Method for detecting biological activity of vaccine

Abstract

The invention provides a method for detecting the biological activity of a vaccine. Through co-culture of the NFAT-Jurkat cell line over-expressing the TCR and the CD8 and the HepG2 cell line expressing the antigen, the biological activity of a vaccine drug can be rapidly detected, presentation of the antigen and activation of specific T cells can be quantitatively detected, and meanwhile the method has the advantages of being convenient to detect, high in throughput and low in cost.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for detecting the biological activity of a vaccine. Background Art

[0002] At present, there are two main methods for in vitro detection of biological activity of vaccine drugs in different drug forms, including peptides, mRNA vaccines, DNA vaccines and DC vaccines: one is to detect the abundance of antigen expression in cells by flow cytometry; the other is to detect the secretion of antigens in the supernatant by enzyme-linked immunosorbent assay (ELISA). However, the above two in vitro detection methods have limitations. First, the antigen to be detected must have developed commercial antibodies. Secondly, there are requirements for the localization of antigen cells. Flow cytometry is best for antigen localization on the cell membrane, and the ELISA method is suitable for secretory proteins. For vaccines that mainly activate T cell immunity, neither method can detect the efficiency of antigen presentation by major histocompatibility complex (MHC) molecules and the strength of specific T cell activation. Therefore, the current detection methods for this type of vaccine are mainly carried out in experimental animals, with long test cycles, low throughput and high costs, which have great limitations.

[0003] Therefore, it is crucial to provide a convenient method for biological detection of vaccines. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a method for detecting the biological activity of a vaccine.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a method for constructing a cell line for detecting the biological activity of a vaccine, the method comprising constructing a cell line expressing a target head that recognizes an antigen.

[0007] Furthermore, the cell line used to express the target head that recognizes the antigen is an immortalized cell line that expresses a reporter gene.

[0008] Furthermore, the cell line also includes a molecule that assists in recognition.

[0009] Furthermore, the target head that recognizes the antigen is selected from TCR.

[0010] Furthermore, the TCR is a TCR targeting a specific antigen.

[0011] Furthermore, the antigens include viral antigens, tumor antigens, autoimmune antigens, and pathogen antigens.

[0012] Furthermore, the antigens include viral antigens and tumor antigens.

[0013] Furthermore, the viral antigen is selected from DNA viral antigens.

[0014] Furthermore, the DNA virus antigen is selected from herpes virus antigens.

[0015] Furthermore, the herpes virus antigen is selected from CMV.

[0016] Furthermore, the tumor antigen is selected from tumor-specific antigens.

[0017] Furthermore, the tumor-specific antigen is selected from MAGE-As.

[0018] Furthermore, the MAGE-As are selected from MAGE-A1.

[0019] Further, CMV_TCR is selected from CMV_TCRα and CMV_TCRβ.

[0020] Furthermore, a cleavage peptide is connected between CMV_TCRα and CMV_TCRβ.

[0021] Furthermore, the sequence of CMV_TCR has at least 90% sequence identity with the sequence shown in SEQ ID NO:2.

[0022] Furthermore, the sequence of CMV_TCR is shown in SEQ ID NO:2.

[0023] Furthermore, the sequence of MAGE-A1_TCR has at least 90% sequence identity with the sequence shown in any one of SEQ ID NOs: 9-11, respectively.

[0024] Furthermore, the sequence of MAGE-A1_TCR is shown in any one of SEQ ID NOs: 9-11.

[0025] Furthermore, the reporter gene includes a luciferase reporter gene, a chloramphenicol acetyltransferase reporter gene, β-galactosidase, secretory human placental alkaline phosphatase, and green fluorescent protein.

[0026] Furthermore, the reporter gene is selected from a luciferase reporter gene.

[0027] Furthermore, the immortalized cell line is selected from an immortalized T cell line.

[0028] Furthermore, the immortalized T cell line is selected from Jurkat cells.

[0029] Furthermore, the auxiliary recognition molecules include one or more of molecules that assist the binding of the target head to the antigen, costimulatory signal molecules and cytokine receptors.

[0030] Furthermore, the auxiliary recognition molecules are selected from molecules that assist the binding between the target head and the antigen.

[0031] Furthermore, the molecules that assist the binding of the target head to the antigen include CD8, CD4, CD3, CD19, and CD20.

[0032] Furthermore, the molecule that assists the binding of the target head to the antigen is selected from CD8.

[0033] Furthermore, the CD8 is selected from CD8α and CD8β.

[0034] Furthermore, a cleavage peptide is connected between the CD8α and CD8β.

[0035] Further, the cleavage peptide is selected from P2A cleavage peptide.

[0036] Furthermore, the sequence of CD8 has at least 90% sequence identity with the sequence shown in SEQ ID NO:1.

[0037] Furthermore, the sequence of CD8 is shown in SEQ ID NO:1.

[0038] Furthermore, the method for constructing a cell line expressing a target head that recognizes an antigen includes a plasmid transfection method, a virus transfection method, a liposome transfection method, and a transposon transfection method.

[0039] Furthermore, the method for constructing a cell line expressing a target head that recognizes an antigen is selected from a plasmid transfection method and a virus transfection method.

[0040] Furthermore, the viral transfection method includes lentiviral transfection method, adenoviral transfection method, and adeno-associated viral transfection method.

[0041] Furthermore, the viral transfection method is selected from a lentiviral transfection method.

[0042] Furthermore, the vectors used in the plasmid transfection method include PZXK and pLVX.

[0043] Furthermore, the method for transferring the plasmid into the cell line is selected from a physical method.

[0044] Furthermore, the physical method is selected from electroporation.

[0045] Furthermore, the vaccine is selected from T cell immune vaccine.

[0046] Furthermore, the types of vaccines include polypeptide vaccines, nucleic acid vaccines, and DC vaccines.

[0047] Furthermore, the nucleic acid vaccine includes mRNA vaccine and DNA vaccine.

[0048] Furthermore, the vaccine is selected from polypeptide vaccines and nucleic acid vaccines.

[0049] Furthermore, the nucleic acid vaccine is selected from mRNA vaccine.

[0050] The second aspect of the present invention provides a cell line for detecting the biological activity of a vaccine, wherein the cell line is a cell line expressing a target head that recognizes an antigen.

[0051] Furthermore, the target head that recognizes the antigen is selected from TCR.

[0052] Furthermore, the cell line used to express the target head that recognizes the antigen is an immortalized cell line that expresses a reporter gene.

[0053] Furthermore, the reporter gene is selected from a luciferase reporter gene.

[0054] Furthermore, the immortalized cell line is selected from an immortalized T cell line.

[0055] Furthermore, the immortalized T cell line is selected from Jurkat cells.

[0056] Furthermore, the cell line also includes a molecule that assists in recognition.

[0057] Furthermore, the auxiliary recognition molecules include one or more of molecules that assist the binding of the target head to the antigen, costimulatory signal molecules and cytokine receptors.

[0058] Furthermore, the auxiliary recognition molecules are selected from molecules that assist the binding between the target head and the antigen.

[0059] Furthermore, the molecule that assists the binding of the target head to the antigen is selected from CD8.

[0060] The third aspect of the present invention provides a method for detecting the biological activity of a vaccine, the method comprising using the cell line described in the second aspect of the present invention to detect the biological activity of the vaccine.

[0061] Furthermore, the method further comprises co-culturing the cell line with a cell line expressing an antigen.

[0062] Furthermore, the method also includes using a detection reagent to detect the biological activity of the vaccine.

[0063] Furthermore, the detection reagent is selected from a luciferase detection reagent.

[0064] Furthermore, the cell lines expressing antigens include natural cell lines and engineered cell lines.

[0065] Furthermore, the antigen-expressing cell line is selected from natural or engineered tumor cell lines.

[0066] Furthermore, the natural or engineered tumor cell line is selected from the HepG2 cell line.

[0067] Furthermore, the antigen-expressing cell line is constructed by co-culture.

[0068] Furthermore, the effector-target ratio of the co-culture is (0.5-20):1.

[0069] Furthermore, the effector-target ratio of the co-culture is (3-10):1.

[0070] Furthermore, the co-cultivation time is 2-48 hours.

[0071] Furthermore, the co-cultivation time is 6 hours.

[0072] Furthermore, the vaccine is selected from T cell immune vaccine.

[0073] Furthermore, the types of vaccines include polypeptide vaccines, nucleic acid vaccines, and DC vaccines.

[0074] Furthermore, the nucleic acid vaccine includes mRNA vaccine and DNA vaccine.

[0075] Furthermore, the vaccine is selected from polypeptide vaccines and nucleic acid vaccines.

[0076] Furthermore, the nucleic acid vaccine is selected from mRNA vaccine.

[0077] Furthermore, the biological activity of the vaccine includes the efficiency of antigen presentation and the degree of T cell activation.

[0078] The fourth aspect of the present invention provides a product for detecting the biological activity of a vaccine, wherein the product comprises the cell line described in the second aspect of the present invention.

[0079] Furthermore, the product also includes a cell line expressing the antigen.

[0080] Furthermore, the product also includes a detection reagent.

[0081] Furthermore, the detection reagent is selected from luciferase.

[0082] Furthermore, the product also includes instructions for use, positive controls, and negative controls.

[0083] Furthermore, the product also includes one or more sterile containers.

[0084] Furthermore, the product includes a kit.

[0085] A fifth aspect of the present invention provides any of the following applications:

[0086] (1) Use of the cell line described in the second aspect of the present invention or the product described in the fourth aspect of the present invention in detecting the biological activity of a vaccine;

[0087] (2) Use of the cell line described in the second aspect of the present invention or the method described in the third aspect of the present invention in quality control of vaccine and drug production and research and development;

[0088] (3) Use of the cell line described in the second aspect of the present invention or the method described in the third aspect of the present invention in screening vaccines.

