CN113308491B - Recombinant plasmid and recombinant cell for co-expressing NFAT and human DNAM-1 protein, and construction method and application thereof - Google Patents
Recombinant plasmid and recombinant cell for co-expressing NFAT and human DNAM-1 protein, and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant plasmid and a recombinant cell for co-expressing NFAT and human DNAM-1 protein, and a construction method and application thereof, and relates to the technical field of biological medicines. The invention provides a construction method of the recombinant plasmid, the recombinant plasmid obtained by construction, and a recombinant cell and a monoclonal recombinant cell constructed on the basis of the recombinant plasmid. The recombinant plasmid bidirectional promoter provided by the invention starts the expression of the DNAM-1 gene and the NFAT-Luciferase gene, and the gene expression is not influenced mutually. The recombinant cell is constructed based on the human immune type cell Jurkat, is more similar to an NK cell model, does not introduce viruses or cause cells to generate inflammation-related passages when the recombinant cell is constructed, and is simple to operate. The recombinant cell or the monoclonal recombinant cell can also be used for detecting and/or screening anti-human DNAM-1 antibodies.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant plasmid and a recombinant cell for co-expressing NFAT and human DNAM-1 protein, and a construction method and application thereof.
Background
DNAM-1(DNAX access polypeptide-1, CD226, one of the activated receptors for NK cells), originally called T cell lineage specific activation antigen (TLISA1), was involved in regulating the differentiation of cytotoxic T Cells (CTL). It was subsequently discovered that it is also expressed on platelets and can participate in platelet activation and aggregation, and is also named platelet T cell activation antigen (PTA1), and further studies have found that NK cell surface is also expressed, and that proteins with multiple Ig-like domains, formally named DNAM-1 at the international conference on leukocyte differentiation antigen in 2000, can bind to MHC-I (major histocompatibility complex) molecules. DNAM-1 has been shown to positively regulate NK cell activation and proliferation, and is usually expressed on the surface of NK cells infiltrated by tumor tissues, and is a marker of NK cell activation. The functional disorder of the immune system caused by virus infection or autoimmune disease and malignant tumor of human body can be improved by enhancing the function of DNAM-1, and the prognosis of the disease is improved. As DNAM-1 positively regulates the activity of NK cells, enhancing the DNAM-1 molecular function on the surface of NK cells can enhance the anti-tumor capability of NK cells. The anti-human DNAM-1 antibody improves the function of DNAM-1 by specifically binding DNAM-1 molecules, so as to enhance the anti-tumor efficacy of NK cells, thereby maximally mobilizing the reaction of the immune system of a patient to kill tumor cells to achieve the purpose of treating tumors, and therefore, the development of an immunotherapy medicament taking DNAM-1 as a target and the activation of DNAM-1 have important values.
The conventional method for determining the activation of DNAM-1 mainly comprises the following steps: the development of therapeutic antibody is carried out by using a method related to the regulation and control of antibody CD155, but the method can not only activate NK cells by combining with CD226 protein and inhibit the functions of the NK cells by combining with TIGIT protein because CD155 is a competitive receptor, and can not obtain DNAM-1 molecular antibody. Therefore, there is a strong need in the art for a detection method capable of directly recognizing an antibody binding to DNAM-1 protein, thereby screening anti-DNAM-1 antibodies.
Disclosure of Invention
In view of the above, the present invention provides a recombinant plasmid and a recombinant cell co-expressing NFAT and human DNAM-1 protein, and a construction method and an application thereof, wherein the recombinant plasmid and the recombinant cell can start expression of DNAM-1 gene and NFAT-Luciferase gene by bidirectional promoter, do not affect each other during gene expression, and can directly recognize and bind DNAM-1 protein antibody, thereby screening anti-DNAM-1 antibody.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a construction method of a recombinant plasmid for co-expressing NFAT and human DNAM-1 protein, which comprises the following steps: (1) inserting the coding gene sequence of NFAT-reverse between SpeI and BstBI enzyme cutting sites of a vector PB-713B-1, and carrying out small-extraction preparation of endotoxin-free plasmids to obtain PB-NFAT-Luc-713 plasmids;
(2) after codon optimization is carried out on the gene sequence of the human DNAM-1, the gene sequence is inserted between Bsu36I and NdeI restriction enzyme sites of the PB-NFAT-Luc-713 plasmid, and endotoxin-free plasmid preparation is carried out to obtain the recombinant plasmid PB-NFAT-Luc-CD 226.
Preferably, the 5 'end of the NFAT-reverse encoding gene sequence in step (1) comprises a SpeI enzyme cutting site and a Luc sequence, and the 3' end comprises a BstBI enzyme cutting site.
