WO2022002015A1 - 多肽、hla-dr蛋白及其制备方法和应用 - Google Patents

多肽、hla-dr蛋白及其制备方法和应用 Download PDF

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WO2022002015A1
WO2022002015A1 PCT/CN2021/102956 CN2021102956W WO2022002015A1 WO 2022002015 A1 WO2022002015 A1 WO 2022002015A1 CN 2021102956 W CN2021102956 W CN 2021102956W WO 2022002015 A1 WO2022002015 A1 WO 2022002015A1
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seq
amino acid
hla
protein
polypeptide
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马灵芝
刘阳
张宏航
孙玉龙
王弢
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江苏为真生物医药技术股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • the invention belongs to the technical field of biological immunity, and relates to a polypeptide, an HLA-DR protein and a preparation method and application thereof.
  • MHC The major histocompatibility complex
  • the gene group has more than 100 gene loci, and its encoded products include MHC class I molecules and MHC class II molecules.
  • MHC class II molecules are molecules capable of presenting antigens and play a role in regulating immune responses and immune cell development.
  • MHC class II molecules are a group of highly polymorphic transmembrane glycoproteins consisting of non-covalently linked alpha and beta chains that are presented on the surface of antigen-presenting cells (APCs) of the immune system and can present processed foreign antigens
  • APCs antigen-presenting cells
  • Antigen receptors given to helper T cells lead to activation and differentiation of helper T cells, which play an important role in inducing immune responses.
  • HLA class II (HLA-II) molecules or proteins present peptide antigens from extracellular proteins, including proteins from extracellular pathogens, while HLA class I proteins present intracellular proteins or peptides from pathogens.
  • HLA class II proteins loaded on the cell surface interact with CD4+ helper T cells. This interaction leads to the recruitment of phagocytes, local inflammation and/or humoral responses through the activation of B cells.
  • Human MHC antigen is usually called human leukocyte antigen (HLA).
  • HLA II has many kinds, such as DR, DQ, DP, etc. Among them, the DR allele is the most and the most abundant.
  • Sepsis has definite diagnostic criteria, but the prognosis of patients varies, which may be closely related to immune status, especially immunosuppression.
  • HLA-DR human leukocyte antigen-DR
  • the expression rate of human leukocyte antigen-DR (HLA-DR) in CD14 cells has been widely used in the judgment of sepsis.
  • Many researchers have regarded the expression rate of HLA-DR as less than or equal to 30%.
  • the decreased expression of HLA-DR indicates that the body is in a state of immunosuppression, leading to aggravation of infection.
  • the method of measuring the expression rate of membrane proteins by flow cytometry is complicated and expensive, and a fast and simple method is urgently needed.
  • soluble forms exist in peripheral blood, and recent studies have shown that increased levels of soluble HLA-DR are associated with transplant-related complications and autoimmune diseases. The latter mechanism of action may be attributed to the belief that bacterial and some virus-derived superantigens are pathogenic agents that induce polyclonal T cell activation, as is the pathogenic mechanism of sepsis. Soluble HLA-DR molecules can act as ligands for superantigens, and therefore, may play a role in the "detoxification" of superantigens, which can warn of sepsis earlier than membrane-bound HLA-DR.
  • the gene encoding the ⁇ chain of HLA-DR lacks polymorphism, while the gene encoding the ⁇ chain has many subtypes. 1001 and 1402 etc. Due to the existence of a wide variety of HLA-DR molecules, each HLA-DR molecule can bind to a variety of peptide fragments, thus ensuring that T cells can immune responses to all antigens, which is a useful tool for the detection of soluble HLA-DR bands. come difficult. Whether the selected antibody can recognize all or most of the HLA-DR molecules, and whether the calibrator used for quantification can accurately quantify the HLA-DR molecules is difficult.
  • HLA-DR purification was performed by immunoaffinity chromatography.
  • Such methods have the following shortcomings: (1) The content of HLA-DR molecules on the cell membrane surface is very small, and even cannot be detected by Western blotting, so a large number of cultured cells becomes a limitation in the purification process.
  • HLA-DR molecules purified from cell lines have most of the antigen binding sites. All have antigen peptide binding, which is different from soluble HLA-DR.
  • leucine zipper assembly method specifically use RT-PCR to amplify the signal peptides and extracellular sequences of DR ⁇ and DR ⁇ from 721.221 cells, and use overlapping PCR to separate the DR ⁇ and DR ⁇ fragments with DR ⁇ and DR ⁇ fragments respectively.
  • the leucine zipper sequences of Fos and Jun are connected to form DR ⁇ -Fos and DR ⁇ -Jun structures.
  • DR ⁇ and DR ⁇ can combine with the leucine zipper of Fos and Jun to form a DR1 molecule, and then DR ⁇ - Fos is linked to the Fc segment of human IgG1 to form a DR ⁇ -Fos-Fc recombination sequence, and two homologous DR1 molecules can form dimers through the disulfide bond of the Fc segment of IgG1.
  • DR ⁇ -Fos-Fc and DR ⁇ -Jun were respectively inserted into the two multi-cloning sites of the baculovirus expression vector pFastBacTMDual to construct a recombinant vector pFastBacTMDual+[DR1/Fc].
  • the presenting polypeptide assembly method is adopted, and such methods are specifically cloning a single ⁇ -chain extracellular segment and a ⁇ -chain extracellular segment, and expressing in the E. coli system respectively.
  • Inclusion body proteins were purified separately.
  • the prokaryotically expressed ⁇ and ⁇ chains were assembled in vitro with the assistance of human myelin basic protein (b-MBP(83-102)Y), and purified with a heterodimer-specific monoclonal antibody (L243). product.
  • the first object of the present invention is to provide a polypeptide;
  • the second object of the present invention is to provide the polypeptide as a connecting peptide (named Linker) of the ⁇ chain and the ⁇ chain of the HLA-DR protein.
  • the third object of the present invention is to provide an HLA-DR protein, which is soluble;
  • the fourth object of the present invention is to provide a full-length polynucleotide encoding the HLA-DR protein
  • the fifth object of the present invention is to provide a recombinant vector or recombinant cell that encodes the full-length polynucleotide of the HLA-DR protein;
  • the sixth object of the present invention is to provide a method for preparing the HLA-DR protein;
  • the seventh object of the present invention The purpose is to provide the use of the HLA-DR protein in the preparation of a sepsis diagnostic reagent.
  • the eighth object of the present invention is to provide a kit for quantitatively detecting sHLA-DR comprising the HLA-DR protein.
  • the present invention provides a polypeptide, which is a polypeptide of 8-48 amino acid residues (named Linker) composed of glycine (G) and serine (S) interconnected by peptide bonds.
  • Linker a polypeptide of 8-48 amino acid residues
  • G glycine
  • S serine
  • the polypeptide is a polypeptide of 16-32 amino acid residues composed of glycine and serine connected to each other by peptide bonds.
  • the polypeptide is a polypeptide of 20 amino acid residues composed of glycine and serine connected to each other by peptide bonds.
  • amino acid sequence of the polypeptide is selected from one of the following sequences:
  • the present invention provides the use of the above-mentioned polypeptides as connecting peptides of ⁇ chain and ⁇ chain of HLA-DR protein in constructing HLA-DR protein.
  • the present invention also provides an HLA-DR protein
  • the HLA-DR protein structural composition includes an ⁇ chain, a ⁇ chain and the above-mentioned polypeptide; the C-terminus of the ⁇ -chain is connected to the N-terminus of the polypeptide; The C-terminus of the polypeptide is linked to the N-terminus of the beta chain.
