recombinant human CD8 alpha protein of any of the above.

[0017] Further, the polynucleotide has a nucleotide sequence as shown in any one of SEQ ID NO. 16-21.

[0018] Further, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 16, and encodes the recombinant human CD8 alpha protein with an amino acid sequence as shown in SEQ ID NO. 7.

[0019] Further, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 17, and encodes the recombinant human CD8 alpha protein with an amino acid sequence as shown in SEQ ID NO. 8.

[0020] Further, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 18, and encodes the recombinant human CD8 alpha protein with an amino acid sequence as shown in SEQ ID NO. 9.

[0021] Further, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 19, and encodes the recombinant human CD8 alpha protein with an amino acid sequence as shown in SEQ ID NO. 10.

[0022] Further, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 20, and encodes the recombinant human CD8 alpha protein with an amino acid sequence as shown in SEQ ID NO. 11.

[0023] Further, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 21, and encodes the recombinant human CD8 alpha protein with an amino acid sequence as shown in SEQ ID NO. 12.

[0024] The application also provides an expression vector comprising any of the above polynucleotides.

[0025] Further, the expression vector is a recombinant plasmid comprising any of the above polynucleotides.

[0026] Further, the plasmid comprises a pcDNA3.4 plasmid or the like.

[0027] Further, the expression vector is a pcDNA3.4 plasmid linked to any of the above polynucleotides.

[0028] The application also provides an expression system, which is a mammalian cell comprising the above expression vector.

[0029] Further, the mammalian cell includes HEK293 cell.

[0030] The application also provides the use of the polynucleotide, the expression vector, or the expression system in the preparation of the recombinant human CD8α protein.

[0031] Further, the use includes culturing the expression system, or transfecting the expression vector into mammalian cells and culturing the transfected cells to obtain the recombinant human CD8α protein.

[0032] The application also provides a method for preparing the recombinant human CD8α protein, which includes culturing the expression system, or transfecting the expression vector into mammalian cells and culturing the transfected cells to obtain the recombinant human CD8α protein.

[0033] Further, the method for preparing the recombinant human CD8α protein further includes a purification step, which includes:

[0034] purifying the recombinant human CD8α protein of SEQ ID NO. 7 or SEQ ID NO. 10 using His tag affinity chromatography;

[0035] or purifying the recombinant human CD8α protein of SEQ ID NO. 8 or SEQ ID NO. 11 using Protein A affinity chromatography;

[0036] or purifying the recombinant human CD8α protein of SEQ ID NO. 9 or SEQ ID NO. 12 using Protein B affinity chromatography.

[0037] The application also provides the use of the recombinant human CD8α protein in the preparation of anti-CD8α antibody.

[0038] Further, the use includes immunizing animals with the recombinant human CD8α protein as an antigen, obtaining serum, and preparing polyclonal antibody.

[0039] Further, the use includes emulsifying the recombinant human CD8α protein, immunizing New Zealand rabbits, immunizing the rabbits in multiple points on the back, collecting and separating serum containing polyclonal antibody, and purifying and preparing polyclonal antibody.

[0040] Further, the multiple immunizations include four immunizations.

[0041] The application also provides polyclonal antibody against CD8α, which is obtained by immunizing animals with the recombinant human CD8α protein of SEQ ID NO. 11 as an antigen.

[0042] Further, the titer of the polyclonal antibody is 1:750-1:768000.

[0043] Further, the titer of the polyclonal antibody is 1:750.

[0044] Further, the titer of the polyclonal antibody is 1:3000.

[0045] Further, the titer of the polyclonal antibody is 1:12000.

[0046] Further, the titer of the polyclonal antibody is 1:48000.

[0047] Further, the titer of the polyclonal antibody is 1:192000.

[0048] Further, the titer of the polyclonal antibody is 1:768000.

[0049] The application also provides the use of the polyclonal antibody as a raw material for preparing a CD8a protein detection reagent.

[0050] The application also provides the use of the polyclonal antibody in the preparation of a CD8a protein detection product.

[0051] 3. Beneficial effects

[0052] Compared with the prior art, the application has the beneficial effects that:

[0053] (1) The recombinant human CD8a protein and its preparation method and application provided by the application include a signal peptide and a purification tag, and can be used to guide the efficient secretion expression of the recombinant human CD8a protein in mammalian cells. As shown in the examples, the application selects a combination of two signal peptides and three purification tags, and the experimental results show that the production process constructed using the combination of signal peptides B and C-human Fc tag can realize the efficient secretion expression of the recombinant human CD8a protein in mammalian cells, effectively solving the production problem of the difficult expression of the recombinant protein.