[0089] Furthermore, the vaccine is selected from T cell immune vaccine.

[0090] Furthermore, the types of vaccines include polypeptide vaccines, nucleic acid vaccines, and DC vaccines.

[0091] Furthermore, the vaccine screening includes vaccine optimization, TCR screening, and CAR molecule design.

[0092] Furthermore, the vaccine optimization includes antigen screening, enhancing element screening, cap structure screening, base modification screening, UTR region screening, PolyA structure screening, codon optimization sequence screening, circular mRNA structure screening, self-replicating mRNA screening, delivery system screening, and cationic liposome screening.

[0093] Furthermore, the TCR screening includes TCR affinity screening, TCR specificity screening, TCR on / offtarget evaluation, TCR response strength screening, TCR stability screening, endogenous TCR knockout efficiency evaluation, and exogenous TCR expression evaluation.

[0094] Advantages and beneficial effects of the present invention:

[0095] The present invention can quickly detect the biological activity of vaccine drugs by co-culturing the NFAT-Jurkat cell line overexpressing TCR and CD8 with the HepG2 cell line expressing the antigen, and can quantitatively detect the presentation of antigens and the activation of specific T cells, while having the advantages of convenient detection, high throughput and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 This is a functional assay diagram of the NFAT-Jurkat reporter cell line;

[0097] Figure 2 is the structure diagram of pIPM-CD8αβ, pIPM-CMVpp65 and pIPM-CMV_TCR plasmids;

[0098] Figure 3 This is a graph showing the efficiency of NFAT-Jurkat transiently overexpressing CD8 and CMV_TCR;

[0099] Figure 4It is an in vitro test diagram of the biological activity of epitope peptides and mRNA;

[0100] Figure 5 is the structure diagram of pLVX-CD8 and pLVX-CMV_TCR plasmids;

[0101] Figure 6 This is a graph showing the efficiency of lentiviral infection of NFAT-Jurkat;

[0102] Figure 7 This is the detection diagram of CD8 and CMV_TCR expression in NFAT-Jurkat monoclonal cell line;

[0103] Figure 8 It is an in vitro test diagram of the biological activity of epitope peptides and mRNA;

[0104] Fig. 9 is the structure diagram of pIPM-MAGE-A1 and pIPM-MAGE-A1_TCR plasmids;

[0105] Fig.10 This is a graph showing the efficiency of NFAT-Jurkat cells transiently overexpressing CD8 and MAGE-A1_TCR;

[0106] Fig.11 are 3 MAGE-A1_TCR affinity detection graphs;

[0107] Fig.12 This is a graph of in vitro activity assays of MAGE-A1 scanning peptides, mRNA, and LNPs;

[0108] Fig.13 is a diagram of the plasmid structures of pZXK-CD8 and pZXK-MAGE-A1_TCR-1367;

[0109] Fig.14 This is the positive group detection diagram of NFAT-Jurkat-CD8-MAGE-A1_TCR mixed clone strain;

[0110] Fig.15 This is the NFAT-Jurkat-CD8-MAGE-A1_TCR monoclonal strain selection diagram;

[0111] Fig.16 This is a graph of in vitro activity assays of MAGE-A1 scanning peptides and mRNA;

[0112] Fig.17 This is a graph showing the in vitro activity detection of MAGE-A1-LNP. DETAILED DESCRIPTION

[0113] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0114] The present invention provides a method for constructing a cell line for detecting the biological activity of a vaccine, the method comprising constructing a cell line expressing a target head for recognizing an antigen.

[0115] In one embodiment, biological activity refers to the specific ability or potential of a biological product to achieve a defined biological effect, and the biological effect of a specific cell line can be used to assess the corresponding biological activity of the biological product.

[0116] In one embodiment, the target head that recognizes an antigen includes any target head that can recognize an antigen and transmit a signal. Examples of target heads that recognize antigens include, but are not limited to, natural T cell receptors TCR, B cell receptors BCR, NK cell receptors KIR, and artificially modified antigen-recognizing receptors such as CAR molecules.

[0117] In a specific embodiment, the target head that recognizes the antigen is selected from the natural T cell receptor TCR.

[0118] The TCR is a TCR directed against a specific antigen.

[0119] In one embodiment, the antigen includes but is not limited to viral antigens, tumor antigens, autoimmune antigens, pathogen antigens.

[0120] In a preferred embodiment, the antigen is selected from a viral antigen and a tumor antigen.

[0121] In one embodiment, the viral antigens include but are not limited to DNA viral antigens and RNA viral antigens.

[0122] In a preferred embodiment, the viral antigen is selected from DNA viral antigens.

[0123] In one embodiment, the DNA virus antigens include but are not limited to herpes virus antigens, adenovirus antigens, hepatitis B virus antigens, and smallpox virus antigens.

[0124] In a more preferred embodiment, the DNA virus antigen is selected from herpes virus antigens.

[0125] In one embodiment, the herpes virus antigens include but are not limited to cytomegalovirus antigen (CMV), varicella-zoster virus antigen, and Epstein-Barr virus antigen.

[0126] In a specific embodiment, the herpes virus antigen is selected from cytomegalovirus antigen (CMV).

[0127] In one embodiment, the tumor antigen includes but is not limited to tumor specific antigen and tumor associated antigen. Among them, tumor specific antigen (TSA) is an antigen that is unique to tumor cells or only exists in certain tumor cells but not in normal cells.

[0128] In a preferred embodiment, the tumor antigen is selected from tumor-specific antigens.

[0129] In one embodiment, the tumor-specific antigens include but are not limited to melanoma family antigens MAGE-As (Melanoma antigen), prostate-specific antigen (PSA), and carcinoembryonic antigen (CEA).

[0130] In a more preferred embodiment, the tumor-specific antigen is selected from melanoma antigen family antigens (MAGE-As).

[0131] In one embodiment, the MAGE-As include but are not limited to MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, and MAGE-A12.

[0132] In a specific embodiment, the MAGE-As are selected from MAGE-A1.

[0133] In one embodiment, the reporter gene includes but is not limited to luciferase (LUC) reporter gene, chloramphenicol acetyltransferase (CAT) reporter gene, β-galactosidase (β-gal), secretory human placental alkaline phosphatase (SEAP), green fluorescent protein (GFP).

[0134] In a specific embodiment, the reporter gene is selected from a luciferase reporter gene.

[0135] The immortalized cell line is selected from immortalized T cell lines.

[0136] In one embodiment, the immortalized T cell lines include but are not limited to Jurkat cells and SupT1 cells.

[0137] In a specific embodiment, the immortalized T cell line is selected from Jurkat cells.

[0138] The auxiliary recognition molecules include one or more of molecules that assist the binding of the target head with the antigen, costimulatory signal molecules and cytokine receptors.

[0139] In one embodiment, the molecules that assist in binding between the target head and the antigen include, but are not limited to, CD8, CD4, CD3, CD19, and CD20.

[0140] In one embodiment, the co-stimulatory signal molecules include but are not limited to CD28, 41BB, CD40L, LFA-A, and CD2.

[0141] In one embodiment, cytokine receptors include, but are not limited to, interleukin family receptors, interferon receptors, TNF superfamily receptors, chemokine receptors.

[0142] In a preferred embodiment, the molecules that assist in recognition are selected from molecules that assist in binding of the target head to the antigen.

[0143] In a specific embodiment, the molecule that assists in binding of the target head to the antigen is selected from CD8.

[0144] In one embodiment, the CD8 includes CD8α and CD8β, CD8α and CD8α, CD8β and CD8β.

[0145] In a specific embodiment, said CD8 is selected from CD8α and CD8β.

[0146] In one embodiment, a cleavage peptide is linked between CD8α and CD8β.

[0147] In a preferred embodiment, the cleavage peptide is selected from the 2A cleavage peptide.

[0148] In one embodiment, the 2A cleavage peptide includes a P2A cleavage peptide, a T2A cleavage peptide, an E2A cleavage peptide, and a F2A cleavage peptide.

[0149] In a specific embodiment, the 2A cleavage peptide is selected from a P2A cleavage peptide.

[0150] In one embodiment, the types of vaccines include polypeptide vaccines, nucleic acid vaccines, and DC vaccines.

[0151] Among them, nucleic acid vaccines include mRNA vaccines and DNA vaccines.

[0152] In one embodiment, the mRNA vaccine also includes an mRNA vaccine constructed by delivery technologies such as viral vectors, mechanical transfection, and non-viral vectors.

[0153] In a preferred embodiment, the delivery technology is selected from non-viral vectors.

[0154] In a specific embodiment, the non-viral vector is selected from lipid nanoparticles (LNP), and LNP may include ionizable cationic phospholipids, neutral auxiliary phospholipids, cholesterol, and polyethylene glycol-modified phospholipids (PEGylated lipid), that is, the mRNA vaccine also includes mRNA-LNP vaccine.

[0155] In one embodiment, the method for constructing a cell line expressing a target head that recognizes an antigen includes, but is not limited to, plasmid transfection, virus transfection, liposome transfection, and transposon transfection.

[0156] In a specific embodiment, the method for constructing a cell line expressing a target head that recognizes an antigen is selected from a plasmid transfection method and a viral transfection method.