  • the invention creatively designs a soluble HLA-DR protein, which connects the ⁇ chain and the ⁇ chain constituting the HLA-DR protein through a specific polypeptide consisting of 8 to 48 amino acids containing glycine and serine.
  • the designed HLA-DR proteins can be expressed in tandem in mammalian cells and can mimic soluble sHLA-DR expression.
  • the HLA-DR protein of the present invention is similar in structure to natural sHLA-DR, can be recognized by antibodies such as L234 and CR3/43, and has a more stable conformation than the membrane protein mHLA-DR.
  • the HLA-DR protein of the present invention can be expressed in eukaryotes The system performs soluble and high-efficiency expression, and is expected to have good applications in the field of medical biology and biomedical development.
  • the ⁇ chain includes an ⁇ 1 domain polypeptide; the C-terminus of the ⁇ 1 domain polypeptide is connected to the N-terminus of the polypeptide;
  • the amino acid sequence of the ⁇ 1 domain polypeptide includes the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence shown in SEQ ID NO: 8 through substitution, deletion or addition of one or several amino acids and is identical to the ⁇ 1 domain polypeptide. Functional amino acid sequence.
  • the ⁇ 1 domain polypeptide amino acid sequence SEQ ID NO: 8 is as follows:
  • amino acid sequence that is substituted by one or several amino acids in the amino acid sequence shown in SEQ ID NO: 8 and has the same function as the ⁇ 1 domain polypeptide may include the following amino acid sequences:
  • the ⁇ chain further includes an ⁇ 2 domain polypeptide, the C-terminus of the ⁇ 1 domain polypeptide is connected to the N-terminus of the ⁇ 2 domain polypeptide, and the ⁇ 2 domain polypeptide is connected.
  • the C-terminus is connected with the N-terminus of the polypeptide;
  • the amino acid sequence of the ⁇ 2 domain polypeptide includes the amino acid sequence shown in SEQ ID NO: 9 or the amino acid sequence shown in SEQ ID NO: 9 through substitution, deletion or addition of one or several amino acids and is identical to the ⁇ 2 domain polypeptide.
  • the ⁇ 2 domain polypeptide amino acid sequence SEQ ID NO: 9 is as follows:
  • amino acid sequence that is substituted with one or several amino acids in the amino acid sequence shown in SEQ ID NO: 9 and has the same function as the ⁇ 2 domain polypeptide may include the following amino acid sequences:
  • the ⁇ chain includes a ⁇ 1 domain polypeptide and a ⁇ 2 domain polypeptide; the C-terminus of the polypeptide is connected to the N-terminus of the ⁇ 1 domain polypeptide, and the ⁇ 1 domain polypeptide is connected. The C-terminus of the polypeptide is connected to the N-terminus of the ⁇ 2 domain polypeptide;
  • the amino acid sequence of the ⁇ 1 domain polypeptide includes the amino acid sequence shown in SEQ ID NO: 10 or the amino acid sequence shown in SEQ ID NO: 10 through substitution, deletion or addition of one or several amino acids and is identical to the ⁇ 1 domain polypeptide. Functional amino acid sequence.
  • the ⁇ 1 domain polypeptide amino acid sequence SEQ ID NO: 10 is as follows:
  • amino acid sequence that has the same function as the ⁇ 1 domain polypeptide by substituting one or several amino acids in the amino acid sequence shown in SEQ ID NO: 10 may include the following amino acid sequences:
  • the amino acid sequence of the ⁇ 2 domain polypeptide includes the amino acid sequence shown in SEQ ID NO: 11 or the amino acid sequence shown in SEQ ID NO: 11 through substitution, deletion or addition of one or several amino acids and is consistent with the ⁇ 2 domain
  • the amino acid sequence of the polypeptide has the same function, or the amino acid sequence that is a partial fragment of the amino acid sequence shown in SEQ ID NO: 11 and has the partial function of the ⁇ 2 domain polypeptide (for example, the underlined part of the following ⁇ 2 domain amino acid sequence).
  • the ⁇ 2 domain polypeptide amino acid sequence SEQ ID NO: 11 is as follows:
  • amino acid sequence that is substituted by one or several amino acids in the amino acid sequence shown in SEQ ID NO: 11 and has the same function as the ⁇ 2 domain polypeptide may include the following amino acid sequences:
  • the ⁇ chain further comprises a connecting peptide; the C-terminus of the ⁇ 2 domain polypeptide is connected with the N-terminus of the connecting peptide;
  • the amino acid sequence of the connecting peptide includes the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence that is substituted, deleted or added with one or several amino acids in the amino acid sequence shown in SEQ ID NO: 12 and has the same function as the connecting peptide. .
  • the connecting peptide amino acid sequence SEQ ID NO: 12 is as follows:
  • the amino acid sequence of this HLA-DR protein includes SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: One of the sequences shown in 40.
  • HLA-DR protein preferably, the amino acid sequence of this HLA-DR protein is as shown in SEQ ID NO:13:
  • the present invention also provides a full-length polynucleotide encoding the above-mentioned HLA-DR protein.
  • nucleotide sequence of the full-length polynucleotide encoding the HLA-DR protein whose amino acid sequence is SEQ ID NO: 13 of the present invention is shown in SEQ ID NO: 51.
  • the present invention also provides recombinant plasmids or recombinant cells comprising the above-mentioned full-length polynucleotides.
  • the present invention also provides a method for preparing an HLA-DR protein, the method comprising: cloning the above-mentioned full-length polynucleotide into a plasmid vector, transfecting the plasmid vector into an expression cell for culture, expressing the target protein, and then Protein purification was performed to obtain HLA-DR protein.
  • the expression cells include CHO cells.
  • the spontaneous or artificial assembly method of heterodimers after expression in vitro is cumbersome and the yield is extremely low.
  • the present invention adopts the way of tandem expression of ⁇ chain and ⁇ chain, the yield is dozens of times higher than other methods, and the purification process Simple and easy to repeat.
  • the present invention also provides the use of the above-mentioned HLA-DR protein in the preparation of a diagnostic reagent for sepsis.
  • the present invention also provides a quantitative detection kit for soluble human leukocyte antigen-DR (sHLA-DR), the kit comprising the above-mentioned HLA-DR protein.
  • HLA-DR soluble human leukocyte antigen-DR
  • the HLA-DR protein of the present invention can be used as a working calibrator, a calibrator, and a quality control product; the working calibrator is the highest traceable substance in an enterprise, and is used for the traceability and determination of the calibrator/quality control product.
  • Standards are used to generate/standardize master curves to assign values to routine samples (eg, septic plasma samples); quality controls are used to verify the performance of analytical instruments or kits.
  • the kit further comprises biotin-labeled sHLA-DR mouse monoclonal antibody, acridinium ester-labeled sHLA-DR murine monoclonal antibody, streptavidin-labeled immunomagnetic microbeads and buffer. .
  • HLA-DR soluble human leukocyte DR antigen
  • Fig. 1 is the electrophoresis figure of HLA-DR protein obtained in the embodiment of the present invention 1;
  • Figure 2 is the sHLA-DR standard curve obtained in Example 2 of the present invention.
  • HLA-DR human leukocyte antigen-DR
  • the ⁇ chains are mainly DRB1, DRB3, DRB4, DRB5, of which DRB1 has the most variation.
  • the protein sequences of 50 DRB1, 24 DRB3, 19 DRB4, and 22 DRB5 were screened; the sequences with higher homology were obtained by further comparison and screening, and the ⁇ chain amino acid sequence was designed and obtained, with a total of 231 amino acids.
  • nucleotide sequence of the beta chain is designed as shown in SEQ ID NO: 50.