[0054] (2) The recombinant human CD8a protein and its preparation method and application provided by the application can significantly improve the yield of the recombinant human CD8a protein produced in mammalian cells, especially the production process constructed using the combination of signal peptides B and C-human Fc tag, which can produce 331.73 mg / L of the recombinant human CD8a protein, and the SDS-PAGE electrophoresis band of the target protein (recombinant human CD8a protein) is clear and single, effectively reducing the production cost of the recombinant protein.

[0055] (3) The application provides a recombinant human CD8 alpha protein and a preparation method and application thereof. The prepared recombinant human CD8 alpha protein antigen has good immunogenicity. The specific polyclonal antibody can be produced by immunizing animals with the recombinant human CD8 alpha protein antigen prepared by the production process, and the specific polyclonal antibody can be used for the diagnosis of the disease marker CD8 alpha of familial CD8 deficiency, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 is a pcDAN3.4 plasmid map.

[0057] Figure 2 is a schematic diagram of plasmid construction.

[0058] Figure 3 is a recombinant human CD8 alpha protein secretion expression detection result graph.

[0059] Figure 4 is a recombinant human CD8 alpha protein purity detection result graph.

[0060] Figure 5 is a recombinant human CD8 alpha protein yield detection result graph. DETAILED DESCRIPTION

[0061] The application will be further described below in combination with specific examples.

[0062] It should be noted that the terms such as "upper", "lower", "left", "right", "middle" and the like cited in the specification are only for the convenience of clear description, and are not intended to limit the scope of implementation. The change or adjustment of the relative relationship is also regarded as the scope of implementation of the application without substantial change of the technical content.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0064] Unless otherwise specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are conventional products that can be purchased on the market, unless otherwise specified.

[0065] As used herein, the term "about" is used to provide flexibility and inaccuracy associated with a given term, measure or value. The degree of flexibility of a specific variable can be easily determined by a person skilled in the art.

[0066] As used herein, the term "at least one of" is intended to mean one or more of. For example, "at least one of A, B and C" includes at least one of A, at least one of B, at least one of C, at least one of A and B, at least one of A and C, at least one of B and C and at least one of A, B and C.

[0067] Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be construed as having been followed to the rightmost significant figure of the number when it has been followed, even though some of the significant figures within the range might not be to the accurate degree of precision stated for the range. However, some of the numerical parameters are presented only to the nearest integer. Although the numerical ranges and parameters setting forth the broad scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, can contain certain errors associated with testing equipment, its calibration, and / or the tester.

[0068] In the embodiments, two signal peptides and three tags are designed for human T cell surface glycoprotein CD8a, and are used in combination. It is finally found that the signal peptide B and C-human Fc tag can realize efficient secretion expression of the target protein. The recombinant human CD8a protein is successfully obtained through affinity purification. The SDS-PAGE electrophoresis band of the target protein is clear and single, and the yield can reach 331.73 mg / L Cells. The recombinant human CD8a protein antigen prepared by the preparation method (production process) has good immunogenicity. The animals immunized with the recombinant human CD8a protein antigen can produce specific polyclonal antibodies. The preparation method (production process) can be effectively applied to large-scale production of recombinant human CD8a protein antigen, and the antigen is used as an immunogen for immunization, antibody preparation and production, and is further used for diagnosis of the disease marker CD8a of familial CD8 deficiency.

[0069] Embodiment 1

[0070] The embodiment provides a recombinant human CD8a protein amino acid sequence, a nucleotide sequence encoding the recombinant human CD8a protein, and design and construction of an expression vector of the recombinant human CD8a protein.

[0071] The full-length human CD8a protein has 235 aa, the signal peptide has 1-21 aa, the extracellular region has 22-182 aa, the helix region has 183-203 aa, and the cytoplasmic region has 204-235 aa, wherein the 22-182 aa has biological activity. Therefore, the recombinant human CD8a protein includes the human CD8a 22-182 aa (SEQ ID NO. 1, the nucleotide sequence encoding is shown in SEQ ID NO. 13), which can have biological activity.