Preferably, the gene sequence of the human DNAM-1 subjected to codon optimization in the step (2) comprises a Bsu36I enzyme cutting site and an LTR sequence at the 5 'end, and an NdeI enzyme cutting site at the 3' end.
Preferably, the gene sequence of the codon-optimized human DNAM-1 is shown in SEQ ID NO. 2.
The invention also provides the recombinant plasmid PB-NFAT-Luc-CD226 constructed by the construction method.
The invention also provides a recombinant cell J containing the recombinant plasmid PB-NFAT-Luc-CD226urkat-NFAT-Luc-CD226。
The invention also providesThe preparation method of the recombinant cell Jurkat-NFAT-Luc-CD226 comprises the following steps: the recombinant plasmid PB-NFAT-Luc-CD226 is used for transfecting Jurkat cells to obtain the recombinant cell Jurkat-NFAT-Luc-CD226。
The invention also provides a monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 prepared on the basis of the recombinant cell Jurkat-NFAT-Luc-CD 226.
The invention also provides application of the monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 in self-production and/or screening of anti-human DNAM-1 antibodies.
The invention also provides a method for detecting and/or screening the anti-human DNAM-1 antibody, which comprises the following steps: and mixing and incubating the monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 and an anti-human DNAM-1 antibody for 4h, collecting cell suspension, centrifuging to remove supernatant, and mixing with the ONE-Lite substrate and buffer solution 1: 1, mixing the mixed solution, measuring a fluorescence signal on an enzyme labeling instrument, and detecting and/or screening the anti-human DNAM-1 antibody according to the intensity of the fluorescence signal.
The invention also provides an anti-human DNAM-1 antibody obtained by screening by the method, wherein the amino acid sequence of the heavy chain variable region of the anti-human DNAM-1 antibody is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.
The invention provides a construction method of a recombinant plasmid co-expressing NFAT and human DNAM-1 protein, the constructed recombinant plasmid, and a recombinant cell and a monoclonal recombinant cell constructed on the basis of the recombinant plasmid. The recombinant plasmid bidirectional promoter provided by the invention starts the expression of the DNAM-1 gene and the NFAT-Luciferase gene, and the gene expression is not influenced mutually. The recombinant cell Jurkat-NFAT-Luc-CD226 is constructed based on a human immune type cell Jurkat, is more similar to an NK cell model, and when the recombinant cell is constructed, a transposon technology is adopted to transfect plasmids, so that viruses are not introduced, a cell inflammation related passage is not caused, the operation is simple, the operation error of experimenters is not easy to generate, the constructed cell highly expresses DNAM-1 protein on a membrane, and the NFAT is activated to express Luciferase under the combined stimulation of an anti-human DNAM-1 antibody, so that the fluorescence can be detected and the Luciferase can be used for detecting the anti-human DNAM-1 antibody.
In the embodiment of the invention, Jurkat cells are used for transfecting plasmids PB-NFAT-Luc-CD226, a Jurkat monoclonal cell line stably expressing NFAT reporter elements and human DNAM-1 protein is established, an antibody anti-human CD226(DNAM-1) is added, and the binding capacity of the transfected cells to the antibody and the degree of firefly luciferin expression of the cells after being activated by the antibody are detected. The invention compares the self-produced anti-human DNAM-1 antibody with the commercial anti-human DNAM-1 antibody, the isotype antibody and other scFv antibodies, and determines the difference of the luciferase catalysis chemical substrate luminescence of Jurkat-NFAT-Luc-CD226 cell to screen the DNAM-1 antibody. The result shows that the Jurkat-NFAT-Luc-CD226 recombinant cell can be combined with the anti-human DNAM-1 molecular antibody and activates the NKCR pathway to express firefly Luciferase (Luciferase), the recombinant cell can be used for screening and identifying the anti-human DNAM-1 molecular antibody, and the invention screens out the anti-human DNAM-1 antibody.
Drawings
FIG. 1 shows the recombinant plasmid PB-NFAT-Luc-structure of CD 226;
FIG. 2 shows that after Jurkat-NFAT-Luc-CD226 cells and wild type Jurkat cells are respectively added with 11A8 antibody and incubated for 4h, an enzyme-linked immunosorbent assay detects that the antibody activates cells to express fluorescein (Luciferase);
FIG. 3 shows the results of flow-assay of DNAM-1 protein expressed on the cell membrane of Jurkat-NFAT-Luc-CD 226;
FIG. 4 shows the flow assay result of screening monoclonal cells of Jurkat-NFAT-Luc-DNAM-1 to highly express DNAM-1 protein;
FIG. 5 shows the results of luminescence of Jurkat-NFAT-Luc-DNAM-1 cells # 2, #8, #14, #16 and #18 expressing Luciferase monoclonal;
FIG. 6 shows the results of measuring Luciferase luminescence signal intensity of J-DNAM-1- #2 cells under the conditions of commercial anti-human DNAM-1 antibody, antibody No. 9, antibody concentration of 2. mu.g/ml for other scFv antibodies, and incubation time of 4 h;
FIG. 7 shows the results of the J-DNAM-1- #2 cells activating luminescence of expression of Luciferase at the cell antibody co-incubation times of 16h, 8h, 6h, 4h, 2h and 0h for the J-DNAM-1- #2 cells;
FIG. 8 shows the luminescence results of Luciferase assay in J-DNAM-1- #2 cells stimulated by different concentrations of anti-human DNAM-1 antibody;
FIG. 9 shows the result of flow cytometry for identifying the specificity of DNAM-1 protein;
FIG. 10 shows the report function results of anti-human DNAM-1 antibody and homotypic antibody activated J-DNAM-1- #2 cells.