[0157] In one embodiment, the vector used in the viral transfection method can be any vector, including but not limited to pIPM, pLKO.1, pLVX-IRES-ZsGreenl, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCV-MCS-EFl-copGFP, pLVX-ZsGreenl-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdT rack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP- C, pXCl, pBHGE3, pAAV-MCS, pAAV-RC, pHHelper, pAAV-LacZ, pLK0.1-puro, pLK0.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLK0.1-CMV-Neo, pLK0.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1 -puro-CMV-TagRFP,pLKO.1-puro-CMV-TagFP635,pLKO.-puro-UbC-TurboGFP,pLKO.1-puro-UbC-TagFP635,pLKO-pur o-IPTG-lxLacO, pLKO-puro-IPTG-3xLacO, pLPl, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DEST, pLenti6-GW / U6-1aminshrna, pcDNA1,2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP and Lenti6.2 / N-Lumio / V5-GW / lacZ.

[0158] In one embodiment, the method of transferring the plasmid into the cell line includes but is not limited to physical method, chemical method, and biological method.

[0159] Among them, physical methods include but are not limited to electroporation, gene gun, magnetic bead method, and microinjection.

[0160] Chemical methods include, but are not limited to, calcium phosphate precipitation, cationic polymers.

[0161] Biological methods include, but are not limited to, Agrobacterium transfection.

[0162] In a preferred embodiment, the method of transferring the plasmid into the cell line is selected from physical methods.

[0163] In a specific embodiment, the physical method is selected from electroporation.

[0164] In one embodiment, viral transfection methods include but are not limited to lentiviral transfection methods, adenoviral transfection methods, and adeno-associated viral transfection methods.

[0165] In a specific embodiment, the viral transfection method is selected from a lentiviral transfection method, and the lentiviral infection method comprises infecting a plasmid expressing a target head that recognizes an antigen.

[0166] The lentiviral transfection method further comprises the step of packaging the lentivirus before infection.

[0167] In one embodiment, the packaging cells used for lentiviral packaging are 293 cells.

[0168] In one embodiment, 293 cells include but are not limited to 293LT cells, 293T cells, 293T / 17 cells, 293T / 17SF cells, 293H cells, 293A (Adherent) cells, 293E cells, 293-6E cells, 293F cells, 293FT cells, 293FTM cells, 293S (suspension) cells, 293SG cells, and 293SGGD cells.

[0169] In a specific embodiment, the 293 cells are selected from 293FT cells.

[0170] The lentiviral transfection method also includes a step of screening single clones after infection.

[0171] In one embodiment, flow cytometry is used to screen single clones.

[0172] In one embodiment, monoclonal cells are selected by flow cytometry after staining with a CMVpp65 tetramer flow cytometry antibody that can be recognized by CMV_TCR.

[0173] The present invention provides a method for detecting the biological activity of a vaccine, the method further comprising co-culturing the cell line with a cell line expressing an antigen.

[0174] In one embodiment, the cell line expressing the antigen includes any natural cell line, engineered cell line.

[0175] In a preferred embodiment, the cell line expressing the antigen is selected from a natural or engineered tumor cell line, and the natural or engineered tumor cell line includes but is not limited to HepG2 cell line, Raji cell line, WIL2-S cell line, SU-DHL-10 cell line, BJAB cell line, SU-DHL-4 cell line, P30 / OHK cell line, P116 cell line, MUTZ-3 cell line, SUPB15 cell line, SNK6 cell line, SK-MEL-28 cell line, C8 161 cell line, RPMI-7951 cell line, HeLa cell line, A549 cell line, MCF7 cell line, PC12 cell line, HT1080 cell line, H460 cell line, H146 cell line, T-47D cell line, ZR-75-1 cell line, MKN-45 cell line, SGC-7901 cell line, CT26 cell line, HCT-8 cell line, L-428 cell line, L1326 cell line, Caov-3 cell line, PA-1 cell line.

[0176] In a specific embodiment, the natural or engineered tumor cell line is selected from the HepG2 cell line.

[0177] In one embodiment, the effector-target ratio of the co-culture is (0.5-20):1.

[0178] In a preferred embodiment, the effector-target ratio of the co-culture is (3-10):1.

[0179] In one embodiment, the effector-target ratio (E:T) refers to the ratio of effector cells to target cells. In the present application, effector cells refer to NFAT-Jurkat cells expressing TCR and CD8, and target cells refer to HepG2 cells expressing antigens.

[0180] The culture medium used in the co-culture is any culture medium suitable for cell growth.

[0181] In a preferred embodiment, any medium suitable for cell growth is selected from a basal medium.

[0182] In one embodiment, the basal culture medium includes but is not limited to DMEM (Dulbecco's Modified Eagle Medium), DMEM / Nutrient Mixture F-12 (DMEM / F-12), Advanced DMEM / F-12, 1640 medium, IMDM (Iskoff's Modified Dulbecco's Medium), MEM (Minimum Essential Medium), BME (Basal Medium Eagle), KnockOutDMEM, KnockOutDMEM / F12, AdvancedDMEM / F12, neurobasal.

[0183] In a specific embodiment, the basal culture medium is selected from 1640 medium.

[0184] The present invention provides a product for detecting the biological activity of a vaccine, wherein the product comprises the above cell line.

[0185] In one embodiment, the product also includes one or more sterile containers. Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers can be plastic, glass, laminated paper, metal foil, or other materials.

[0186] The present invention provides application of the cell line or the method in screening vaccines.

[0187] The vaccine screening includes vaccine optimization, TCR screening, and CAR molecule design.

[0188] In one embodiment, the vaccine optimization includes but is not limited to antigen screening, enhancing element screening, cap structure screening, base modification screening, UTR region screening, PolyA structure screening, codon optimized sequence screening, circular mRNA structure screening, self-replicating mRNA screening, delivery system screening, and cationic liposome screening.

[0189] In one embodiment, screening cationic liposomes refers to screening the biological activity of mRNA vaccines prepared with different cationic liposomes.

[0190] In one embodiment, screening for codon-optimized sequences refers to screening for the biological activity of different sequences after codon optimization of nucleic acid vaccines.

[0191] In one embodiment, screening vaccine cap structures refers to screening the biological activity of mRNA vaccines with different cap structures.

[0192] In one embodiment, screening for vaccine base modifications refers to screening the biological activity of mRNA vaccines with different base modifications.

[0193] In one embodiment, screening circular mRNA structures refers to the detection of biological activity of circular mRNA vaccines.

[0194] In one embodiment, screening for self-replicating mRNA refers to the detection of biological activity of self-replicating mRNA vaccines.

[0195] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0196] Example 1

[0197] 1. Experimental Materials

[0198] 1) Cell lines and culture media: NFAT-Jurkat cell line was purchased from Nanjing Novozymes Biotech Co., Ltd.; HepG2 cell line was purchased from Zhejiang Meisen; RPMI-1640 culture medium was purchased from Thermo Fisher Scientific, catalog number 11875-093; fetal bovine serum was purchased from Cellsera, catalog number AU-FBS / FS.

[0199] 2) Plasmids including pIPM-CD8, pIPM-CMVpp65 and pIPM-G44-01M (pIPM-CMV_TCR) were commissioned to Nanjing GenScript Biotechnology Co., Ltd.

[0200] CD8α chain-P2A-CD8β chain (CD8) CDS sequence:

[0201]

[0202] CMV_TCRα chain-P2A-CMV_TCRβ (CMV_TCR) CDS sequence:

[0203]

[0204] CMVpp65 epitope CDS sequence:

[0205]

[0206] 3) mRNA preparation kit: EasyCap T7 Co-transcription Kit with CAG Trimer was purchased from Nanjing Novogene Biotech Co., Ltd. with the catalog number DD4203.

[0207] 4) Electroporation kit: Neon TM The 100 μL electroporation kit was purchased from Thermo Fisher Scientific with the catalog number MPK10096.

[0208] 5) NFAT-Jurka stimulation reagents include: PMA + ionomycin (PMA and Ionomycin) purchased from Shenzhen Dakoway Biotechnology Co., Ltd., with a catalog number of 2030421; CMVpp65 epitope peptide sequence NLVPMVATV (SEQ ID NO: 4), purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0209] 6) Flow cytometry antibodies include: T-Select HLA-A*02:01CMVpp65 Tetramer-NLVPMVATV-PE purchased from MBL, catalog number TS-0010-1C; BV510 Mouse Anti-Human CD8 purchased from BD Biosciences, catalog number 563919.

[0210] 7) Luciferase Assay Kit: Bio-Lite Luciferase Assay System was purchased from Nanjing Novozymes Biotech Co., Ltd. with the product number DD1201-02.

[0211] 2. Experimental methods;

[0212] Step 1: NFAT-Jurkat cells transiently overexpress CD8 and CMV_TCR

[0213] NFAT-Jurkat cell line culture: cultured with 1640 medium containing 10% FBS, maintaining the cell density in the range of 5E5-5E6, and cultured the cells for 2-3 generations before testing their functions.

[0214] NFAT-Jurkat cell function detection: 3E3, 6E3, 3E4, 6E4 and 3E5 were respectively placed in a 96-well white-bottom cell culture plate. The culture medium was 1640 culture medium with 10% FBS. After stimulation with 1 μg / mL PMA and 10 ng / mL Ionomycin for 6 h, the supernatant was discarded after centrifugation at 350 g for 5 min, and 100 μL of luciferase detection reagent (Bio-Lite Luciferase Assay System) was added. After blowing evenly, the cell plate was placed in a microplate reader to detect the fluorescence value.

[0215] Plasmid construction process: Nanjing GenScript Biotechnology Co., Ltd. was commissioned to clone the sequences encoding CMV epitope, CD8α chain-P2A-CD8β chain and CMV_TCRα chain-P2A-CMV_TCRβ into the middle of the BamHI and SacI restriction endonuclease cutting sites of the in vitro transcription plasmid vector. The plasmid structure diagram is shown in Figure 2 .