[0072] In this embodiment, two signal peptides are designed. The signal peptide A is the signal peptide of the human CD8a protein, i.e., the human CD8a 1-21 aa (SEQ ID NO. 2, the nucleotide sequence encoding is shown in SEQ ID NO. 14); the signal peptide B is a signal peptide in the prior application of the applicant and granted, which is disclosed in the Chinese patent for invention with the publication number CN116102625B, and has 25 aa (SEQ ID NO. 3, the nucleotide sequence encoding is shown in SEQ ID NO. 15).

[0073] In this embodiment, three purification tags are designed, which are C-His tag (SEQ ID NO. 4), C-human Fc tag (SEQ ID NO. 5) or C-mouse Fc tag (SEQ ID NO. 6), respectively.

[0074] In this embodiment, the human CD8a 22-182 aa is combined with two signal peptides and three purification tags, and six recombinant human CD8a proteins are obtained, i.e.,

[0075] signal peptide A+CD8a 22-182 aa+C-His (SEQ ID NO. 7);

[0076] signal peptide A+CD8a 22-182 aa+C-human Fc (SEQ ID NO. 8);

[0077] signal peptide A+CD8a 22-182 aa+C-mouse Fc (SEQ ID NO. 9);

[0078] signal peptide B+CD8a 22-182 aa+C-His (SEQ ID NO. 10);

[0079] signal peptide B+CD8a 22-182 aa+C-human Fc (SEQ ID NO. 11);

[0080] signal peptide B+CD8a 22-182 aa+C-mouse Fc (SEQ ID NO. 12).

[0081] In this embodiment, the nucleotide sequences encoding the above-mentioned six kinds of recombinant human CD8a proteins are respectively as follows:

[0082] Signal peptide A + CD8a 22-182aa + C-His-DNA (SEQ ID NO. 16);

[0083] Signal peptide A + CD8a 22-182aa + C-human Fc-DNA (SEQ ID NO. 17);

[0084] Signal peptide A + CD8a 22-182aa + C-mouse Fc-DNA (SEQ ID NO. 18);

[0085] Signal peptide B + CD8a 22-182aa + C-His-DNA (SEQ ID NO. 19);

[0086] Signal peptide B + CD8a 22-182aa + C-human Fc-DNA (SEQ ID NO. 20);

[0087] Signal peptide B + CD8a 22-182aa + C-mouse Fc-DNA (SEQ ID NO. 21).

[0088] The above-mentioned amino acid and nucleotide sequences are shown in Table 2.

[0089] In this embodiment, the recombinant human CD8a protein vector is a recombinant plasmid including the above-mentioned nucleotide sequences encoding the six kinds of recombinant human CD8a proteins, and the plasmid is pcDNA3.4 (purchased from Suzhou Hongxun, as shown in Figure 1 ).

[0090] In this embodiment, the construction method of the recombinant human CD8a protein vector, i.e., the pcDNA3.4 plasmid including the above-mentioned nucleotide sequences encoding the six kinds of recombinant human CD8a proteins, is as follows: Figure 2The polynucleotide sequences of the present embodiment were synthesized by Suzhou Hongxun. During the synthesis, the enzyme cutting site Xbal and the Kozak sequence were added in front of the 5' end of the polynucleotide sequences SEQ ID NO. 16-21, and the stop codon and enzyme cutting site Eco32I were added at the 3' end. The synthesized polynucleotide sequences and pcDNA3.4 plasmid were double-digested with restriction endonuclease Xbal and Eco32I, followed by agarose gel electrophoresis identification, and the target fragment was recovered by gel cutting. The recovered target fragment and pcDNA3.4 were ligated to obtain recombinant plasmids 1-6 (Table 1) with one of the signal peptides A and B, and one of the purification tags of C-His tag, C-human Fc tag and C-mouse Fc tag. The above-mentioned recombinant plasmids 1-6 were transformed into TOP competent cells, respectively, and plated for 16 h of culture. Single colonies were picked for plasmid miniprep, and the extracted plasmid was double-digested with restriction endonuclease Xbal and Eco32I, followed by agarose gel electrophoresis identification. The positive clones were sent to Kings River Biotech Co., Ltd. for sequencing. The recombinant clones with correct sequences were subjected to plasmid maxi-prep, and the extracted recombinant plasmid was stored at -20°C for standby.