Detailed Description
The invention provides a construction method of a recombinant plasmid for co-expressing NFAT and human DNAM-1 protein, which comprises the following steps: (1) inserting the coding gene sequence of NFAT-reverse between SpeI and BstBI enzyme cutting sites of a vector PB-713B-1, and carrying out small-extraction preparation of endotoxin-free plasmids to obtain PB-NFAT-Luc-713 plasmids;
(2) after codon optimization is carried out on the gene sequence of the human DNAM-1, the gene sequence is inserted between Bsu36I and NdeI restriction enzyme sites of the PB-NFAT-Luc-713 plasmid, and endotoxin-free plasmid preparation is carried out to obtain the recombinant plasmid PB-NFAT-Luc-CD 226.
The coding gene sequence of NFAT-reverse is inserted between SpeI and BstBI restriction enzyme cutting sites of a vector PB-713B-1, and endotoxin-free plasmid is prepared to obtain PB-NFAT-Luc-713 plasmid. The source of the NFAT-reverse coding gene sequence is not specially limited, and the NFAT-reverse coding gene sequence is preferably synthesized by a whole-gene synthesis technology, then a SpeI enzyme cutting site is added at the 5 'end, a BstBI enzyme cutting site is added at the 3' end, then the synthesized NFAT-reverse gene (SpeI-Luc-NFAT-BstBI-reverse, SEQ ID NO.1) is constructed to a vector PB-713B-1 (purchased from SBI company, LOT:190316-004) through an upstream SpeI enzyme cutting site and a downstream BstBI enzyme cutting site, and then small-extraction preparation of endotoxin-free plasmids (completed by trusted Jinzhi Biotechnology Limited) is carried out to obtain PB-NFAT-Luc-713 plasmid.
After codon optimization is carried out on the gene sequence of the human DNAM-1, the gene sequence is inserted between Bsu36I and NdeI restriction enzyme sites of the PB-NFAT-Luc-713 plasmid, endotoxin-free plasmid preparation is carried out, and the recombinant plasmid PB-NFAT-Luc-CD226 is constructed. The invention preferably takes human as host, carries out codon optimization on DNAM-1 gene sequence and adds Bsu36I enzyme cutting site and LTR at 5' end(containing one SalI enzyme digestion site) sequence, adding NdeI enzyme digestion site at the 3' end, then conventionally synthesizing the DNAM-1 gene sequence, constructing the synthesized DNAM-1 gene (Bsu36I-LTR-SalI-DNAM-1-NdeI3, SEQ ID NO.2) on a vector PB-NFAT-Luc-713 through an upstream Bsu36I enzyme digestion site and a downstream NdeI enzyme digestion site, and carrying out large-scale extraction preparation of an endotoxin-free plasmid (finished by the Kingjinzhi Biotech Co., Ltd.), thus obtaining the PB-NFAT-LucThe CD226 plasmid (FIG. 1).
The invention also provides the recombinant plasmid PB-NFAT-Luc-CD226 shown in the figure 1, which is constructed by the construction method. The recombinant plasmid bidirectional promoter provided by the invention can be used for promoting the co-expression of the DNAM-1 gene and the NFAT-Luciferase gene, and the gene expression is not influenced mutually.
The invention also provides a recombinant cell Jurkat-NFAT-Luc-CD226 containing the recombinant plasmid PB-NFAT-Luc-CD226, and the recombinant cell Jurkat-NFAT-Luc-CD226 co-expresses NFAT and human DNAM-1 protein.