[0216] Preparation of mRNA: linearize the plasmid with BspQI, react at 50℃ for 3h, and terminate the enzyme digestion with 80℃ for 20min. Purify the linearized plasmid with DNA magnetic beads, and use the purified linearized plasmid as a template to prepare mRNA with EasyCap T7 Co-transcription Kit. The prepared mRNA can be used for electrotransfer or stored at -80℃.

[0217] mRNA electroporation and electroporation efficiency verification process: Take 2E6 NFAT-Jurkat cells, resuspend them in 100μL R solution in the electroporation kit, add 7.5μg CD8-mRNA and 7.5μg CMV_TCR-mRNA and mix well, insert the electroporation gun into the electroporation cup pre-added with 3-5mL E2 solution (electroporation kit) and turn on the electroporator. The electroporation conditions are 1400mV, 20ms, 2plus. After electroporation, resuspend the cells in 1640 complete culture medium. After 24h, take some electroporated cells for CMVpp65 tetramer staining.

[0218] Tetramer staining: Resuspend the test cells of about 3E5-10E5 in 100μL of PBS with 2μL of Tetramer dye, stain for 15-30min in the dark, wash with 1mL PBS and detect on the microscope.

[0219] Step 2: In vitro activity assay of CMVpp65 epitope peptides and mRNA

[0220] NFAT-Jurkat cell line culture: cultured with 1640 medium containing 10% FBS, maintaining the cell density in the range of 5E5-5E6, and cultured the cells for 2-3 generations before testing their functions.

[0221] HepG2 cell line culture: cultured with DMEM medium containing 10% FBS. When the cells grew to 90% density, the HepG2 cells were digested with 0.25% EDTA-trypsin and then passaged at a ratio of 1:5. After 2-3 generations of cell culture, they were used for electroporation or peptide loading.

[0222] Epitope peptide detection: HepG2 cells were incubated with CMVpp65 peptide at a concentration of 1 μg / mL overnight. On the second day, the HepG2 cells were digested and recounted, and 1E5 (E:T=3:1), 6E4 (E:T=5:1) and 3E4 (E:T=10:1) peptide-loaded HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate. 3E5 electroporated CMV_TCR-transfected NFAT-Jurkat cells were added. The culture medium was 200 μL of 1640 culture medium containing 10% FBS. After 6 hours of co-culture, 100 μL of luciferase detection reagent was added, and the fluorescence value was detected in an ELISA instrument.

[0223] mRNA detection: The prepared mRNA needs to be tested with Neon TM 100μL electroporation kit electroporation, specific operation with 100μL Neon TM4E5 HepG2 cells resuspended in 100μL R solution in the electroporation kit were added with 5μg CMV-mRNA and mixed well. After adding 3-5mL E2 solution (electroporation kit) to the electroporation cup, it was placed in the cup slot of the electroporator. The cell suspension mixed with mRNA was carefully aspirated with a 100μL electroporation gun tip to avoid bubbles. The electroporation gun was inserted into the E2 solution of the electroporator and electroporated under the voltage conditions of 1350mV, 10ms, and 4plus. After the electroporated HepG2 cells were cultured overnight in 10% FBS 1640 medium, the HepG2 cells were digested and recounted, and 1E5 (E:T=3:1), 6E4 (E:T=5:1) and 3E4 (E:T=10:1) peptide-loaded HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate, and 3E5 electroporated NFAT-Jurkat cells were added. The culture medium was 200 μL of 1640 medium containing 10% FBS. After co-culture for 6 hours, 100 μL of luciferase detection reagent was added, and the fluorescence value was detected in a microplate reader.

[0224] 3. Experimental results

[0225] Figure 1 The results showed that 3E5 NFAT-Jurkat cells / well were stimulated with PMA and Ionomycin for 6 h, and the fluorescence value detected by the microplate reader increased from 2 to 4362, proving that the NFAT-Jurkat reporter cells functioned normally.

[0226] Figure 2 The plasmid structure used for in vitro transcription to prepare mRNA for CD8 α and β chains ( Figure 2 A), CMV_TCRα chain and β chain ( Figure 2 B) or epitope encoding sequence of CMVpp65 that can be presented by HLA-A*02:01 ( Figure 2 C) The coding region is inserted between the 5'UTR and 3'UTR, a Kozak sequence is inserted before the coding region to mediate translation initiation, and a T7 promoter is present before the 5'UTR for transcription initiation.

[0227] Figure 3 NFAT-Jurkat cells electroporated with CD8 and CMV_TCR are shown. After 24 hours of culture, the flow cytometry of CMVpp65-Tetramer staining shows that the proportion of double-positive cells for CD8 and CMVpp65-Tetramer is 23.53% (right). NFAT-Jurkat cells electroporated with only CD8 have no double-positive cell population (left), proving that mRNA overexpression is successful.

[0228] Figure 4The results showed that after co-culture of NFAT-Jurkat cells electroporated with CD8 and CMV_TCR and HepG2 cells loaded with CMVpp65 peptide or electroporated with CMV-mRNA, the fluorescence value detection was upregulated at three effector-target ratios (NFAT-Jurkat cells were fixed at 3E5, and HepG2 cells were added with 1E5, 6E4 and 3E4 cells at effector-target ratios of 3:1, 5:1 and i0:1, respectively), proving that the cells can be used to detect the biological activity of peptides and mRNA, including antigen presentation and activation of specific T cells.

[0229] Example 2

[0230] 1. Experimental Materials

[0231] 1) Cell lines and culture media: NFAT-Jurkat cell line was purchased from Nanjing Novozymes Biotech Co., Ltd.; HepG2 cell line was purchased from Zhejiang Meisen; 293FT cell line was purchased from Beina Biotech; RPMI-1640 culture medium was purchased from Thermo Fisher Scientific with a catalog number of 11875-093; DMEM culture medium was purchased from ThermoFisher Scientific with a catalog number of C12430500BT; and fetal bovine serum was purchased from Cellsera with a catalog number of AU-FBS / FS.

[0232] 2) The raw materials required for plasmid construction include: PLVX-3xFlag-IRES-mcherry plasmid was purchased from Nanjing GenScript Biotechnology Co., Ltd.; restriction endonucleases XhoI and BamHI were purchased from New England Biolabs; PCR reagent KOD-Plus-Neo was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., with the catalog number KOD-401; T4 DNA ligase was purchased from New England Biolabs; UE DNA gel recovery kit was purchased from Suzhou Uni-Easy Biotechnology Co., Ltd.; endotoxin-free plasmid extraction kit (enhanced) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0233] PCR primers

[0234] CD8AB-XhoI-Forward:

[0235]

[0236] CD8AB-BamHI-Reverse:

[0237]

[0238] CMV_TCR-XhoI-Forward:

[0239]

[0240] CMV_TCR-BamHI-Reverse: CGGGATCCTCAGCTGTTCTTCTTCTICACCATGGCCAT (SEQ IDNO: 8)

[0241] 3) Materials for constructing lentiviral cell lines include: second-generation lentiviral packaging plasmids psPAX2 and pMD2.G were purchased from Nanjing GenScript Biotechnology Co., Ltd.; Puromycin was purchased from MedChemExpress with the catalog number HY-B1743A; LipofectamineTM3000 transfection reagent was purchased from Thermo Fisher Scientific with the catalog number L3000015; Opti-MEM reduced serum medium was purchased from Thermo Fisher Scientific with the catalog number 31985062.

[0242] 4) Electroporation kit: Neon TM The 100 μL electroporation kit was purchased from Thermo Fisher Scientific with the catalog number MPK10096.

[0243] 5) NFAT-Jurkat stimulation reagents include: PMA + ionomycin (PMA and Ionomycin) purchased from Shenzhen Dakoway Biotechnology Co., Ltd., catalog number 2030421; CMVpp65 epitope peptide sequence NLVPMVATV, purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0244] 6) Flow cytometry antibodies include: T-Select HLA-A*02:01CMVpp65 Tetramer-NLVPMVATV-PE purchased from MBL, catalog number TS-0010-1C; BV510 Mouse Anti-Human CD8 purchased from BD Biosciences, catalog number 563919.

[0245] 7) Luciferase Assay Kit: Bio-Lite Luciferase Assay System was purchased from Nanjing Novozymes Biotech Co., Ltd. with the product number DD1201-02.

[0246] 2. Experimental methods

[0247] Step 1: Construction of NFAT-Jurkat-CD8-CMV_TCR stable cell line

[0248] Construction of plasmid: primers were designed with plasmids pIPMK-CD8 and pIPMK-CMV_TCR as templates, CD8 and CMV_TCR were amplified by PCR reaction respectively, PCR products were subjected to nucleic acid gel electrophoresis to cut CD8 and CMV_TCR corresponding bands, PCR products were recovered with UE DNA gel recovery kit to obtain CD8 and CMV_TCRPCR products with restriction sites on both sides, then pLVX plasmid (vector) and PCR products were digested with restriction endonucleases XhoI and BamHI to expose sticky ends, digested vector fragments and PCR products were purified by gel back, and then CD8 and CMV_TCR after digestion were connected to the vector with T4 ligase, and the ligated products were transformed into competent cells, and after plating growth in a culture plate with resistance, single clones were selected for sequencing, and after the monoclonal strain with correct sequencing was selected for expansion culture, the plasmid was extracted with an endotoxin-free plasmid extraction kit for mRNA preparation. The structure of the plasmid is shown in the figure below. Figure 5 shown.