[0091] Table 1: Plasmids constructed and their main components

[0092] Number Signal peptide Region Tag Plasmid 1 A 22-182 aa C-His Plasmid 2 A 22-182 aa C-human Fc Plasmid 3 A 22-182 aa C-mouse Fc Plasmid 4 B 22-182 aa C-His Plasmid 5 B 22-182 aa C-human Fc Plasmid 6 B 22-182 aa C-mouse Fc

[0093] Table 2: Amino acid and polynucleotide sequences

[0094]

[0095]

[0096]

[0097]

[0098] Example 2

[0099] The present embodiment provides a recombinant human CD8a protein expression system and a method for constructing the same in Example 1, and uses the same to express a recombinant human CD8a protein.

[0100] In the present embodiment, the recombinant human CD8a protein expression system is a mammalian cell including the expression vector (recombinant plasmid) in Example 1, specifically a HEK293 cell, which is used to express a recombinant human CD8a protein using a mammalian cell eukaryotic system.

[0101] In this embodiment, the recombinant human CD8a protein expression system is obtained by transiently transfecting HEK293 cells with the recombinant plasmid in Example 1 using cationic liposome method (FectoPRO), and the recombinant human CD8a protein is expressed by the expression system. The specific transfection and culture operation is as follows:

[0102] (1) One day before transfection, the density of HEK293 cells is adjusted to 2 x 10 6 cells / mL with preheated OPM-293CD05 Medium (Shanghai OPM), and the cells are placed in a shaking incubator with relative humidity ≥ 80%, 8% CO2, and 37°C, and shaken at 95 rpm;

[0103] (2) The next day, the cell density is adjusted to 3 x 10 6 cells / mL with preheated OPM-293CD05 Medium for standby;

[0104] (3) The transfection mixture is configured as follows: 75 μg of recombinant plasmid (self-prepared), 50 μL of FectoPRO (Polyplus SA), and 5 mL of OPM-CDTrans293 (Shanghai OPM);

[0105] (4) After the mixture is placed at room temperature for 5 min, 50 mL of cells with a density of 3 x 10 6 cells / mL are added, and the cells are cultured in a shaking incubator with relative humidity ≥ 80%, 8% CO2, and 37°C, and shaken at 95 rpm;

[0106] (5) After 24 h of transfection, 3 mL of OPM-293ProFeed (Shanghai OPM) is added, and the culture is continued for 96 h;

[0107] (6) The supernatant is collected by centrifugation at 12,000 rpm for 10 min for SDS-PAGE verification and affinity purification.

[0108] In this embodiment, the purification of the recombinant human CD8a protein includes:

[0109] (1) Purification process of C-His tag

[0110] ​His tag affinity chromatography was used to separate and purify the target protein with C-His tag. The filler used was Ni-Smart (Changzhou Tian Dihuan, item number: SA036500). The supernatant was filtered with a 0.45 μm filter (Merck Millipore, item number: SLHPR33RB) and then combined with a 5 mL Ni-Smart chromatography column previously equilibrated with PBS. Then, 5 column volumes of PBS containing 10 mM imidazole were used to wash away the impurities. Finally, 3 column volumes of PBS containing 500 mM imidazole were used to elute the target protein, and the collected liquid was analyzed by SDS-PAGE.

[0111] (2) Purification process of C-human Fc tag

[0112] Protein A affinity chromatography was used to separate and purify the target protein with C-human Fc tag. The filler used was Protein A affinity chromatography medium (Nanjing Kings River, item number: L00433). The supernatant was filtered with a 0.45 μm filter (Merck Millipore) and then combined with a 5 mL Protein A chromatography column previously equilibrated with PBS. Then, 5 column volumes of PBS containing 1 M NaCl were used to wash away the impurities. Finally, 3 column volumes of 0.1 M glycine were used to elute the target protein, and 1 M Tris-HCl (pH 8.5) was added to the collected liquid to neutralize it to neutral. The neutralized sample was analyzed by SDS-PAGE.