The invention also provides a preparation method of the recombinant cell Jurkat-NFAT-Luc-CD226, which comprises the following steps: the recombinant plasmid PB-NFAT-Luc-CD226 is used for transfecting Jurkat cells to obtain the recombinant cell Jurkat-NFAT-Luc-CD226。
The transfection according to the invention is preferably carried out according to the instructions of the electrotransformation instrument Celetrix, CTX-1500A), and specifically comprises: washing and centrifuging Jurkat cells by 1640 culture medium without fetal bovine serum (REF:10099-141, brand: gibco) and penicillin-streptomycin mixed liquor (Cat: SV30010 brand: HyClone), then re-suspending by 120 microliter of 1640 culture medium (fetal bovine serum-free and penicillin-streptomycin mixed liquor), adding plasmids, gently mixing uniformly, transferring the cell suspension into an electric shock cup (120 microliter Celetric Cat. NO.1204) matched with an electric rotor, placing the electric shock cup on ice for 5mins, performing electric shock operation, and then placing the electric shock cup on ice for cooling for 5 mins; all cell suspensions were then transferred to 75CM2 cell FLASKs (CORNING flag, 430641) containing 25ml volumes of 1640medium (10% fetal bovine serum) and cultured in 37 ℃ incubator (ESCD, CCL-240B-8) for 48 h. At this time, the transfected cells were designated Jurkat-NFAT-Luc-CD 226. The Jurkat cells (Clone E6-1) of the present invention are preferably purchased from Shanghai cell Bank of Chinese academy of sciences.
The recombinant cell Jurkat-NFAT-Luc-CD226 is constructed based on human immune type cell Jurkat and is more similar to an NK cell model, and a transposon technology is adopted to transfect plasmids during construction, so that viruses cannot be introduced, inflammation-related passages of cells cannot be caused, the operation is simple, and errors in operation of experimenters cannot be easily generated.
After the recombinant cell Jurkat-NFAT-Luc-CD226 is obtained, the puromycin drug screening is preferably carried out on the recombinant cell Jurkat-NFAT-Luc-CD226 in sequence, whether an NFAT report element and a DNAM-1 protein are expressed or not is identified, and when the NFAT report element and the DNAM-1 protein are both expressed, the recombinant cell Jurkat-NFAT-Luc-CD226 is constructed and completed.
The invention also provides a monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 prepared on the basis of the recombinant cell Jurkat-NFAT-Luc-CD 226.
The invention preferably uses a flow sorter to prepare the monoclonal recombinant cell Jurkat-NFAT-Luc-CD226, and more preferably comprises: after the recombinant cell Jurkat-NFAT-Luc-CD226 was washed out of the medium with PBS containing 0.5% BSA, the antibody PE anti-human CD226(DNAM-1) was added and incubated at room temperature in the dark for 20 min. After washing the fluorescent antibody with PBS, it was resuspended at 1X 10 in PBS6Cell concentration per ml, flow cytometry monoclonal DNAM-1 positive cells. The cell cloned by the cell verified to be highly expressed by the NFAT reporter element and the DNAM-1 protein is named as a monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 (or Jurkat-NFAT-Luc-DNAM-1 monoclonal cell).
The monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 has high DNAM-1 protein expression level, and can be activated by an anti-human DNAM-1 antibody with the concentration of 2 mu g/ml to express high Luciferase.
The invention also provides application of the monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 in detection and/or screening of anti-human DNAM-1 antibodies.
The invention also provides a method for detecting and/or screening the anti-human DNAM-1 antibody, which comprises the following steps: and mixing and incubating the monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 and an anti-human DNAM-1 antibody for 4h, collecting cell suspension, centrifuging to remove supernatant, and mixing with the ONE-Lite substrate and buffer solution 1: 1, mixing the mixed solution, measuring a fluorescence signal on an enzyme labeling instrument, and detecting and/or screening the anti-human DNAM-1 antibody according to the intensity of the fluorescence signal.
The ratio of the monoclonal recombinant cell Jurkat-NFAT-Luc-CD226 to the anti-human DNAM-1 antibody is preferably 1 × 105Cell: 1μg, and further comprising resuspending the monoclonal recombinant cells Jurkat-NFAT-Luc-CD226 in 1640medium prior to the mixed incubation. The temperature of the mixed incubation according to the invention is preferably 37 ℃. After removing the supernatant, the invention adds 1: 1, and the volume ratio of the mixed solution to the 1640medium for resuspension is preferably 1: 5. In the present invention, after the mixing, the cell pellet is preferably transferred to a 96-well plate. An enzyme-linked immunosorbent assay (MD SpectraMax i3X) (absorbance, LM 1: 450, LM 2: 630, plate type: 96-hole standard pore plate, reading height: 14.6 mm) detects the intensity of a luminescent signal activated by the antibody, and the anti-human DNAM-1 antibody is detected and screened according to the existence and the intensity of a fluorescent signal.
The invention has the following advantages that 1: 1 the source of the mixture is not particularly limited, and is preferably from novispan, having the following trade number: DD1203-01, wherein the Buffer solution is One-Lite Luciferase Assay Buffer 1, and the substrate is: One-Lite Luciferase Assay Substrate (lysophilized).