[0249] Lentivirus packaging: 293FT cells were cultured in a T175 culture flask until the cell confluence reached 50-60%, and Lip03000 and P3000 were prepared separately. After preparation, the mixture was allowed to stand at room temperature for 5 minutes. The mixture was evenly mixed and allowed to stand at room temperature for 30 minutes. Then, the mixture was slowly added with a pipette to the cell culture flask containing the virus to be packaged and cultured in a CO2 incubator (Lipo3000 formula: 500 μL Opti-MEM plus 25 μL Lip03000, P3000 formula: 500 μL Opti-MEM, 50 μL P3000, 6.25 μg pLVX-CD8 or pLVX-CMV_TCR plasmid, 12.5 μg psPAX, and 6.25 μg pMD2.G). Collect the supernatant 48h and 72h after plasmid transfection (add 20mL of fresh culture medium after collecting the supernatant 48h), centrifuge at 4500rpm and 4℃ for 5min to remove cell debris and other precipitates, then use a 0.22μM vacuum filter bottle to remove impurities from the supernatant, add 1 / 5 of the total volume of PEG 8000 concentration reagent to the filtered solution, shake to mix, place in a 4℃ refrigerator for overnight concentration and sedimentation, then transfer to a 50mL centrifuge tube, centrifuge at 4500rpm and 4℃ for 50min, discard the supernatant, turn the centrifuge tube upside down on a paper towel for 2-3min, control the liquid as much as possible, add 200μL of pre-cooled D-PBS to resuspend, and then dispense into 15mL centrifuge tubes, 50μL / tube, store at -80℃ or use directly to infect target cells.

[0250] Lentivirus infection: 3E5 NFAT-Jurkat cells were inoculated into a 12-well plate, 1 mL of culture medium, 5 μL of polybrene infection reagent and 50 μL of purified lentivirus were added, mixed and cultured in a CO2 incubator, and the culture medium was replaced within 12-24 hours. After expanded culture, mixed clones with a high proportion of CD8 and CMV_TCR positive cells were detected by flow cytometry for monoclonal screening.

[0251] Monoclonal screening: The expanded NFAT-Jurkat cells were sorted into 96-well plates, cultured in a CO2 incubator, and the monoclonal strains with the best expression of CD8 and CMV_TCR were selected by flow cytometry for expansion or preservation.

[0252] Tetramer staining: Resuspend about 3-10E5 cells in 100 μL of PBS with 2 μL Tetramer dye, stain for 15-30 minutes in the dark, wash with 1 mL PBS and then detect on a microscope.

[0253] Step 2: In vitro activity assay of CMVpp65 epitope peptides and mRNA

[0254] NFAT-Jurkat cell line culture: cultured with 1640 medium containing 10% FBS, maintaining the cell density in the range of 5E5-5E6, and cultured the cells for 2-3 generations before testing their functions.

[0255] HepG2 cell line culture: cultured with DMEM medium containing 10% FBS. When the cells grew to 90% density, the HepG2 cells were digested with 0.25% EDTA-trypsin and then passaged at a ratio of 1:5. After 2-3 generations of cell culture, the cells were used for electroporation or peptide loading.

[0256] Epitope peptide detection: HepG2 cells were incubated with 1 μg / mL CMVpp65 peptide overnight. On the second day, the HepG2 cells were digested and recounted, and 6E4 (E:T=5:1) peptide-loaded HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate. 3E5 electroporated NFAT-Jurkat cells were added. The culture medium was 200 μL of 1640 culture medium containing 10% FBS. After 6 hours of co-culture, 100 μL of luciferase detection reagent was added, and the fluorescence value was detected in a microplate reader.

[0257] mRNA detection: The prepared mRNA needs to be tested with Neon TM 100μL electroporation kit electroporation, specific operation with 100μL Neon TM4E5 HepG2 cells resuspended in 100μL R solution in the electroporation kit were added with 5μg CMV-mRNA and mixed well. After adding 3-5mL E2 solution to the electroporation cup, it was placed in the cup slot of the electroporator. The cell suspension mixed with mRNA was carefully aspirated with a 100μL electroporation gun tip to avoid bubbles. The electroporation gun was inserted into the E2 solution of the electroporator and electroporated under the voltage conditions of 1350mV, 10ms, and 4plus. After the electroporated HepG2 cells were cultured overnight in 10% FBS 1640 medium, the HepG2 cells were digested and recounted, and 6E4 (E:T=5:1) peptide-loaded HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate, and 3E5 NFAT-Jurkat-CD8-CMV_TCR cells were added. The culture medium was 200 μL of 1640 medium containing 10% FBS. After co-culture for 6 hours, 100 μL of luciferase detection reagent was added, and the fluorescence value was detected in a microplate reader.

[0258] 3. Experimental results

[0259] Figure 5 Displays the plasmid structure for lentiviral overexpression, CD8α chain-P2A-β chain ( Figure 5 A) or CMV_TCRα chain-P2A-β chain ( Figure 5 B) After the enhancer is cloned, it is integrated with the Puromycin resistance gene between the 3'LTR of the HIV virus and can be packaged into a lentivirus for the construction of a gene overexpression cell line.

[0260] Figure 6 The CD8 and CMVpp65-Tetramer flow cytometry results of NFAT-Jurkat cells infected with lentivirus are shown. The proportion of CD8-positive cells is 77%, and the proportion of CMV_TCR-positive cells is 20%.

[0261] like Figure 7 As shown, among the monoclonal cell lines, monoclonal line No. 18 had the best overexpression effect, and the flow cytometry staining results showed that the proportion of cells double-positive for CD8 and CMV_TCR could reach more than 90%.

[0262] Figure 8 The results showed that after NFAT-Jurkat-CD8-CMV_TCR was co-cultured with HepG2 cells loaded with CMVpp65 peptide or electroporated with CMV-mRNA at an effector-target ratio of 5:1 for 6 hours, the fluorescence value was detected to be upregulated, indicating that NFAT-Jurkat-CD8-CMV_TCR can be used to detect the biological activity of peptides and mRNA, including antigen presentation and activation of specific T cells.

[0263] Example 3

[0264] 1. Experimental Materials

[0265] 1) Cell lines and culture media: NFAT-Jurkat cell line was purchased from Nanjing Novozymes Biotech Co., Ltd.; HepG2 cell line was purchased from Zhejiang Meisen; RPMI-1640 medium used for cell culture was purchased from Thermo Fisher Scientific, catalog number 11875-093; fetal bovine serum was purchased from Cellsera, catalog number AU-FBS / FS.

[0266] 2) The raw materials required for plasmid construction include: pIPM-MAGE-A1 and pIPM-MAGE-A1_TCR plasmids were commissioned to Nanjing GenScript Biotechnology Co., Ltd. for construction.

[0267] MAGE-A1_TCR-1367 sequence:

[0268]

[0269] MAGE-A1_TCR-1405 sequence:

[0270]

[0271]

[0272] MAGE-A1_TCR-1705 sequence:

[0273]

[0274] MAGE-A1 epitope CDS sequence:

[0275]

[0276] 3) mRNA preparation kit: EasyCap T7 Co-transcription Kit with CAG Trimer was purchased from Nanjing Novogene Biotech Co., Ltd. with the catalog number DD4203.

[0277] 4) Electroporation kit: Neon TM The 100 μL electroporation kit was purchased from Thermo Fisher Scientific with the catalog number MPK10096.

[0278] 5) NFAT-Jurkat stimulation reagents include: PMA + ionomycin (PMA and Ionomycin) purchased from Shenzhen Dakoway Biotechnology Co., Ltd., catalog number 2030421; the HLA-A*02:01 restricted epitope of MAGE-A1 is KVLEYVIKV (SEQ ID NO: 13), and a 30-mer scanning peptide is used, the sequence of which is

[0279] YEFLWGPRALAETSYVKVLEYVIKVSARVR (SEQ ID NO: 14) was purchased from Nanjing GenScript Biotech Co., Ltd.

[0280] 6) Flow cytometry antibodies include: HLA-A*02:01MAGE-A1 Tetramer-KVLEYVIKV-APC purchased from MBL with the catalog number TB-M070-2; BV510 Mouse Anti-Human CD8 purchased from BD Biosciences with the catalog number 563919.

[0281] 7) Luciferase Assay Kit: Bio-Lite Luciferase Assay System was purchased from Nanjing Novozymes Biotech Co., Ltd. with the product number DD1201-02.

[0282] 2. Experimental methods

[0283] Step 1: NFAT-Jurkat cells transiently overexpress CD8 and MAGE-A1_TCR

[0284] Plasmid construction: Nanjing GenScript Biotechnology Co., Ltd. was commissioned to clone the sequences encoding CD8α chain-P2A-CD8β chain and three MAGE-A1_TCR sequences into the middle of the BamHI and SacI restriction endonuclease sites of the in vitro transcription plasmid vector. The structure of pIPM-CD8 plasmid is shown in Figure 2 The three MAGE-A1_TCR sequences were identified by Matthias et al. in 2015 from CD8+T cells that can recognize the HLA-A*02 restricted MAGE-A1 epitope. Among them, MAGE-A1_TCR-1367 and MAGE-A1_TCR-1705 have higher affinity, while MAGE-A1_TCR-1405 has slightly lower affinity. The pIPMK-MAGE-A1_TCR plasmid structure is shown in Fig. 9 shown.

[0285] Preparation of mRNA: linearize the plasmid with BspQI, terminate the enzyme digestion after reacting at 50℃ for 3h, purify the linearized plasmid with DNA magnetic beads, use the purified linearized plasmid as a template, and prepare mRNA with EasyCap T7 Co-transcription Kit kit. The prepared mRNA is used for electrotransfer or stored at -80℃.