[0113] (3) Purification process of C-mouse Fc tag

[0114] Protein G affinity chromatography was used to separate and purify the target protein with C-mouse Fc tag. The filler used was Protein G Resin (Nanjing Kings River, item number: L00664). The supernatant was filtered with a 0.45 μm filter (Merck Millipore) and then combined with a 5 mL Protein G chromatography column previously equilibrated with PBS. Then, 5 column volumes of PBS containing 1 M NaCl were used to wash away the impurities. Finally, 3 column volumes of 0.1 M glycine were used to elute the target protein, and 1 M Tris-HCl (pH 8.5) was added to the collected liquid to neutralize it to neutral. The neutralized sample was analyzed by SDS-PAGE.

[0115] Example 3

[0116] This example compares the combination of the above two signal peptides with three purification tags, i.e. under the guidance of different preparation methods (production process), the secretion expression level and yield of recombinant human CD8a protein in HEK293.

[0117] (1) Sample pretreatment

[0118] The samples collected in Example 2 were concentrated and desalted using a 15 mL, 3 kDa ultrafiltration tube (Millipore, Catalog No.: UFC9003), and the buffer was replaced with 0.2 M PBS (pH 7.4). The centrifugation conditions were 4000 rpm for 10 min.

[0119] (2) SDS-PAGE analysis

[0120] The level and yield of secretory expression of the target protein were analyzed by SDS-PAGE, and the specific steps were as follows: a 4-20% gradient gel was selected, and 10 μL of sample was taken for electrophoresis The electrophoresis was performed at a constant voltage of 150 V, and the bromophenol blue was terminated at the bottom of the gel.

[0121] (3) Comparison of SDS-PAGE results

[0122] Under the same conditions, the combinations of different signal peptides and purification tags in the present application were compared, and the determination results of the secretory expression level of the target protein were as shown in Figure 3 , the determination results of the protein purity were as shown in Figure 4 , and the determination results of the protein yield were as shown in Figure 5 . Among them, the determination results of the secretory expression level of the protein showed that the SDS-PAGE electrophoresis bands of the target protein in the supernatant expressed by plasmid 4, plasmid 5 and plasmid 6 were stronger than those of plasmid 1, plasmid 2 and plasmid 3 Figure 3 , which indicated that the secretory expression effect of signal peptide B was better than that of signal peptide A. The determination results of the protein purity showed that the purity of plasmid 2 and plasmid 5 was the best Figure 4 , which indicated that the purification process using C-human Fc tag could obtain target protein with high purity. The combination of signal peptide B and C-human Fc tag (plasmid 5) greatly improved the secretory expression level of recombinant human CD8a protein in mammalian cells, and the final yield of the protein was as high as 331.73 mg / L Cells Figure 5 .

[0123] Example 4

[0124] The present embodiment provides the use of recombinant human CD8a protein in the preparation of polyclonal antibodies (polyclonal serum) against CD8a.

[0125] The purified recombinant human CD8a protein was used as an antigen to prepare polyclonal serum, and New Zealand white rabbits were immunized with the antigen and adjuvant. The obtained rabbit antiserum was purified, and the antibody titer against CD8a protein antigen was detected by indirect ELISA.

[0126] (1) Immunization procedure

[0127] Example 3 Four New Zealand rabbits (No. K0005-K0008 or K0009-K0012) were immunized with 200 μg of the pre-treated recombinant human CD8a protein as an immunogen, by multiple-point immunization on the back. The first immunization was performed by mixing the recombinant human CD8a protein with complete Freund's adjuvant, and the second immunization was performed by mixing the recombinant human CD8a protein with incomplete Freund's adjuvant two weeks later. The third immunization was performed by mixing the recombinant human CD8a protein with incomplete Freund's adjuvant two weeks later, and the fourth immunization was performed by the same procedure.

[0128] (2) Indirect ELISA for determination of the titer of rabbit antisera

[0129] Five days after the fourth immunization, the rabbit ear marginal vein was bled, and the rabbit antisera were obtained by centrifugation at 4000 rpm for 10 min. The untagged CD8a antigen protein (purchased from Creative Biolabs, USA, Catalog No. GPX04-127J) was diluted with 50 mM carbonate buffer (pH 9.6) to 100 μL / well and 100 ng / well, and was coated on an enzyme-linked plate at 4°C overnight. The next day, 1% bovine serum albumin was used for blocking, and the blocking was performed at 37°C for 2 h. The rabbit antisera were diluted by four-fold gradient dilution as a primary antibody (1:750 dilution in the first well, and then dilution by the ratio, and two wells were measured for each concentration), and 1% BSATBS solution was used as a negative control. After incubation at 37°C for 1 h, 1:5000 goat anti-rabbit secondary antibody was added, and color development was performed, and the OD 450 signal value of the enzyme-linked plate was detected.