In the embodiment of the present invention, the antibody No. 9 is screened, and the method for preparing the antibody No. 9 preferably includes: the hybridoma technology is utilized to carry out fusion screening and monoclone on the mouse spleen cell of the immune DNAM-1 antigen and the SP2.0 cell to obtain the antibody capable of secreting the anti-human DNAM-1, and the sequence is sent to Jinzhi for codon optimization and then is constructed in a CHO cell to secrete and express the anti-human DNAM-1 antibody. Collecting and enriching the culture medium supernatant for culturing the cell line to obtain secreted antibody, wherein the antibody No. 9 is collected antibody with higher concentration.
The invention also provides an anti-human DNAM-1 antibody obtained by screening by using the method, wherein the amino acid sequence of the heavy chain variable region of the anti-human DNAM-1 antibody is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4. In the embodiment of the invention, the obtained anti-human DNAM-1 antibody (antibody No. 9) is verified, and the antibody No. 9 has higher luminous intensity than commercial 11A8 antibody Luciferase fluorescence value when activating J-DNAM-1- #2 cells, so that the antibody can be combined with DNAM-1 molecules and has better effect.
The following examples are provided to illustrate the recombinant plasmid and recombinant cell co-expressing NFAT and human DNAM-1 protein, and the construction method and application thereof in detail, but they should not be construed as limiting the scope of the present invention.
The complete culture Medium is RPMI1640Medium and HEPES (GIBCO, 22400-.
Example 1Jurkat-NFAT-LucConstruction of-CD 226 cells
1. Primitive cell
The original Jurkat cells (Clone E6-1) are purchased from Shanghai cell bank of Chinese academy of sciences, and are subcultured and stored in a laboratory, and the cell using rate of cell activity is more than 95%.
2. Plasmid construction
The NFAT-reverse is synthesized by adopting a whole gene synthesis technology, a SpeI enzyme cutting site is added at the 5 'end of the NFAT-reverse, a BstBI enzyme cutting site is added at the 3' end of the NFAT-reverse, then the synthesized NFAT-reverse gene (SEQ ID NO.1) is constructed to a vector PB-713B-1 (purchased from SBI company, LOT: 190316-.
Using human as host to make codon optimization of DNAM-1 gene sequence, adding Bsu36I enzyme cutting site and LTR (containing a SalI enzyme cutting site to remove UTR region) sequence at its 5 'end, adding NdeI enzyme cutting site at 3' end, conventionally synthesizing DNAM-1 gene sequence, making the synthesized DNAM-1 gene (SEQ ID NO.2) pass through upstream Bsu36I enzyme cutting site and lower partThe upstream NdeI restriction site was constructed on the vector PB-NFAT-Luc-713(Ampicillin), and the endotoxin-free plasmid was prepared (completed by King Kouzi Biotech Co., Ltd.) to obtain PB-NFAT-L as shown in FIG. 1uc-CD226 plasmid.
3. Plasmid transfected cells
Operating with reference to the instructions of the electrotransfer instrument Celetrix, CTX-1500A), Jurkat cells were treated with fetal bovine serum free (REF:10099-141 brand: gibco), penicillin-free streptomycin mixture (Cat: SV30010 brand: HyClone) 1640 culture medium, adding plasmid, gently mixing, transferring the cell suspension to an electric shock cup (120. mu.l Celetric Cat. NO.1204) matched with an electrotransfer, placing the cell suspension on ice for 5mins, performing electric shock operation, and then placing the electric shock cup on ice for cooling for 5 mins. All cell suspensions were then transferred to 75CM2 cell FLASKs (CORNING flag, 430641) containing 25ml volumes of 1640medium (10% fetal bovine serum) and cultured in 37 ℃ incubator (ESCD, CCL-240B-8) for 48 h. At this time, the transfected cells were designated Jurkat-NFAT-Luc-CD 226.
4. Screening of Jurkat-NFAT-Luc-CD226 cells
(1) Puromycin (Cat: ant-pr-1, brand: Invivogen) drug sieve, Jurkat-NFAT-Luc-CD226 cell electric transfer 48h, adjusting its cell density to 3X 105Then 0.5. mu.g/mL puromycin was added for drug screening. And (4) at the interval of 48h for each passage, and gradually and slightly increasing the drug screening concentration of puromycin if the cell viability is more than 70% during the passage. The concentration of the drug sieve is stably used when the concentration of the puromycin reaches 3.0 mu g/mL.