[0286] mRNA electroporation and electroporation efficiency verification process: 2E6 NFAT-Jurkat cells were taken, resuspended in 100μL R solution in the electroporation kit, and 7.5μg CD8-mRNA and 7.5μg MAGE-A1_TCR-mRNA were added and mixed. The electroporation gun was inserted into the electroporation cup pre-added with 3-5mL E2 solution (electroporation kit) and the electroporator was turned on. The electroporation conditions were 1400mV, 20ms, 2plus. After electroporation, the cells were resuspended in 1640 complete culture medium. After 24h, some electroporated cells were taken for MAGE-A1 tetramer staining.

[0287] Tetramer staining: Resuspend about 3-10E5 test cells in 100 μL of PBS with 2 μL of Tetramer dye, stain for 15-30 minutes in the dark, wash with 1 mL of PBS and then detect on a microscope.

[0288] Step 2: 3-strip MAGE-A1_TCR affinity detection

[0289] NFAT-Jurkat cell line culture: cultured with 1640 medium containing 10% FBS, maintaining the cell density in the range of 5E5-5E6, and cultured the cells for 2-3 generations before testing their functions.

[0290] HepG2 cell line culture: cultured with DMEM medium containing 10% FBS. When the cells grew to 90% density, the HepG2 cells were digested with 0.25% EDTA-trypsin and then passaged at a ratio of 1:5. After 2-3 generations of cell culture, the cells were used for electroporation or peptide loading.

[0291] Affinity detection: HepG2 cells were incubated with 5 μg / mL concentration of MAGE-A1 scanning peptide overnight. On the second day, the HepG2 cells were digested and re-counted, and 6E4 (E:T=5:1) peptide-loaded HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate. 3E5 electroporated NFAT-Jurkat cells were added. The culture medium was 200 μL of 1640 culture medium containing 10% FBS. After co-culture for 6 hours, 100 μL of luciferase detection reagent was added and the fluorescence value was detected in a microplate reader.

[0292] Step 3: In vitro activity assay of MAGE-A1 scanning peptides and mRNA

[0293] NFAT-Jurkat cell line culture: cultured with 1640 medium containing 10% FBS, maintaining the cell density in the range of 5E5-5E6, and cultured the cells for 2-3 generations before testing their functions.

[0294] HepG2 cell line culture: cultured with DMEM medium containing 10% FBS. When the cells grew to 90% density, the HepG2 cells were digested with 0.25% EDTA-trypsin and then passaged at a ratio of 1:5. After 2-3 generations of cell culture, the cells were used for electroporation or peptide loading.

[0295] Scanning peptide detection: HepG2 cells were incubated with 5 μg / mL MAGE-A1 scanning peptide overnight. On the second day, the HepG2 cells were digested and recounted, and 6E4 (E:T=5:1) peptide-loaded HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate. 3E5 electroporated NFAT-Jurkat cells were added, and the culture medium was 200 μL 1640 culture medium containing 10% FBS. After co-culture for 6 hours, 100 μL luciferase detection reagent was added, and the fluorescence value was detected in a microplate reader.

[0296] mRNA detection: The prepared mRNA needs to be tested with Neon TM 100μL electroporation kit electroporation, specific operation with 100μL Neon TM 4E5 HepG2 cells resuspended in 100μL R solution in the electrotransfer kit were added with 5μg of mRNA encoding full-length MAGE-A1 and mixed. After adding 3-5mL E2 solution (electrotransfer kit) to the electrotransfer cup, it was placed in the cup slot of the electrotransfer instrument. The cell suspension mixed with mRNA was carefully aspirated with a 100μL electrotransfer gun tip to avoid bubbles. The electrotransfer gun was inserted into the E2 solution of the electrotransfer instrument and electrotransferred under the voltage conditions of 1350mV, 10ms, and 4plus. After the electroporated HepG2 cells were cultured overnight in 10% FBS 1640 medium, the HepG2 cells were digested and recounted, and 6E4 (E:T=5:1) peptide-loaded or electroporated mRNA HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate. After culturing with 100 μL 1640 culture medium for 24 h, 3E5 electroporated CD8 and MAGE-A1_TCR NFAT-Jurkat cells were added and co-cultured for 6 h. The culture medium was 200 μL 1640 medium containing 10% FBS. After co-culturing for 6 h, 100 μL of luciferase detection reagent was added, and the fluorescence value was detected in a microplate reader.

[0297] Step 4: MAGE-A1-LNP in vitro activity detection

[0298] NFAT-Jurkat cell line culture: cultured with 1640 medium containing 10% FBS, maintaining the cell density in the range of 5E5-5E6, and cultured the cells for 2-3 generations before testing their functions.

[0299] HepG2 cell line culture: cultured with DMEM medium containing 10% FBS. When the cells grew to 90% density, the HepG2 cells were digested with 0.25% EDTA-trypsin and then passaged at a ratio of 1:5. After 2-3 generations of cell culture, the cells were used for electroporation or peptide loading.

[0300] Preparation of mRNA-LNP: mRNA was diluted with citrate buffer and filtered through a 0.22 μm filter, lipid was solvented with anhydrous ethanol and filtered through a 0.22 μm filter, coated into mRNA-LNP using Ignite Instrument, and the prepared mRNA-LNP was diluted with Tris-HCL buffer and concentrated by ultrafiltration tube.

[0301] mRNA-LNP detection: 6E4 HepG2 cells were plated into a 96-well plate and resuspended with 50 μL 1640 complete medium; LNP was diluted to 1 μg / mL with serum-free 1640 medium at 50 μL / well, and the diluted LNP was added to the cells, cultured overnight in a CO2 cell culture incubator, and 100 μL of fresh medium was replaced the next day; 3E5 NFAT-Jurkat cells electroporated with CD8 and MAGE-A1_TCR were added and co-cultured for 6 hours, and the culture medium was 200 μL 1640 medium containing 10% FBS. After 6 hours of co-culture, 100 μL of luciferase detection reagent was added, and the fluorescence value was detected in an ELISA instrument.

[0302] 3. Experimental results

[0303] Fig. 9 Demonstrated for in vitro preparation of MAGE-A1_TCR ( Fig. 9 B) and MAGE-A1( Fig. 9 A) The plasmid structure of the mRNA, MAGE-A1_TCR-1367, MAGE-A1_TCR-1405 and MAGE-A1_TCR-1705 were inserted between the BamHI and SacI restriction sites, the coding region was inserted between the 5'UTR and 3'UTR, a Kozak sequence was inserted before the coding region to mediate translation initiation, and a T7 promoter was present before the 5'UTR for transcription initiation.

[0304] Fig.10After electroporation of mRNA encoding CD8 and three commercial MAGE-A1_TCRs, the results of flow cytometry Tetramer detection showed that the proportion of Tetramer-positive population in the CD8-positive population was 65.85%, 44.79% and 73.81% in NFAT-Jurkat cells transiently overexpressing MAGE-A1_TCR-1367, MAGE-A1_TCR-1405 and MAGE-A1_TCR-1705, respectively.

[0305] Fig.11 After 6 hours of co-culture of NFAT-Jurkat cells transiently expressing CD8 and three MAGE-A1_TCRs with HepG2 cells loaded with MAGE-A1 scanning peptides, the fluorescence values ​​were upregulated. The intensity of NFAT-Jurkat activated by MAGE-A1_TCR-1367 and MAGE-A1_TCR-1705 was higher, indicating that this method can be used to screen the affinity of TCR.

[0306] Fig.12 After 6 hours of co-culture with HepG2 cells loaded with MAGE-A1 scanning peptide, electroporated with MAGE-A1 full-length mRNA, or incubated with MAGE-A1-LNP, the fluorescence value was upregulated. NFAT-Jurkat cells transiently expressing CD8 and MAGE-A1_TCR-1367 can be used to detect the biological activity of peptides and mRNA.

[0307] Example 4

[0308] 1. Experimental Materials

[0309] 1) Cell lines and culture media: NFAT-Jurkat cell line was purchased from Nanjing Novozymes Biotech Co., Ltd.; HepG2 cell line was purchased from Zhejiang Meisen; 293FT cell line was purchased from Beina Biotech; RPMI-1640 culture medium was purchased from Thermo Fisher Scientific with a catalog number of 11875-093; DMEM culture medium was purchased from ThermoFisher Scientific with a catalog number of C12430500BT; and fetal bovine serum was purchased from Cellsera with a catalog number of AU-FBS / FS.

[0310] 2) The raw materials required for plasmid construction include: pZXK plasmid purchased from Nanjing GenScript Biotechnology Co., Ltd.; restriction endonucleases NheI and BamHI purchased from New England Biolabs; PCR reagent KOD-Plus-Neo purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., catalog number KOD-401; T4 DNA ligase purchased from New England Biolabs; UE_DNA gel recovery kit purchased from Suzhou Uni-Easy Biotechnology Co., Ltd.; endotoxin-free plasmid extraction kit (enhanced) purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0311] PCR primers

[0312] CD8-PCR-F1: GTGTCCCCTGGATACCCGTATTCTAGAGCTAGCGCCACCATGGCGCTGCCCGTC (SEQ ID NO: 15)

[0313] CD8-PCR-R1: CCGGTCATGGTCAATTGGGCCGCGGATCCTTACTTGTAAAACTGCTTCATGAAGCGC (SEQ ID NO: 16)

[0314] MAGE-A1_TCR-PCR-F1: GTGTCCCTGGATACCCGTATTCTAGAGCTAGCGCCACCATGGGAATCAGACTGCTG (SEQ ID NO: 17)

[0315] MAGE-A1_TCR-PCR-R1: CCGGTCATGGTCAATTGGGCCGCGGATCCTCAGCTGGACCACAGTCTCAGGGTCATC (SEQ ID NO: 18)

[0316] 3) Materials for constructing lentiviral cell lines include: second-generation lentiviral packaging plasmids psPAX2 and pMD2.G were purchased from Nanjing GenScript Biotechnology Co., Ltd.; Puromycin was purchased from MedChemExpress with the catalog number HY-B1743A; LipofectamineTM 3000 transfection reagent was purchased from Thermo Fisher Scientific with the catalog number L3000015; Opti-MEM reduced serum medium was purchased from Thermo Fisher Scientific with the catalog number 31985062.