  • IL-2 signal peptide MYRMQLLSCIALSLALVTNS; enzyme cleavage site: BamHI and XhoI
  • the full-length nucleotide sequence of the target protein is shown in SEQ ID NO: 51.
  • the designed expression unit was sent to the gene synthesis company to synthesize the sequence, and the synthesized expression unit was inserted into the pVAX1 vector to obtain the HLA-DR- ⁇ -L20- ⁇ -pVAX1 recombinant plasmid and the corresponding glycerin bacteria HLA-DR- ⁇ -L20 - ⁇ -pVAX1-DH5 ⁇ .
  • the expression plasmid HLA-DR- ⁇ -L20- ⁇ -pVAX1 containing the target gene was extracted with a plasmid kit, and the DNA was precipitated with ethanol. After natural air-drying in a biological safety cabinet, the DNA was dissolved in sterile water, and the DNA concentration was determined with Nanodrop.
  • Freshly thawed cells were thawed for two or more passages before transfection.
  • Day -1 Divide cells: the day before transfection (Day -1), divide the ExpiCHO-S TM culture from step (1) to a final density of 3 ⁇ 10 6 to 4 ⁇ 10 Cells were grown overnight at 6 viable cells/mL.
  • Day 0 Transfected cells: The next day (day 0), the viable cell density and the percentage of viability were determined.
  • the cell density should reach about 7 ⁇ 10 6 to 10 ⁇ 10 6 viable cells/mL.
  • the survival rate should be 95% to 99% before continuing transfection.
  • ExpiCHO TM expression medium (4) with fresh preheated to 37 [deg.] C in the first step (2) of the cells were diluted to a final density of 6 ⁇ 10 6 viable cells / mL. Gently shake the flask to mix the cells.
  • step 5d Incubate the ExpiFectamine TM CHO/plasmid DNA complex (step 5d) at room temperature for 1 to 5 minutes, then slowly transfer the solution to the flask in step (4), and shake the flask gently during the addition.
  • the cells were cultured on an orbital shaker (37°C incubator under humidified air with 8% CO 2 ).
  • M Protein Marker
  • 1 10ug HLA-DR- ⁇ -L20- ⁇ -pVAX1 (B1) protein
  • 2 20ug HLA-DR- ⁇ -L20- ⁇ -pVAX1 (B1) protein.
  • the sHLA-DR protein concentration was determined to be 1.2 mg/mL using a BCA protein concentration assay kit.
  • the quantitative detection kit uses the double antibody sandwich method to detect the content of soluble human leukocyte antigen-DR (sHLA-DR) in plasma.
  • the kit uses a pair of monoclonal antibodies against sHLA-DR (commonly purchased commercially), one of which is labeled with biotin, and the other is labeled with acridine ester.
  • Specimen/calibration solution/quality control solution reacts with biotin-labeled monoclonal antibody, acridine ester-labeled monoclonal antibody and immunomagnetic particles coated with streptavidin to form an immune complex. The amount of this complex formed It is proportional to the content of the antigen to be tested.
  • the acridinium ester in the complex releases photons under the excitation of the substrate solution, and the relative light intensity RLU emitted within 3 seconds is automatically monitored, which is proportional to the concentration of sHLA-DR, and the instrument automatically fits and calculates the concentration of sHLA-DR .
  • the protein prepared by the present invention is used as a standard substance, a calibration substance and a quality control substance, wherein the standard substance is used to generate a quantitative standard curve.
  • the working calibrator as the highest traceable substance of the enterprise, is used to detect routine samples, generate a standard curve, and determine the value of routine samples.
  • the quality control substance is the known concentration content, and the instrument measurement is used to determine whether the measurement result of the instrument is accurate.
  • Instrument preparation reaction cups, micropipettes and tips, Cosman automatic magnetic particle chemiluminescence instrument SMART6500/500S.
  • Reagent preparation Prepare pre-excitation solution, excitation solution and washing solution (containing phosphate buffer, surfactant), and install it on the instrument together with the kit components.
  • the luminescence value in Table 4 is the luminescence value obtained by diluting the target protein obtained by different design schemes in Table 3 into different concentrations (10000ng/mL, 5000ng/mL, etc.) as the sample in the quantitative detection system; wherein the membrane protein is the control group.
  • the protein activity accounts for more than 20% of the natural protein activity, which can be used as the calibrator of the detection system; more preferably, it is 16-32 amino acids; especially when the length of Linker is 20
  • the protein activity accounts for 75% of the natural protein activity, and the HLA-DR protein expression amount is as high as 5mg/30mL cells. The higher the protein activity is, the closer it is to the natural protein, and the more it can reflect the actual situation when testing the sample.
  • Example 3 it can be seen that the Linker with a length of 20 amino acids has the best effect.
  • the composition of S) affects the activity of HLA-DR protein, and 6 groups of experiments were designed, as shown in Table 5 below:
  • Example 4 it can be seen that the Linker composed of 5 GGGS cycles has the best effect.
  • 4 groups of experiments were designed, as shown in Table 7 below:
  • the antigenic activity of the target protein obtained by 5 GGSG cycles is the best, the antigenic activity accounts for 98% of the natural protein activity, and the HLA-DR protein expression is as high as 4mg/30mL cells.
  • the ⁇ 1 functional region is necessary, and the ⁇ 2 region contributes to the formation of antigen conformation.
  • the antigenic activity of the HLA-DR protein obtained by constructing the full-length peptide of the extracellular segment of the ⁇ 1 domain + ⁇ 2 domain was the best, and the antigenic activity accounted for 96% of the natural protein activity, and the HLA-DR protein expression was as high as 5mg/30mL cells.
  • D2 (SEQ ID NO: 33) 37% 80% extracellular secretion 2.6mg
  • D3 (SEQ ID NO: 34) 0% 80% extracellular secretion 1.3mg
  • the HLA-DR protein antigenic activity obtained by constructing the ⁇ 1 domain polypeptide + ⁇ 2 domain polypeptide, ⁇ 1 domain polypeptide + ⁇ 2 domain polypeptide + the full-length peptide of the connecting peptide public region is the best, especially if it contains public domain
  • the antigenic activity of the protein constructed by the ⁇ chain of the full-length peptide in the region accounts for 96% of the activity of the natural protein, and the expression level of HLA-DR protein is as high as 5 mg/30 mL of cells.
  • Example 8 Effect of partial amino acid mutation in ⁇ chain and ⁇ chain on the activity of fusion protein
  • the optimal Linker, ⁇ chain and ⁇ chain were determined according to Examples 3 to 7, and the obtained HLA-DR protein (ie: B1 group) was constructed from this, and its amino acid sequence is shown in SEQ ID NO: 13. Seven groups of experiments were designed to investigate the effect of amino acid mutations in the ⁇ chain and ⁇ chain on the activity of the fusion protein, as shown in Table 13 below:
  • Embodiment 9 The present invention compares with existing method
  • the yield of the protocol of the present invention is 10 7 times that of the native membrane protein; it is more than 300 times that of the in vitro assembly protocol by leucine zipper in DR ⁇ and DR ⁇ .
  • B1 protein and native protein at the same concentration were placed in a 37°C incubator for accelerated destruction by heat for 3 days;
  • the control group was 1ug/mL of B1 group protein and natural protein and placed in a refrigerator at 4 degrees for 3 days.
  • the quantitative detection kit in Example 2 was used to verify the changes in the protein concentrations of the two groups; the protein concentrations were calculated according to the standard curve in Figure 2.