(2) Identification of the expression of NFAT reporter element by Jurkat-NFAT-Luc-CD226 cells
Respectively taking 1 × 105Jurkat-NFAT-Luc-CD226 cells (two cells, one Control without antibody activation named Control and one experimental group with 11A8 antibody) and Jurkat-WT cells were collected, centrifuged at 400g for 5min, the supernatant was discarded, the cells were resuspended in 500. mu.l 1640medium (10% fetal bovine serum), and then added to a labeled 24-well plate for antibody co-incubation, and each cell was incubated with antibodyMu.g of Biotin anti-human CD226(DNAM-1 Clone:11A8, 2. mu.g/ml) antibody was added to the wells, incubated at 37 ℃ for 4h in an incubator, the cell suspension was collected after the incubation was completed, centrifuged and the supernatant removed (a volume of 10. mu.l was left to avoid cell loss), 100. mu.l of ONE-Lite substrate and 1: 1 mix and transfer the cell suspension to a 96-well plate. In addition, a 96-well plate is provided with ONE ONE-Lit cell added with 100 mu le1: 1 control wells of cocktail reagent.
The intensity of the luminescence signal after activation by the antibody was detected by a microplate reader (MD SpectraMax i3X) (absorbance, LM 1: 450, LM 2: 630, plate: 96-well standard well plate, reading height: 14.6 mm). As shown in FIG. 2, the 11A8 antibody stimulated Jurkat-NFAT-Luc-CD226 to produce higher luminescent signal intensity compared to wild-type cells.
(3) DNAM-1 protein expression identification of Jurkat-NFAT-Luc-CD226 cells
Jurkat-NFAT-Luc-CD226 cells were subjected to one week of drug screening with puromycin (Invivogen brand), and then expression of DNAM-1 protein in Jurkat-NFAT-Luc-CD226 cells was identified by flow cytometry, and 1X 10 cells were used6After the culture medium was washed with 0.5% BSA in PBS (Hyclone, SH30256.01) (the same applies below), 1. mu.l of PE anti-human CD226(DNAM-1) antibody was added thereto, and the cells were incubated at room temperature in the dark for 20 min. After washing the fluorescent antibody with PBS, the sample was resuspended in 400. mu.l of PBS, and the fraction of positive cells expressing DNAM-1 was examined by flow cytometry (BD LSRFORTESSA). As a result, as shown in FIG. 3, the Jurkat-NFAT-Luc-CD226 cell significantly expressed the DNAM-1 protein, compared to the wild-type Jurkat cell. At this time, it was indicated that Jurkat-NFAT-Luc-CD226 cells were successfully constructed, and they were able to be produced by a single clone.
Example 2 preparation and characterization of Jurkat-NFAT-Luc-CD226 monoclonal
1. The Jurkat-NFAT-Luc-CD226 cell clone with clone number 35B8, which was successfully constructed in example 1, was used. The procedure was performed with reference to the flow Sorter (SONY) instructions, taking Jurkat-NFAT-Luc-CD226 and Jurkat-WT cells (negative control), washing the medium with PBS containing 0.5% BSA, adding PE anti-human CD226(DNAM-1) antibody, and incubating at room temperature in the dark for 20 min. After washing the fluorescent antibody with PBS, it was resuspended in PBSTo 1X 106Cell concentration per ml, flow cytometry monoclonal DNAM-1 positive cells. The cells obtained by cloning the cells highly expressing the NFAT reporter element and the DNAM-1 protein were designated as Jurkat-NFAT-Luc-DNAM-1 monoclonal cells, and the results were examined by the following method.
2. Jurkat-NFAT-Luc-DNAM-1 monoclonal cell DNAM-1 protein expression identification
3. Identification of the expression of NFAT reporter element in Jurkat-NFAT-Luc-DNAM-1 monoclonal cells
Respectively taking 1 × 105Jurkat-NFAT-Luc-DNAM-1 monoclonal cells # 2, #8, #14, #16, #18 and Jurkat-WT cells, 400g centrifuged for 5min and the supernatant discarded, 500. mu.l of 1640medium (10% fetal bovine serum) resuspended cells and antibody co-incubated in a cell transfer labelled 24 well plate, 1. mu.g of Biotin anti-human CD226(DNAM-1) antibody was added to each well, incubation at 37 ℃ was carried out for 4h, cell suspension was collected after incubation was completed, centrifuged and the supernatant was removed (10. mu.l volume could be left to avoid cell loss), 100. mu.l of ONE-Lite substrate was added with 1 of buffer: 1 mix and transfer the cell pellet to a 96-well plate. The intensity of the luminescence signal after activation by the antibody was detected by a microplate reader (MD SpectraMax i3X) (absorbance, LM 1: 450, LM 2: 630, plate: 96-well standard well plate, reading height: 14.6 mm). As a result, as shown in FIG. 5, the monoclonal cells Jurkat-NFAT-Luc-DNAM-1#2, #8, #14, #16 and #18 were activated by the anti-human DNAM-1 antibody at a concentration of 2. mu.g/ml to express high Luciferase。
At this time, it is indicated that the Jurkat-NFAT-Luc-DNAM-1 monoclonal cell was successfully constructed, and Jurkat-NFAT-Luc-DNAM-1#2 monoclonal cell was selected to continue the subsequent experiment, and in the subsequent examples, Jurkat-NFAT-Luc-DNAM-1#2 monoclonal cell was named J-DNAM-1- #2 cell.