[0317] 4) Electroporation kit: Neon TMThe 100 μL electroporation kit was purchased from Thermo Fisher Scientific with the catalog number MPK10096.

[0318] 5) NFAT-Jurkat stimulation reagents include: PMA + ionomycin (PMA and Ionomycin) purchased from Shenzhen Dakoway Biotechnology Co., Ltd., catalog number 2030421; the HLA-A*02:01 restricted epitope of MAGE-A1 is KVLEYVIKV, using a 30-mer scanning peptide with the sequence of YEFLWGPRALAETSYVKVLEYVIKVSARVR, purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0319] 6) Flow cytometry antibodies include: HLA-A*02:01MAGE-A1 Tetramer-KVLEYVIKV-APC purchased from MBL with the catalog number TB-M070-2; BV510Mouse Anti-Human CD8 purchased from BD Biosciences with the catalog number 563919.

[0320] 7) Luciferase Assay Kit: Bio-Lite Luciferase Assay System was purchased from Nanjing Novozymes Biotech Co., Ltd. with the product number DD1201-02.

[0321] 2. Experimental methods

[0322] Step 1: Construction of NFAT-Jurkat-CD8-MAGE-A1_TCR stable cell line

[0323] Plasmid construction: Primers were designed to use plasmids pIPMK-CD8 and pIPMK-MAGE-A1_TCR-1367 as templates, CD8 and MAGE-A1_TCR-1367 were amplified by PCR reaction, and the PCR products were subjected to nucleic acid gel electrophoresis to cut the corresponding bands of CD8 and MAGE-A1TCR. The PCR product was recovered by a DNA gel recovery kit to obtain PCR products of CD8 and MAGE-A1_TCR with restriction sites on both sides. Then, the pZXK plasmid (vector) and the PCR product were digested with restriction endonucleases NheI and BamHI to expose the sticky ends. The digested vector fragments and PCR products were purified by gel retrieval. Then, the digested CD8 and MAGE-A1_TCR were connected to the vector with T4 ligase. The ligated products were transformed into competent cells. After plating and growing in a culture plate with resistance, single clones were selected for sequencing. After the monoclonal strain with correct sequencing was selected for expansion culture, the plasmid was extracted by an endotoxin-free plasmid extraction kit for mRNA preparation. The structure of the plasmid is shown in the figure below. Fig.13 shown.

[0324] Lentivirus packaging: 293FT cells were cultured in a T175 culture flask until the cell confluence reached 50-60%, and Lipo3000 and P3000 were prepared separately. After preparation, they were allowed to stand at room temperature for 5 minutes. The two were mixed evenly and allowed to stand at room temperature for 30 minutes. Then, they were slowly added dropwise with a pipette to the cell culture flask to be packaged with the virus and cultured in a CO2 incubator (Lipo3000 formula: 500μL Opti-MEM plus 25μL Lip03000, P3000 formula: 500μL Opti-MEM, 50μL P3000, pZXK-CD8 or pZXK-MAGE-A1_TCR-1367 plasmid 6.25μg, psPAX12.5μg and pMD2.G6.25μg). Collect the supernatant 48h and 72h after plasmid transfection (add 20mL of fresh culture medium after collecting the supernatant 48h), centrifuge at 4500rpm and 4℃ for 5min to remove cell debris and other precipitates, then use a 0.22μM vacuum filter bottle to remove impurities from the supernatant, add 1 / 5 of the total volume of PEG8000 concentration reagent to the filtered solution, shake to mix, place in a 4℃ refrigerator for overnight concentration and sedimentation, then transfer to a 50mL centrifuge tube, centrifuge at 4500rpm and 4℃ for 50min, discard the supernatant, turn the centrifuge tube upside down on a paper towel for 2-3min, control the liquid as much as possible, add 200μL of pre-cooled D-PBS to resuspend, and then divide into 1.5mL centrifuge tubes, 50μL / tube, store at -80℃ or use directly to infect target cells.

[0325] Lentivirus infection: 3E5 NFAT-Jurkat cells were inoculated into a 12-well plate, 1 mL of culture medium, 5 μL of polybrene infection reagent and 50 μL of purified lentivirus were added, mixed and cultured in a CO2 incubator, and the culture medium was replaced within 12-24 hours. After expanded culture, mixed clones with a high proportion of CD8 and MAGE-A1_TCR positive cells were detected by flow cytometry for monoclonal screening.

[0326] Monoclonal screening: The expanded NFAT-Jurkat cells were sorted into 96-well plates, cultured in a CO2 incubator, and the monoclonal strains with the best expression of CD8 and MAGE-A1_TCR were selected by flow cytometry for expansion or preservation.

[0327] Tetramer staining: Resuspend about 3-10E5 cells in 100 μL of PBS with 2 μL Tetramer dye, stain for 15-30 minutes in the dark, wash with 1 mL PBS and then detect on a microscope.

[0328] Step 2: In vitro activity assay of MAGE-A1 scanning peptides and mRNA

[0329] NFAT-Jurkat cell line culture: cultured with 1640 medium containing 10% FBS, maintaining the cell density in the range of 5E5-5E6, and cultured the cells for 2-3 generations before testing their functions.

[0330] HepG2 cell line culture: cultured with DMEM medium containing 10% FBS. When the cells grew to 90% density, the HepG2 cells were digested with 0.25% EDTA-trypsin and then passaged at a ratio of 1:5. After 2-3 generations of cell culture, the cells were used for electroporation or peptide loading.

[0331] Scanning peptide detection: HepG2 cells were incubated with 5 μg / mL MAGE-A1 scanning peptide overnight. On the second day, the HepG2 cells were digested and recounted, and 6E4 (E:T=5:1) peptide-loaded HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate. 3E5 NFAT-Jurkat-CD8-MAGE-A1_TCR cells were added. The culture medium was 200 μL 1640 culture medium containing 10% FBS. After co-culture for 6 hours, 100 μL luciferase detection reagent was added and the fluorescence value was detected in a microplate reader.

[0332] mRNA detection: The prepared mRNA needs to be tested with Neon TM 100μL electroporation kit electroporation, specific operation with 100μL Neon TM 4E5 HepG2 cells resuspended in 100μL R solution in the electrotransfer kit were mixed with 5μg mRNA encoding full-length MAGE-A1. After adding 3-5mL E2 solution (electrotransfer kit) to the electrotransfer cup, it was placed in the electrotransfer cup slot. The cell suspension mixed with mRNA was carefully aspirated with a 100μL electrotransfer gun tip to avoid bubbles. The electrotransfer gun was inserted into the E2 solution of the electrotransfer instrument and electrotransferred under the voltage conditions of 1350mV, 10ms, and 4plus. After the electrotransferred HepG2 cells were cultured overnight in 10% FBS 1640 medium, the HepG2 cells were digested and recounted. 6E4 (E:T=5:1) peptide-loaded or electrotransferred mRNA HepG2 cells were plated in each well of a 96-well flat-bottom cell culture plate. After culturing for 24h with 100μL 1640 culture medium, 3E5

[0333] NFAT-Jurkat-CD8-MAGE-A1_TCR cells were co-cultured for 6 h in 200 μL 1640 medium containing 10% FBS. After 6 h of co-culture, 100 μL luciferase detection reagent was added and the fluorescence value was detected in a microplate reader.

[0334] Step 3: In vitro activity detection of MAGE-A1-LNP

[0335] NFAT-Jurkat cell line culture: cultured with 1640 medium containing 10% FBS, maintaining the cell density in the range of 5E5-5E6, and cultured the cells for 2-3 generations before testing their functions.

[0336] HepG2 cell line culture: cultured with DMEM medium containing 10% FBS. When the cells grew to 90% density, the HepG2 cells were digested with 0.25% EDTA-trypsin and then passaged at a ratio of 1:5. After 2-3 generations of cell culture, the cells were used for electroporation or peptide loading.

[0337] Preparation of mRNA-LNP: mRNA was diluted with citrate buffer and filtered through a 0.22 μm filter, lipid was solvented with anhydrous ethanol and filtered through a 0.22 μm filter, coated into mRNA-LNP using Ignite Instrument, and the prepared mRNA-LNP was diluted with Tris-HCL buffer and concentrated by ultrafiltration tube.

[0338] mRNA-LNP detection: 6E4 HepG2 cells were plated into a 96-well plate and resuspended with 50 μL 1640 complete medium; LNP was diluted to 1 ng / mL, 5 ng / mL, 25 ng / mL and 125 ng / mL with 50 μL serum-free 1640 medium, and the diluted LNP was added to the cells, cultured overnight in a CO2 cell culture incubator, and 100 μL fresh medium was replaced the next day; 3E5 NFAT-Jurkat cells electroporated with CD8 and MAGE-A1_TCR were added and co-cultured for 6 hours, and the culture medium was 200 μL 1640 medium containing 10% FBS. After 6 hours of co-culture, 100 μL luciferase detection reagent was added, and the fluorescence value was detected in an ELISA instrument.

[0339] 3. Experimental results

[0340] Fig.13 Displays the plasmid structure used for lentiviral overexpression of CD8 and MAGE-A1_TCR-1367, CD8α chain-P2A-β chain ( Fig.13 B) or MAGE-A1_TCR-1367( Fig.13 A) After the enhancer is cloned, it is integrated with the Puromycin resistance gene between the 3'LTR of the HIV virus and can be packaged into a lentivirus for the construction of a gene overexpression cell line.