  • Recovery rate experimental group concentration / control group concentration (the closer the recovery rate is to 100%, the better the stability)

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Abstract

本发明提供了一种多肽、HLA-DR蛋白及其制备方法和应用。所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的8～48个氨基酸残基的多肽；该HLA-DR蛋白结构组成包括α链、β链和多肽；α链的C端与多肽的N端相连接；多肽的C端与所述β链的N端相连接。该蛋白质是将构成HLA-DR蛋白的α链和β链通过特定的由8～48个包含甘氨酸和丝氨酸以肽键相互连接构成的多肽连接。设计的HLA-DR蛋白能够在哺乳动物细胞中串联表达，可以模拟sHLA-DR表达，其与天然sHLA-DR结构类似，与膜蛋白mHLA-DR相比构象更稳定，能够使用真核表达系统进行可溶性高效表达，有望在医学生物学领域和生物医药开发中有很好的应用。

Description

多肽、HLA-DR蛋白及其制备方法和应用
交叉参考相关引用
本申请要求2020年06月29日递交的申请号为202010602241.9、发明名称为“多肽、HLA-DR蛋白及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物免疫技术领域，涉及一种多肽、HLA-DR蛋白及其制备方法和应用。
背景技术
主要组织相容性复合体(major histocompatibility complex，MHC)是一个与机体免疫反应相关的基因群，该基因群有100多个基因位点，其编码产物包括MHC I类分子和MHC II类分子，是一些能够提呈抗原的分子，在调节免疫应答和免疫细胞发育中发挥作用。MHC II类分子是呈现在免疫系统抗原呈递细胞(APC)表面的由α链和β链非共价链相连组成的一组高度多态性的跨膜糖蛋白，可提呈经过加工的外来抗原给辅助性T细胞的抗原受体，导致辅助性T细胞激活和分化，从而在诱发免疫应答中起重要的作用。
HLA II类(HLA-II)分子或蛋白质呈递来自细胞外蛋白质的肽抗原，包括细胞外病原体的蛋白质，而HLA I类蛋白质呈递细胞内蛋白质或病原体的肽。细胞表面上加载的HLA II类蛋白与CD4+辅助T细胞相互作用。该相互作用通过激活B细胞导致吞噬细胞的募集，局部炎症和/或体液反应。人MHC抗原通常称为人类白细胞抗原(HLA)，HLA II有DR，DQ，DP等许多种，其中DR等位基因最多，含量最丰富。
脓毒血症已有明确的诊断标准但患者预后各异，可能与免疫状态尤其是免疫抑制密切相关。CD14细胞的人类白细胞抗原-DR(HLA-DR)表达率作为一个新的细菌感染指标，已经比较普遍地应用于脓毒血症的判断，很多研究者将HLA-DR表达率小于或等于30％作为重症脓毒血症的一个指标，HLA-DR表达率下降提示机体处于免疫抑制状态，导致感染加重。但是用流式检测膜蛋白表达率的方法操作复杂，成本较高，急需一种快速简便的方法检测。
除膜结合型HLA-DR外，外周血中还存在可溶性形式，最近的研究表明，可溶性HLA-DR水平的增加与移植相关并发症和自身免疫性疾病有关。后者的作用机制可能是 认为细菌和部分病毒衍生的超抗原是诱导多克隆T细胞活化的致病剂，脓毒症的致病机制也是如此。而可溶性HLA-DR分子可以作为超抗原的配体发挥作用，因此，可能在超级抗原的“解毒”中发挥作用，可以比膜结合型HLA-DR更早的预警脓毒症。
编码HLA-DR的α链的基因缺乏多态性，而编码β链的基因则存在着许多亚型，目前己经发现的亚型有HLA-DRBP 0101，0102，0401，0404，0405，0410，1001和1402等。由于存在类型繁多的HLA-DR分子，而每一种HLA-DR分子都能与多种肽片段结合，从而保证了T细胞能对所有的抗原发生免疫反应，这就为检测可溶性HLA-DR带来困难。所选抗体能否识别全部或者绝大多数HLA-DR分子，用来定量的校准品能否精确定量HLA-DR分子都存在困难。
相关文献报道在原核和真核蛋白表达系统中报道了克隆和表达MHC II类分子的各种尝试。但是由于重组杂二聚体再折叠困难，得率极低。
现有技术中，有采用膜蛋白提取法，此类方法具体为大量培养、收集经鉴定的HLA-DR阳性细胞(比如人B淋巴瘤细胞系Raji以及LG2细胞)，用裂解液裂解细胞，并用免疫亲和层析法进行HLA-DR纯化。此类方法存在如下不足之处：(1)细胞膜表面HLA-DR分子的含量很少，甚至无法通过Western杂交进行检测，因此大量培养细胞则成为纯化过程的限制点。(2)在纯化过程中，操作复杂且免疫亲和柱存在非特异吸附导致微量的杂蛋白混入，纯度不够；(3)从细胞系中纯化获得的HLA-DR分子，大部分抗原结合位点都有抗原肽结合，这与可溶性HLA-DR存在差异。
现有技术中，有采用亮氨酸拉链组装法，此类方法具体采用RT-PCR方法从721.221细胞中扩增DRα和DRβ的信号肽及胞外序列，运用重叠PCR将DRα和DRβ片段分别与Fos和Jun的亮氨酸拉链序列相连，形成DRα-Fos和DRβ-Jun结构，DRα和DRβ可凭借Fos和Jun的亮氨酸拉链结合形成DR1分子，再通过EcoR I酶切位点将DRα-Fos和人IgG1的Fc段相连，形成DRα-Fos-Fc重组序列，2个同源的DR1分子可通过IgG1的Fc段的二硫键结合形成二聚体。分别将DRα-Fos-Fc及DRβ-Jun插入杆状病毒表达载体pFastBacTMDual的2个多克隆位点处，构建出重组载体pFast BacTMDual+[DR1/Fc]，对构建的载体进行PCR及限制性内切酶酶切鉴定和测序。采用脂质体转染方法将表达载体转入昆虫细胞系Sf9中，通过双抗夹心ELISA及Westernblot检测HLA-DR1的表达。此类方法存在如下不足之处：(1)方法繁琐；(2)DRα和DRβ为体外组装，得率低，成本高；(3)所得产物为二聚体，与天然可溶性HLA-DR存在差异。
现有技术中，有采用呈递多肽组装法，此类方法具体为克隆单个α链胞外段和β链胞 外段，并分别在大肠杆菌系统中表达。分别纯化包涵体蛋白。在来自人髓鞘碱性蛋白(b-MBP(83-102)Y)辅助下将原核表达的α链和β链体外组装，并用异二聚体特异性单克隆抗体(L243)纯化组装成功的产物。此类方法存在如下不足之处：(1)原核表达，缺乏复杂的N-糖基化O-糖基化等多种翻译后修饰，与天然蛋白构象差异很大；(2)体外重组，方法繁琐且不可控，产率低，方法可操作性差。
由于现有HLA-DR类分子的提取和制备方法存在诸多缺点如：操作复杂、原料来源较少、得率低、成本高以及潜在携带外源病毒及其他病原微生物的危险，很难实现产业化，迄今为止，未发现国内外关于可溶HLA-DR蛋白定量检测相关产品的报道。
发明内容
基于现有技术存在的缺陷，本发明的第一目的在于提供一种多肽；本发明的第二目的在于提供该多肽作为HLA-DR蛋白的α链和β链的连接肽(命名Linker)在构建HLA-DR蛋白中的应用；本发明的第三目的在于提供一种HLA-DR蛋白，该蛋白具备可溶性；本发明的第四目的在于提供一种编码该HLA-DR蛋白的全长多核苷酸；本发明的第五目的在于提供包含编码该HLA-DR蛋白全长多核苷酸的重组载体或重组细胞；本发明的第六目的在于提供该HLA-DR蛋白的制备方法；本发明的第七目的在于提供该HLA-DR蛋白在制备用于脓毒血症诊断试剂中的用途。本发明的第八目的在于提供包含该HLA-DR蛋白的定量检测sHLA-DR的试剂盒。