Example 3 self-production anti-human DNAM-1 protein No. 9 antibody identification and other scFv antibody comparisons
Preparation of antibody No. 9: the hybridoma technology is utilized to carry out fusion screening and monoclone on the mouse spleen cell of the immune DNAM-1 antigen and the SP2.0 cell to obtain the antibody capable of secreting the anti-human DNAM-1, and the sequence is sent to Jinzhi for codon optimization and then is constructed in a CHO cell to secrete and express the anti-human DNAM-1 antibody. Collecting and enriching the culture medium supernatant for culturing the cell line to obtain secreted antibody, wherein the antibody No. 9 is collected antibody with higher concentration.
Take 6X 105J-DNAM-1- #2 cells, which were equally divided into six portions, were labeled: first, second, third, fourth, second 0 and second 1. The above 6 groups of cells were resuspended in 500. mu.l of 1640medium (10% fetal bovine serum) and transferred to a 24-well plate for culture, and then 1. mu.g of Biotin anti-human CD226(DNAM-1) Clone was added to the well (r): 11A8 antibody, adding 1 μ g of antibody No. 9 into well No.3, adding 1 μ g of antibody N-FC-4 (a scFv antibody, which acts as an irrelevant control antibody) into well No.3, adding 1 μ g of antibody 4-nono-N (a scFv antibody, which acts as an irrelevant control antibody) into well No.4, adding 1 μ g of antibody NM3-FC (a scFv antibody, which acts as an irrelevant control antibody) into well No. fifthly, adding no antibody (negative control) into well No.4, incubating at 37 ℃ for 4h, collecting cell suspension after incubation, centrifuging and removing supernatant (10 μ l of volume can be left, avoiding cell loss), adding 100 μ l of ONE-Lite substrate and buffer solution 1: 1 mix and transfer the cell pellet to a 96-well plate. The intensity of the luminescence signal after activation by the antibody was detected by a microplate reader (MD SpectraMax i3X) (absorbance, LM 1: 450, LM 2: 630, plate: 96-well standard well plate, reading height: 14.6 mm). As shown in FIG. 6, the antibody 9 showed higher fluorescence intensity in activating J-DNAM-1- #2 cells than that of the commercial 11A8 antibody Luciferase, whereas the other scFv isotype antibodies (N-FC-4, 4-nono-N, NM3-FC) did not significantly stimulateThe result of the luminescence of the Luciferase is expressed actively, which shows that the antibody capable of being combined with the DNAM-1 molecule is successfully screened out.
And simultaneously, carrying out antibody sequence sequencing and sequence analysis on the No. 9 antibody obtained by screening, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3: EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARYWRYDGYAMDYWGQGTSVTVSS, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4: IVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIKR are provided.
TABLE 1 analysis of light chain variable region sequences
| Structural domains | Sequence fragment | Position of |
| LFR1 | IVMTQTPKFLLVSAGDRVTITCKAS | 1-25 |
| CDR-L1 | QSVSND | 26-31 |
| LFR2 | VAWYQQKPGQSPKLLIY | 32-48 |
| CDR-L2 | YA | 49-50 |
| LFR3 | SNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC | 51-87 |
| CDR-L3 | QQDYSSPYT | 88-96 |
| LFR4 | FGGGTKLEIKR | 97-107 |
TABLE 2 analysis of sequences of variable regions of heavy chains
| Structural domains | Sequence fragment | Position of |
| HFR1 | EVQLQESGPSLVKPSQTLSLTCSVT | 1-25 |
| CDR-H1 | GDSITSGY | 26-33 |
| HFR2 | WNWIRKFPGNKLEYMGY | 34-50 |
| CDR-H2 | ISYSGST | 51-57 |
| HFR3 | YYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYC | 58-95 |
| CDR-H3 | ARYWRYDGYAMDY | 96-108 |
| HFR4 | WGQGTSVTVSS | 109-119 |
Example 4 reporter Gene experiment optimization
Optimal time optimization of J-DNAM-1- #2 cell antibody incubation
1. Firstly, respectively taking 1 × 105Cell number of J-DNAM-1- #2 cells (#2 cells 6 samples were taken) were divided into the following 6 groups: the method comprises the following steps of (1) incubating the antibodies for 16h, incubating the antibodies for 8h, incubating the antibodies for 6h, incubating the antibodies for 4h, incubating the antibodies for 2h and incubating the antibodies for 0 h.