[0341] Fig.14 The CD8 and MAGE-A1-Tetramer flow cytometry results of lentivirus-infected NFAT-Jurkat cells were shown, and the double positive rate of CD8 and MAGE-A1_TCR was 76.57%.

[0342] Fig.15 Among the 18 monoclonal strains selected, the double positive rate of monoclonal cells No. 4, 5, 6, 10 and 12 can reach more than 99%. The above monoclonal strains are cryopreserved, and monoclonal cells No. 4, 5 and 12 are selected for functional verification.

[0343] Fig.16 After 6 hours of co-culture of the constructed NFAT-Jurkat-CD8-MAGE-A1_TCR monoclonal strains 4 (mono4), 5 (mono5) and 12 (mono12) with HepG2 cells loaded with MAGE-A1 scanning peptide or electroporated with MAGE-A1 full-length mRNA, the fluorescence values ​​were all upregulated, indicating that the NFAT-Jurkat-CD8-MAGE-A1_TCR stably transfected cell line can be used to detect the biological activity of peptides and mRNA.

[0344] like Fig.17 As shown, LNPs with concentrations of 1 ng / mL, 5 ng / mL, 25 ng / mL and 125 ng / mL were incubated in vitro to present MAGE-A1 on the surface of HepG2 cells. Activation could be detected after co-culture with NFAT-Jurkat-CD8-MAGE-A1_TCR cells, and the LUC fluorescence reading was upregulated with increasing LNP concentrations. The results indicate that the NFAT-Jurkat-CD8-MAGE-A1_TCR stably transfected cell line can be used for in vitro detection of the biological activity of LNPs.

[0345] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a cell line for detecting the biological activity of a vaccine, It is characterized in that The method comprises constructing a cell line expressing a target head that recognizes an antigen; Preferably, the cell line used to express the target head that recognizes the antigen is an immortalized cell line that expresses a reporter gene; Preferably, the cell line further comprises a molecule that assists in recognition.

2. The construction method according to claim 1, It is characterized in that The target head for recognizing antigen is selected from TCR; Preferably, the TCR is a TCR targeting a specific antigen; Preferably, the antigens include viral antigens, tumor antigens, autoimmune antigens, pathogen antigens; Preferably, the antigen is selected from viral antigens and tumor antigens; Preferably, the viral antigen is selected from DNA viral antigens; Preferably, the DNA viral antigen is selected from herpes virus antigens; Preferably, the herpes virus antigen is selected from CMV; Preferably, the tumor antigen is selected from tumor-specific antigens; Preferably, the tumor-specific antigen is selected from MAGE-As; Preferably, the MAGE-As are selected from MAGE-A1; Preferably, CMV_TCR is selected from CMV_TCRα and CMV_TCRβ; Preferably, a cleavage peptide is connected between CMV_TCRα and CMV_TCRβ; Preferably, the sequence of CMV_TCR has at least 90% sequence identity with the sequence shown in SEQ ID NO:2; Preferably, the sequence of CMV_TCR is as shown in SEQ ID NO: 2; Preferably, the sequence of MAGE-A1_TCR has at least 90% sequence identity with the sequence shown in any one of SEQ ID NOs: 9-11, respectively; Preferably, the sequence of MAGE-A1_TCR is shown in any one of SEQ ID NOs: 9-11.

3. The construction method according to claim 1, It is characterized in that The reporter genes include luciferase reporter gene, chloramphenicol acetyltransferase reporter gene, β-galactosidase, secretory human placental alkaline phosphatase, and green fluorescent protein; Preferably, the reporter gene is selected from a luciferase reporter gene; Preferably, the immortalized cell line is selected from an immortalized T cell line; Preferably, the immortalized T cell line is selected from Jurkat cells.

4. The construction method according to claim 1, It is characterized in that The auxiliary recognition molecules include one or more of molecules that assist the binding of the target head to the antigen, costimulatory signal molecules, and cytokine receptors; Preferably, the auxiliary recognition molecule is selected from molecules that assist the binding of the target head to the antigen; Preferably, the molecules that assist the binding of the target head and the antigen include CD8, CD4, CD3, CD19, and CD20; Preferably, the molecule that assists the binding of the target head to the antigen is selected from CD8; Preferably, the CD8 is selected from CD8α and CD8β; Preferably, a cleavage peptide is connected between the CD8α and CD8β; Preferably, the cleavage peptide is selected from the group consisting of P2A cleavage peptide; Preferably, the sequence of CD8 has at least 90% sequence identity with the sequence shown in SEQ ID NO: 1; Preferably, the sequence of CD8 is as shown in SEQ ID NO:

1.

5. The method according to claim 1, It is characterized in that The method for constructing a cell line expressing a target head that recognizes an antigen includes a plasmid transfection method, a virus transfection method, a liposome transfection method, and a transposon transfection method; Preferably, the method for constructing a cell line expressing a target head that recognizes an antigen is selected from a plasmid transfection method and a viral transfection method; Preferably, the viral transfection method includes lentiviral transfection method, adenoviral transfection method, and adeno-associated viral transfection method; Preferably, the viral transfection method is selected from lentiviral transfection method; Preferably, the vectors used in the plasmid transfection method include PZXK and pLVX; Preferably, the method of transferring the plasmid into the cell line is selected from physical methods; Preferably, the physical method is selected from electroporation; Preferably, the vaccine is selected from T cell immune vaccines; Preferably, the types of vaccines include polypeptide vaccines, nucleic acid vaccines, and DC vaccines; Preferably, the nucleic acid vaccine includes mRNA vaccine and DNA vaccine; Preferably, the vaccine is selected from polypeptide vaccines and nucleic acid vaccines; Preferably, the nucleic acid vaccine is selected from mRNA vaccines.

6. A cell line for detecting the biological activity of vaccines, It is characterized in that The cell line is a cell line expressing a target head that recognizes an antigen; Preferably, the target head that recognizes the antigen is selected from TCR; Preferably, the cell line used to express the target head that recognizes the antigen is an immortalized cell line that expresses a reporter gene; Preferably, the reporter gene is selected from a luciferase reporter gene; Preferably, the immortalized cell line is selected from an immortalized T cell line; Preferably, the immortalized T cell line is selected from Jurkat cells; Preferably, the cell line further comprises a molecule that assists in recognition; Preferably, the auxiliary recognition molecules include one or more of molecules that assist the binding of the target head to the antigen, costimulatory signal molecules and cytokine receptors; Preferably, the auxiliary recognition molecule is selected from molecules that assist the binding of the target head to the antigen; Preferably, the molecule that assists the binding of the target head to the antigen is selected from CD8.

7. A method for detecting the biological activity of a vaccine, It is characterized in that The method comprises using the cell line of claim 6 to detect the biological activity of the vaccine; Preferably, the method further comprises co-culturing the cell line with a cell line expressing the antigen; Preferably, the method further comprises detecting the biological activity of the vaccine using a detection reagent; Preferably, the detection reagent is selected from a luciferase detection reagent; Preferably, the cell line expressing the antigen includes a natural cell line and an engineered cell line; Preferably, the antigen-expressing cell line is selected from a natural or engineered tumor cell line; Preferably, the natural or engineered tumor cell line is selected from the HepG2 cell line; Preferably, the antigen-expressing cell line is constructed by co-incubation; Preferably, the effector-target ratio of the co-culture is (0.5-20):1; Preferably, the effector-target ratio of the co-culture is (3-10):1; Preferably, the co-cultivation time is 2-48 hours; Preferably, the co-cultivation time is 6 hours; Preferably, the vaccine is selected from T cell immune vaccines; Preferably, the types of vaccines include polypeptide vaccines, nucleic acid vaccines, and DC vaccines; Preferably, the nucleic acid vaccine includes mRNA vaccine and DNA vaccine; Preferably, the vaccine is selected from polypeptide vaccines and nucleic acid vaccines; Preferably, the nucleic acid vaccine is selected from mRNA vaccines; Preferably, the biological activity of the vaccine includes antigen presentation efficiency and T cell activation degree.

8. A product for detecting the biological activity of vaccines, It is characterized in that The product comprises the cell line of claim 6; Preferably, the product further comprises a cell line expressing the antigen; Preferably, the product further comprises a detection reagent; Preferably, the detection reagent is selected from luciferase; Preferably, the product further includes instructions for use, positive controls, and negative controls; Preferably, the product further comprises one or more sterile containers; Preferably, the product comprises a kit.

9. Any of the following applications: (1) Use of the cell line according to claim 6 or the product according to claim 8 in detecting the biological activity of a vaccine; (2) Use of the cell line of claim 6 or the method of claim 7 in quality control of vaccine and drug production and development; (3) Use of the cell line according to claim 6 or the method according to claim 7 in screening vaccines; Preferably, the vaccine is selected from T cell immune vaccines; Preferably, the types of vaccines include polypeptide vaccines, nucleic acid vaccines, and DC vaccines.

10. The use according to claim 9, It is characterized in that The vaccine screening includes vaccine optimization, TCR screening, and CAR molecule design; Preferably, the vaccine optimization includes antigen screening, enhancement element screening, cap structure screening, base modification screening, UTR region screening, PolyA structure screening, codon optimization sequence screening, circular mRNA structure screening, self-replicating mRNA screening, delivery system screening, and cationic liposome screening; Preferably, the TCR screening includes TCR affinity screening, TCR specificity screening, TCR on / off target evaluation, TCR response strength screening, TCR stability screening, endogenous TCR knockout efficiency evaluation, and exogenous TCR expression evaluation.

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