本发明的目的通过以下技术手段得以实现：
一方面，本发明提供一种多肽，所述多肽是由甘氨酸(G)和丝氨酸(S)以肽键相互连接构成的8～48个氨基酸残基的多肽(命名为Linker)。
上述的多肽中，优选地，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的16～32个氨基酸残基的多肽。
上述的多肽中，优选地，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的20个氨基酸残基的多肽。
上述的多肽中，优选地，所述多肽的氨基酸序列选自如下序列中的一种：
GGGSGGGSGGGSGGGSGGGS(SEQ ID NO：1)，
GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：2)，
GGSGGSGGSGGSGGSGGSGG(SEQ ID NO：3)，
GSGSGSGSGSGSGSGSGSGS(SEQ ID NO：4)，
GGSGGGSGGGSGGGSGGGSG(SEQ ID NO：5)，
GSGGGSGGGSGGGSGGGSGG(SEQ ID NO：6)，
SGGGSGGGSGGGSGGGSGGG(SEQ ID NO：7)。
另一方面，本发明提供上述多肽作为HLA-DR蛋白的α链和β链的连接肽在构建HLA-DR蛋白中的应用。
再一方面，本发明还提供一种HLA-DR蛋白，该HLA-DR蛋白结构组成包括α链、β链和上述多肽；所述α链的C端与所述多肽的N端相连接；所述多肽的C端与所述β链的N端相连接。
由于现有HLA-DR类分子的提取方法存在着操作复杂、原料来源较少、得率低、成本高以及潜在携带外源病毒及其他病原微生物的危险，很难实现产业化；基于此，本发明创造性的设计了一种可溶HLA-DR蛋白质，该蛋白质是将构成HLA-DR蛋白的α链和β链通过特定的由8～48个包含甘氨酸和丝氨酸的氨基酸组成的多肽连接，本发明设计的HLA-DR蛋白质能够在哺乳动物细胞中串联表达，可以模拟可溶性sHLA-DR表达。本发明的HLA-DR蛋白质与天然sHLA-DR结构类似，能够被L234、CR3/43等抗体识别，与膜蛋白mHLA-DR相比构象更稳定，本发明的HLA-DR蛋白质可以使用真核表达系统进行可溶性高效表达，有望在医学生物学领域和生物医药开发中有很好的应用。
上述的HLA-DR蛋白中，优选地，所述α链包括α1结构域多肽；所述α1结构域多肽的C端与所述多肽的N端相连接；
所述α1结构域多肽的氨基酸序列包括SEQ ID NO：8所示氨基酸序列或与SEQ ID NO：8所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与α1结构域多肽具有相同功能的氨基酸序列。
α1结构域多肽氨基酸序列SEQ ID NO：8如下：
Figure PCTCN2021102956-appb-000001
与SEQ ID NO：8所示氨基酸序列中经过取代一个或几个氨基酸且与α1结构域多肽具有相同功能的氨基酸序列可以包括如下氨基酸序列：
SEQ ID NO：41：
Figure PCTCN2021102956-appb-000002
上述的HLA-DR蛋白中，优选地，所述α链还包括α2结构域多肽，所述α1结构域多 肽的C端与所述α2结构域多肽的N端相连接，所述α2结构域多肽的C端与所述多肽的N端相连接；
所述α2结构域多肽的氨基酸序列包括SEQ ID NO：9所示氨基酸序列或与SEQ ID NO：9所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与α2结构域多肽具有相同功能的氨基酸序列，或为SEQ ID NO：9所示氨基酸序列的部分片段且具备α2结构域多肽部分功能的氨基酸序列(例如下述α2结构域多肽氨基酸序列中划线部分)。
α2结构域多肽氨基酸序列SEQ ID NO：9如下：
Figure PCTCN2021102956-appb-000003
与SEQ ID NO：9所示氨基酸序列中经过取代一个或几个氨基酸且与α2结构域多肽具有相同功能的氨基酸序列可以包括如下氨基酸序列：
SEQ ID NO：42：
Figure PCTCN2021102956-appb-000004
SEQ ID NO：43：
Figure PCTCN2021102956-appb-000005
SEQ ID NO：44：
Figure PCTCN2021102956-appb-000006
上述的HLA-DR蛋白中，优选地，所述β链包括β1结构域多肽和β2结构域多肽；所述多肽的C端与所述β1结构域多肽的N端相连接，所述β1结构域多肽的C端与所述β2结构域多肽的N端相连接；
所述β1结构域多肽的氨基酸序列包括SEQ ID NO：10所示氨基酸序列或与SEQ ID NO：10所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与β1结构域多肽具有相同功能的氨基酸序列。
β1结构域多肽氨基酸序列SEQ ID NO：10如下：
Figure PCTCN2021102956-appb-000007
与SEQ ID NO：10所示氨基酸序列中经过取代一个或几个氨基酸且与β1结构域多肽具有相同功能的氨基酸序列可以包括如下氨基酸序列：
SEQ ID NO：45：
FLEQSKSECHFFNGTERVRFLDRYFYNQEEYVRFDSDVGEYRAVTELGRPDAEYWN AQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVHPK所述β2结构域多肽的氨基酸序列包括SEQ ID NO：11所示氨基酸序列或与SEQ ID NO：11所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与β2结构域多肽具有相同功能的氨基酸序列，或为SEQ ID NO：11所示氨基酸序列的部分片段且具备β2结构域多肽部分功能的氨基酸序列(例如下述β2结构域氨基酸序列中划线部分)。
β2结构域多肽氨基酸序列SEQ ID NO：11如下：
Figure PCTCN2021102956-appb-000008
与SEQ ID NO：11所示氨基酸序列中经过取代一个或几个氨基酸且与β2结构域多肽具有相同功能的氨基酸序列可以包括如下氨基酸序列：
SEQ ID NO：46：
Figure PCTCN2021102956-appb-000009
SEQ ID NO：47：
Figure PCTCN2021102956-appb-000010
SEQ ID NO：48：
Figure PCTCN2021102956-appb-000011
上述的HLA-DR蛋白中，优选地，所述β链还包括连接肽；所述β2结构域多肽的C端与所述连接肽的N端相连接；
所述连接肽的氨基酸序列包括SEQ ID NO：12所示氨基酸序列或与SEQ ID NO：12所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与连接肽具有相同功能的氨基酸序列。
连接肽氨基酸序列SEQ ID NO：12如下：
RARSESAQSK
上述的HLA-DR蛋白中，优选地，该HLA-DR蛋白的氨基酸序列包括SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO： 26、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40所示序列中的一种。
上述的HLA-DR蛋白中，优选地，该HLA-DR蛋白的氨基酸序列如SEQ ID NO：13所示：