[0130] (3) Analysis of the results of the titer experiment of rabbit antisera

[0131] Under the same conditions, the signal peptide B with the best secretion-promoting expression effect in the present application was combined with different purification tags for comparison, and the results are shown in Tables 3-5. The titer of the rabbit antisera prepared from the immunogen protein (plasmid 4) composed of the signal peptide B and the C-His purification tag was as high as 1:192000 (Table 3), the titer of the rabbit antisera prepared from the immunogen protein (plasmid 5) composed of the signal peptide B and the C-human Fc purification tag was as high as 1:192000 (Table 4), and the titer of the rabbit antisera prepared from the immunogen protein (plasmid 6) composed of the signal peptide B and the C-mouse Fc purification tag was as high as 1:192000 (Table 5). These results show that the three different purification tags have little effect on the immunization effect, and the three kinds of immunogen proteins prepared thereby have similar immunogenicity and specificity.

[0132] ​Table 3 Results of indirect enzyme-linked immunoassay for detecting titer of rabbit antiserum (plasmid 4, A450 value)

[0133]

[0134] Table 4 Results of indirect enzyme-linked immunoassay for detecting titer of rabbit antiserum (plasmid 5, A450 value)

[0135]

[0136] Table 5 Results of indirect enzyme-linked immunoassay for detecting titer of rabbit antiserum (plasmid 6, A450 value)

[0137]

[0138] In summary, the present application provides a recombinant human CD8a protein, a preparation method and application thereof. In particular, under the combination of signal peptides B and C-human Fc tag, efficient production of the recombinant human CD8a protein is achieved, the SDS-PAGE electrophoresis band of the target protein is clear and single, and the yield can reach 331.73 mg / L Cells. The recombinant human CD8a protein antigen prepared by the preparation method (production process) of the present application has good immunogenicity, and animals immunized with the recombinant human CD8a protein as an antigen can produce specific polyclonal antibodies, which can be used for the diagnosis of the marker CD8a of diseases such as familial CD8 deficiency. Therefore, the preparation method (production process) can be effectively applied to the large-scale production of recombinant human CD8a protein antigen.

Claims

1. A recombinant human-derived CD8α protein, characterized in that, The amino acid sequence of the recombinant human CD8 alpha protein is shown as SEQ ID NO.

11.

2. A polynucleotide, comprising, The polynucleotide encodes the recombinant human CD8 alpha protein as claimed in claim 1.

3. The polynucleotide of claim 2, wherein The nucleotide sequence of the polynucleotide is shown as SEQ ID NO.

20.

4. An expression vector, characterized by, The expression vector comprises the polynucleotide as claimed in claim 2 or 3.

5. The expression vector of claim 4, wherein, The expression vector is a pcDNA3.4 plasmid linked to the polynucleotide as claimed in claim 2 or 3.

6. An expression system comprising, The expression system is a mammalian cell comprising the expression vector as claimed in claim 4 or 5.

7. An expression system according to claim 6, wherein, The mammalian cell is a HEK 293 cell.

8. Use of the polynucleotide as claimed in claim 2 or 3, or the expression vector as claimed in claim 4 or 5, or the expression system as claimed in claim 6 or 7 in the preparation of the recombinant human CD8 alpha protein as claimed in claim 1.

9. The method of producing the recombinant human-derived CD8α protein according to claim 1, characterized by, The method comprises culturing the expression system as claimed in claim 6 or 7, or transfecting the expression vector as claimed in claim 4 or 5 into a mammalian cell and culturing the transfected cell to obtain the recombinant human CD8 alpha protein.

10. The method of claim 9, wherein, The method further comprises a purification step, which comprises purifying the recombinant human CD8 alpha protein as shown in SEQ ID NO. 11 using Protein A affinity chromatography.

11. Use of the recombinant human CD8 alpha protein as claimed in claim 1 in the preparation of an anti-CD8 alpha antibody.

12. A polyclonal antibody against CD8a, characterized in that, The polyclonal antibody is obtained by immunizing an animal with the recombinant human CD8 alpha protein as shown in SEQ ID NO. 11 as an antigen.

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