Figure PCTCN2021102956-appb-000012
再一方面，本发明还提供一种编码上述HLA-DR蛋白的全长多核苷酸。
例如，本发明编码氨基酸序列为SEQ ID NO：13的HLA-DR蛋白的所述全长多核苷酸的核苷酸序列如SEQ ID NO：51所示。
再一方面，本发明还提供包含上述全长多核苷酸的重组质粒或重组细胞。
再一方面，本发明还提供一种HLA-DR蛋白制备方法，该方法包括：将上述全长多核苷酸克隆到质粒载体上，将质粒载体转染至表达细胞中培养，表达目标蛋白，然后进行蛋白纯化得到HLA-DR蛋白。
上述的制备方法中，优选地，所述表达细胞包括CHO细胞。
现有技术中，异源二聚体体外分别表达后自发或人为组装方法繁琐且产率极低，本发明采用α链和β链串联表达的方式，产量高出其他方法几十倍且纯化工艺简单易重复。
再一方面，本发明还提供上述HLA-DR蛋白在制备用于脓毒血症诊断试剂中的用途。
再一方面，本发明还提供一种可溶性人白细胞抗原-DR(sHLA-DR)的定量检测试剂盒，该试剂盒包括上述的HLA-DR蛋白。
本发明的HLA-DR蛋白可以用作工作校准品、校准品、质控品；工作校准品是企业最高溯源物质，用于给校准品/质控品溯源定值。标准品用于生成/标准主曲线，给常规样本定值(例如：脓毒血症血浆样本)；质控品用于验证分析仪器或试剂盒的性能。
上述的试剂盒中，优选地，所述试剂盒还包括生物素标记sHLA-DR鼠单抗、吖啶酯标记sHLA-DR鼠单抗、链霉亲和素标记的免疫磁性微珠和缓冲液。
本发明的有益效果：
(1)通过特定Linker长度和序列的选择，将HLA-DR的α链和β链串联表达，并形成正确的空间结构，抗原活性与天然蛋白相似度95％左右；
(2)选取不同亚型β链的公共序列表达，可同时模拟多种类型的HLA-DR分子；
(3)现有技术是将异源二聚体体外表达后的产物进行自发或人为组装，操作繁琐且产率极低，本发明采用α链和β链串联表达的方式，产量高出其他方法几十倍且工艺简单易重复；
(4)应用：可以用于脓毒症诊断中可溶性人类白细胞DR抗原(sHLA-DR)的定量检测，比细胞膜表面的HLA-DR更早预警脓毒症。
附图说明
图1为本发明实施例1中获得的HLA-DR蛋白的电泳图；
图2为本发明实施例2中获得的sHLA-DR标准曲线。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
实施例1 HLA-DR蛋白制备方法(以SEQ ID NO：13为例)
1、载体构建
1.1、α链氨基酸序列获取
从NCBI数据库中获得了人源的人类白细胞抗原-DR(HLA-DR)的α链氨基酸序列以及基因序列(UniProtKB-P01903(DRA_HUMAN)；Gene ID:3122,updated on 16-Feb-2020)；HLA-DRα链核苷酸序列如SEQ ID NO：49所示。
1.2、β链氨基酸序列获取
β链主要是DRB1，DRB3，DRB4，DRB5这几种，其中DRB1变异最多。根据uniprot数据库，筛选了50份DRB1，24份DRB3，19份DRB4，22份DRB5的蛋白序列；进一步对比筛选获得同源性较高的序列，设计获得了β链氨基酸序列，共231氨基酸。
根据β链氨基酸序列，设计β链核苷酸序列如SEQ ID NO：50所示。
1.3、目的序列合成
目的序列设计：酶切位点+(GCCACC)+IL2信号肽+目的基因+10xhis+TAA+酶切位点
其中IL-2信号肽：MYRMQLLSCIALSLALVTNS；酶切位点：BamHI和XhoI
目的蛋白的全长核苷酸序列如SEQ ID NO：51所示。
将设计的表达单元送交基因合成公司合成了该序列，合成的表达单元插入在pVAX1载体中，获得HLA-DR-α-L20-β-pVAX1重组质粒以及相应甘油菌HLA-DR-α-L20-β-pVAX1-DH5α。
2、真核表达
2.1、表达的质粒制备
用质粒试剂盒提取含有目的基因的表达质粒HLA-DR-α-L20-β-pVAX1，乙醇沉淀DNA，在生物安全柜中自然风干后，用无菌水溶解DNA，用Nanodrop测定DNA浓度。
2.2、CHO细胞株准备
2.2.1、解冻ExpiCHO-S TM细胞
从液氮中取出细胞管，在37℃的水浴中转动冻存管1～2分钟后将细胞全部转移至含有30mL的预热ExpiCHO TM表达培养基的通气式锥形摇瓶中。将细胞置于轨道摇床上(37℃培养箱含8％CO 2，相对湿度≥80％的空气条件下)培养。
解冻三天后，测定活细胞密度和存活率百分比(细胞存活率应≥90％)。继续监测细胞密度和存活率，当细胞密度达到4×10 6～6×10 6个活细胞/mL时，进行细胞传代。
2.3、转染
使新鲜解冻的细胞复苏培养两代或更多代，方可进行转染。
(1)传代并扩增ExpiCHO-S TM细胞，直至细胞密度达到约4×10 6～6×10 6个活细胞/mL。
(2)第-1天：分种细胞：转染前一天(第-1天)，分种第(1)步中的ExpiCHO-S TM培养物，最终密度为3×10 6～4×10 6个活细胞/mL，使细胞过夜生长。
(3)第0天：转染细胞：次日(第0天)，测定活细胞密度和存活率百分比。细胞密度应达到约7×10 6～10×10 6个活细胞/mL。存活率应为95％～99％，方可继续转染。
(4)使用新鲜的预热至37℃的ExpiCHO TM表达培养基将第(2)步中的细胞稀释至最终密度为6×10 6个活细胞/mL。轻轻晃动培养瓶，混匀细胞。
(5)使用冷的试剂(4℃)配制ExpiFectamine TM CHO/质粒DNA复合物。络合过程中无需将试剂置于冰上。只需从冰箱中取出试剂，开始DNA络合即可。
(6)室温孵育ExpiFectamine TM CHO/质粒DNA复合物(第5d步)1～5分钟，然后将 溶液慢慢转移至第(4)步的培养瓶中，在添加过程中轻轻晃动培养瓶。
(7)将细胞置于轨道摇床上(37℃培养箱含8％CO 2的湿化空气条件下)培养。
3、蛋白纯化
将30mLExpiCHO-S TM细胞培养液于4000g离心10分钟，上清经过0.22um过滤澄清去除细胞碎片。澄清的细胞培养液中加入1/25(v/v)500mM咪唑，使用镍柱分离纯化。镍柱使用20mM PB(500mM NaCl 20mM咪唑pH7.0)平衡5个柱体积，然后进样分离，分别以100mM，300mM和500mM咪唑阶段洗脱，收集目标咪唑的洗脱部分作为目标蛋白洗脱峰。即为目的蛋白HLA-DR-α-L20-β-pVAX1(B1)蛋白。蛋白纯化结果如图1所示，SDS-PAGE验证纯度95％以上。
图1中，M：蛋白Marker；1：10ug HLA-DR-α-L20-β-pVAX1(B1)蛋白；2：20ug HLA-DR-α-L20-β-pVAX1(B1)蛋白。
4、蛋白定量：
将上述样本置换透析缓冲液至PBS中，超滤浓缩后采用BCA蛋白浓度测定试剂盒测定sHLA-DR蛋白浓度为1.2mg/mL。
实施例2 sHLA-DR定量检测试剂盒原理及检测步骤
1、检验原理
定量检测试剂盒采用双抗体夹心法检测血浆中可溶性人白细胞抗原-DR(sHLA-DR)的含量。试剂盒采用针对sHLA-DR的一对单克隆抗体(常规市售购买获得)，其中一株抗体标记生物素，另一株抗体标记吖啶酯。标本/校准液/质控液与生物素标记的单克隆抗体、吖啶酯标记的单克隆抗体以及包被有链霉亲和素的免疫磁性微粒反应形成免疫复合物，此复合物的形成量与待测抗原的含量成正比。复合物中的吖啶酯在底物液的激发下释放光子，自动监测3秒钟内发出的相对光强度RLU，与sHLA-DR浓度成一定的比例关系，仪器自动拟合计算sHLA-DR浓度。
其中，本发明制备的蛋白作为标准品、校准品、质控品，其中标准品用于生成定量标准曲线。工作校准品作为企业最高溯源物质，用于检测常规样本，生成标准曲线，给常规样本的定值，质控品是已知的浓度含量，用仪器测量来鉴定该仪器测量结果是否准确。
2、主要组成成分
试剂盒主要成分如下表1所示：
表1：
Figure PCTCN2021102956-appb-000013
3、检测步骤
3.1、前期准备
仪器准备：反应杯、微量移液器和吸头、科斯迈全自动磁微粒化学发光仪SMART6500/500S。
试剂准备：准备预激发液、激发液和洗涤液(含磷酸盐缓冲液、表面活性剂)，并连同试剂盒组分一起安装到仪器上。
3.2、检测步骤
3.2.1、基本操作步骤如表2所示：
表2：
Figure PCTCN2021102956-appb-000014
Figure PCTCN2021102956-appb-000015
3.2.2、全自动化学发光仪操作步骤：开机准备→仪器自检→试剂安装→校准→质控→样本加载→仪器运行测定→结果审核→报告。
4、标准曲线如图2所示，样本浓度计算：仪器软件会自动将样本发光值根据标曲计算出浓度。
实施例3 Linker长度变化对融合蛋白活性影响
本实施例设计了6组不同长度的Linker，如下表3所示：
表3：
Figure PCTCN2021102956-appb-000016
表4：
Figure PCTCN2021102956-appb-000017
表4中发光值，是将表3不同设计方案所获取的目的蛋白稀释成不同浓度(10000ng/mL、5000ng/mL等)作为样本在定量检测体系里的发光值；其中膜蛋白为对照 组。等浓度样本发光值与膜蛋白越相近，蛋白活性越好；蛋白活性是通过计算等浓度样本发光值与膜蛋白发光值比值的平均值来评价。
由上述实验数据可知：Linker长度在8～48个氨基酸时，蛋白活性占天然蛋白活性的20％以上，可以作为检测体系校准品；更优为16～32个氨基酸；尤其当Linker的长度为20个氨基酸时，蛋白活性占天然蛋白活性的75％，HLA-DR蛋白表达量高达5mg/30mL细胞。蛋白活性越高，越接近天然蛋白，检测样本时越能反应实际情况。
实施例4 Linker氨基酸组成的变化对融合蛋白活性影响
根据实施例3可以看出，20个氨基酸长度的Linker效果最佳，在此前提下，基于实施例3中L4组实验，本实施例针对20个氨基酸长度的Linker中甘氨酸(G)和丝氨酸(S)的组成对HLA-DR蛋白活性的影响，设计了6组实验，如下表5所示：
表5：
Figure PCTCN2021102956-appb-000018
表6：
Figure PCTCN2021102956-appb-000019
由上述实验数据可知：Linker中氨基酸由甘氨酸和丝氨酸混合组成相比单一甘氨酸或丝氨酸组成具备更优的抗原活性，且5个GGGS循环和4个GGGGS循环获得的目的 蛋白抗原活性最好，尤其是5个GGGS循环最优，抗原活性占天然蛋白活性的76％，HLA-DR蛋白表达量高达4.5mg/30mL细胞。
实施例5 Linker为5个GGGS循环时，甘氨酸和丝氨酸的组成顺序变化对融合蛋白活性影响
根据实施例4可以看出，5个GGGS循环构成的Linker效果最佳，在此前提下，基于实施例4中L4组实验，本实施例针对GGGS中甘氨酸和丝氨酸的组成顺序变化对HLA-DR蛋白活性的影响，设计了4组实验，如下表7所示：
表7：
编号 蛋白活性 表达方式 表达量(30mL细胞)
L4(SEQ ID NO：17) 78％ 80％胞外分泌 5mg
B1(SEQ ID NO：13) 98％ 80％胞外分泌 4mg
B2(SEQ ID NO：25) 90％ 80％胞外分泌 3mg
B3(SEQ ID NO：26) 110％ 80％胞外分泌 2.5mg
表8：
Figure PCTCN2021102956-appb-000020
由上述实验数据可知：20个氨基酸的Linker组成中，5个GGSG循环获得的目的蛋白抗原活性最好，抗原活性占天然蛋白活性的98％，HLA-DR蛋白表达量高达4mg/30mL细胞。
实施例6 α链序列对融合蛋白活性影响
根据实施例3～5确定Linker最佳为5个GGSG循环(即：SEQ ID NO：5)的前提下，本实施例针对α链序列的变化对HLA-DR蛋白活性的影响，设计了6组实验，如下 表9所示：
表9：
编号 蛋白活性 表达方式 表达量(30mL细胞)
B1(SEQ ID NO：13) 96％ 80％胞外分泌 5mg
C1(SEQ ID NO：27) 0％ 70％胞外分泌 1.5mg
C2(SEQ ID NO：28) 0％ 80％胞外分泌 2mg
C3(SEQ ID NO：29) 36％ 80％胞外分泌 3mg
C4(SEQ ID NO：30) 48％ 80％胞外分泌 1.2mg
C5(SEQ ID NO：31) 76％ 80％胞外分泌 1.8mg
表10：
Figure PCTCN2021102956-appb-000021
由上述实验数据可知：α1功能区域是必要的，α2区域有助于抗原构象生成。α1结构域+α2结构域胞外段全长肽段构建获得的HLA-DR蛋白抗原活性最好，抗原活性占天然蛋白活性的96％，HLA-DR蛋白表达量高达5mg/30mL细胞。
实施例7 β链序列对融合蛋白活性影响
根据实施例3～5确定Linker最佳为5个GGSG循环(即：SEQ ID NO：5)，根据实施例5确定α链为α1结构域多肽(即：SEQ ID NO：8)+α2结构域多肽(即：SEQ ID NO：9)胞外段全长肽段的前提下，本实施设计了4组实验考察β链序列变化对HLA-DR蛋白活性的影响，如下表11所示：
表11：
编号 蛋白活性 表达方式 表达量(30mL细胞)
B1(SEQ ID NO：13) 96％ 80％胞外分泌 5mg
D1(SEQ ID NO：32) 71％ 70％胞外分泌 2mg
D2(SEQ ID NO：33) 37％ 80％胞外分泌 2.6mg
D3(SEQ ID NO：34) 0％ 80％胞外分泌 1.3mg
表12：
Figure PCTCN2021102956-appb-000022
由上述实验数据可知：β1结构域多肽+β2结构域多肽、β1结构域多肽+β2结构域多肽+连接肽公共区全长肽段构建获得的HLA-DR蛋白抗原活性最好，尤其是包含公共区全长肽段的β链构建的蛋白抗原活性占天然蛋白活性的96％，HLA-DR蛋白表达量高达5mg/30mL细胞。
实施例8 α链和β链中部分氨基酸突变对融合蛋白活性影响
根据实施例3～7确定了最佳的Linker、α链和β链，由此构建获得的HLA-DR蛋白(即：B1组)，其氨基酸序列如SEQ ID NO：13所示。设计了7组实验考察α链和β链中氨基酸突变对融合蛋白活性的影响，如下表13所示：
表13：
编号 蛋白活性 表达方式 表达量(30mL细胞)
B1(SEQ ID NO：13) 95％ 80％胞外分泌 5mg
E1(SEQ ID NO：35) 90％ 80％胞外分泌 4.5mg
E2(SEQ ID NO：36) 93％ 80％胞外分泌 4mg
E3(SEQ ID NO：37) 94％ 80％胞外分泌 4.3mg
E4(SEQ ID NO：38) 93％ 80％胞外分泌 3.2mg
E5(SEQ ID NO：39) 90％ 80％胞外分泌 3.8mg
E6(SEQ ID NO：40) 91％ 80％胞外分泌 3mg
表14：
Figure PCTCN2021102956-appb-000023
Figure PCTCN2021102956-appb-000024
注：相同实验组，在不同批操作时，实验结果不会完全一致，会有些许误差，一般产品重复性CV<10％均符合要求，这里默认误差在±10％均是合理的。
由上述实验数据可知：α链和β链部分氨基酸突变(抗原表位除外)对HLA-DR蛋白活性影响较小。
实施例9 本发明与现有方法比较
1、表达量对比实验如下表15和表16所示：
表15：
表达方案 产量
天然膜蛋白 74pg/1.8×10 8个细胞
本发明 4mg/1.8×10 8个细胞
表16：
表达方案 产量
体外重组 12ug/30mL细胞
本发明 4mg/30mL细胞
由表15和表16所示：相比之下，本发明的方案产量是天然膜蛋白产量的10 7倍；是在DRα和DRβ通过亮氨酸拉链体外组装方案的300多倍。
参考文献：
1.HLA-DR分子的分离纯化，贾帅争，孙红琰，刘晓达，杜芝燕，王全立，(军事医学科学院野战输血研究所，北京100850)军事医学科学院院刊2001年3月，第25卷第 1期。
2.Kalandadze A,Galleno M,Foncerrada L,et al.Expression of recombinant HLA-DR2 molecules[J].Journal of Biological Chemistry,1996,271(33):20156-20162。
2、稳定性对比实验：
比较本发明B1组蛋白和天然蛋白的热加速稳定性。
方案：
实验组：将相同浓度(均为1ug/mL)的B1蛋白和天然蛋白放在37℃温箱中热加速破坏3天；
对照组是1ug/mL的B1组蛋白和天然蛋白4度冰箱放置3天。
用实施例2中的定量检测试剂盒验证两组蛋白浓度变化；根据图2标准曲线计算蛋白浓度。
回收率＝实验组浓度/对照组浓度(回收率越接近100％说明稳定性越好)
结果如下表17所示：
表17：
Figure PCTCN2021102956-appb-000025
由表17可以看出：实验数据计算获得的B1组回收率＝94％；对照组膜蛋白回收率＝37％。由此可见，本发明B1组HLA-DR蛋白的稳定性显著性优于天然膜蛋白的稳定性。

Claims (20)

  1. 一种多肽，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的8～48个氨基酸残基的多肽。
  2. 根据权利要求1所述的多肽，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的16～32个氨基酸残基的多肽。
  3. 根据权利要求2所述的多肽，其中，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的20个氨基酸残基的多肽。
  4. 根据权利要求3所述的多肽，其中，所述多肽的氨基酸序列选自如下序列中的一种：
    GGGSGGGSGGGSGGGSGGGS(SEQ ID NO：1)，
    GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：2)，
    GGSGGSGGSGGSGGSGGSGG(SEQ ID NO：3)，
    GSGSGSGSGSGSGSGSGSGS(SEQ ID NO：4)，
    GGSGGGSGGGSGGGSGGGSG(SEQ ID NO：5)，
    GSGGGSGGGSGGGSGGGSGG(SEQ ID NO：6)，
    SGGGSGGGSGGGSGGGSGGG(SEQ ID NO：7)。
  5. 权利要求1～4任一项所述多肽作为HLA-DR蛋白的α链和β链的连接肽在构建HLA-DR蛋白中的应用。
  6. 一种HLA-DR蛋白，该HLA-DR蛋白结构组成包括α链、β链和权利要求1～4任一项所述多肽；所述α链的C端与所述多肽的N端相连接；所述多肽的C端与所述β链的N端相连接。
  7. 根据权利要求6所述的HLA-DR蛋白，其中，所述α链包括α1结构域多肽；所述α1结构域多肽的C端与所述多肽的N端相连接；
    所述α1结构域多肽的氨基酸序列包括SEQ ID NO：8所示氨基酸序列或与SEQ ID NO：8所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与α1结构域多肽具有相同功能的氨基酸序列。
  8. 根据权利要求7所述的HLA-DR蛋白，其中，所述α链还包括α2结构域多肽，所述α1结构域多肽的C端与所述α2结构域多肽的N端相连接，所述α2结构域多肽的C端与所述多肽的N端相连接；
    所述α2结构域多肽的氨基酸序列包括SEQ ID NO：9所示氨基酸序列或与SEQ ID NO：9所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与α2结构域多肽具有相同功能的氨基酸序列，或为SEQ ID NO：9所示氨基酸序列的部分片段且具备α2结构域多肽部分功能的氨基酸序列。
  9. 根据权利要求6所述的HLA-DR蛋白，其中，所述β链包括β1结构域多肽和β2结构域多肽；所述多肽的C端与所述β1结构域多肽的N端相连接，所述β1结构域多肽的C端与所述β2结构域多肽的N端相连接；
    所述β1结构域多肽的氨基酸序列包括SEQ ID NO：10所示氨基酸序列或与SEQ ID NO：10所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与β1结构域多肽具有相同功能的氨基酸序列；
    所述β2结构域多肽的氨基酸序列包括SEQ ID NO：11所示氨基酸序列或与SEQ ID NO：11所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与β2结构域多肽具有相同功能的氨基酸序列，或为SEQ ID NO：11所示氨基酸序列的部分片段且具备β2结构域多肽部分功能的氨基酸序列。
  10. 根据权利要求9所述的HLA-DR蛋白，其中，所述β链还包括连接肽；所述β2结构域多肽的C端与所述连接肽的N端相连接；
    所述连接肽的氨基酸序列包括SEQ ID NO：12所示氨基酸序列或与SEQ ID NO：12所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与连接肽具有相同功能的氨基酸序列。
  11. 根据权利要求6所述的HLA-DR蛋白，其中，该HLA-DR蛋白的氨基酸序列包括SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40所示序列中的一种。
  12. 根据权利要求11所述的HLA-DR蛋白，其中，该HLA-DR蛋白的氨基酸序列包括SEQ ID NO：13所示的序列。
  13. 一种编码权利要求6～12任一项所述HLA-DR蛋白的全长多核苷酸。
  14. 根据权利要求13所述HLA-DR蛋白的全长多核苷酸，其中，编码氨基酸序列为SEQ ID NO：13的HLA-DR蛋白的全长多核苷酸的核苷酸序列如SEQ ID NO：51所 示。
  15. 一种包含权利要求13或14所述全长多核苷酸的重组载体或重组细胞。
  16. 一种HLA-DR蛋白的制备方法，该方法包括：将权利要求13或14所述全长多核苷酸克隆到质粒载体上，将质粒载体转染至表达细胞中培养，表达目标蛋白，然后进行蛋白纯化得到HLA-DR蛋白。
  17. 根据权利要求16所述的制备方法，其中，所述表达细胞包括CHO细胞。
  18. 权利要求6～12任一项所述HLA-DR蛋白在制备用于脓毒血症诊断试剂中的用途。
  19. 一种定量检测sHLA-DR的试剂盒，该试剂盒包括权利要求6～12任一项所述HLA-DR蛋白。
  20. 根据权利要求19所述的试剂盒，其中，所述试剂盒还包括生物素标记sHLA-DR鼠单抗、吖啶酯标记sHLA-DR鼠单抗、链霉亲和素标记的免疫磁性微珠和缓冲液。
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