2. Group I cell antibody co-incubation: taking 1X 10 of J-DNAM-1- #2 cells5The cells were centrifuged at 400g/min for 5min, the supernatant was discarded, 500. mu.l of 1640 complete medium was added to suspend the cells, and the cells were co-incubated in 24-well plates labeled with cell transfer, 1. mu.g of Biotin anti-human CD226(DNAM-1) was added to each well, and the cells were incubated at 37 ℃ in an incubator for 16h, respectively.
3. After the first group cell antibody is incubated for 8h, the second group cell antibody is incubated, and the number of cells and the method are the same as the step 2.
4. And (3) after the cell antibodies of the first group are incubated for 10 hours, the cell antibodies of the third group are incubated, and the cell number and the method are the same as the step 2.
5. After the cell antibody of the first group is incubated for 12h, the cell antibody of the second group is incubated, and the number of cells and the method are the same as those in the step 2.
6. After the first group cell antibody is incubated for 14h, the fifth group cell antibody is incubated again, and the number of cells and the method are the same as those in step 2.
7. After the cell antibody of the first group is incubated for 16h, the cell antibody of the sixth group is incubated, and the cell number and the method are the same as the step 2.
After mixing of the group 6 cell antibody cells, the cell suspension was collected, centrifuged and the supernatant removed (10. mu.l volume was left to avoid cell loss), 100. mu.l of ONE-Lite substrate plus 1: 1 mix and transfer the cell pellet to a 96-well plate. The intensity of the luminescence signal after activation by the antibody was detected by a microplate reader (MD SpectraMax i3X) (absorbance, LM 1: 450, LM 2: 630, plate: 96-well standard well plate, reading height: 14.6 mm). As a result, as shown in FIG. 7, the luminescence value of Luciferase detected by the cells was the highest when the antibody was incubated with the cells for 4 hours under the same concentration of the anti-human DNAM-1 antibody (concentration of 2. mu.g/ml).
Example 5 optimal concentration optimization of antibody activation in J-DNAM-1- #2 cells
5 anti-human DNAM-1 antibody (Clone:11A8) antibody concentration gradients were designed: 0.5. mu.g/ml, 1. mu.g/ml, 2. mu.g/ml, 2.5. mu.g/ml, 3. mu.g/ml.
Firstly, respectively taking 1 × 105Cell number J-DNAM-1- #2 cells (#2 cells 10 samples were taken, one set of two duplicate wells) were divided into the following 5 groups: 0.5 mu g/ml antibody concentration and cell co-incubation, 1 mu g/ml antibody concentration and cell co-incubation, 2 mu g/ml antibody concentration and cell co-incubation, 2.5 mu g/ml antibody concentration and cell co-incubation, and 3 mu g/ml antibody concentration and cell co-incubation. After centrifugation at 400g for 5min, the supernatant was discarded and the corresponding concentration of Biotin anti-human CD226(DNAM-1) Clone was added: 11A8 antibody, 500 u l volume 1640medium (10% fetal bovine serum) heavy suspension, placed in 24 hole plate and incubated at 37 ℃ for 4h, after incubation, cell suspension is collected, centrifuged and supernatant removed (can be 10 u l volume, avoid cell loss), 100 u l ONE-Lite bottom is added1 ratio of substance to buffer: 1 mix and transfer the cell pellet to a 96-well plate. The intensity of the luminescence signal after activation by the antibody was detected by a microplate reader (MD SpectraMax i3X) (absorbance, LM 1: 450, LM 2: 630, plate: 96-well standard well plate, reading height: 14.6 mm). As a result, as shown in FIG. 8, the anti-human DNAM-1 antibody at a concentration of 2. mu.g/ml was stimulated to an optimal concentration.
Example 6 DNAM-1 protein specificity identification
1. Flow cytometry identified the specificity of the DNAM-1 protein.
Take 3X 106J-DNAM-1- #2 cells, which were evenly divided into three portions and labeled: J-DNAM-1- #2+ DNAM-1, J-DNAM-1- #2+ NKG2D, and J-DNAM-1- #2+ NKp 46; then take 3X 106Jurkat cells, which were equally divided into three, labeled: jurkat + DNAM-1, Jurkat- + NKG2D, and Jurkat + NKp 46. After washing the above 6 groups of cells with PBS containing 0.5% BSA, 1. mu.g of the following three flow antibodies were added to each tube: PE anti-human CD226(DNAM-1), FITC anti-human CD314(NKG2D), PE anti-human CD335(NKp46), incubated at room temperature in the absence of light for 20 min. After washing the fluorescent antibodies with PBS, it was resuspended in 400 μ l PBS, the PE channel fluorescence signal intensity was detected by flow cytometry (BD lsrfortestassa), and analyzed by Flowjo software and plotted as PE channel fluorescence MFI values. As a result, as shown in FIG. 9, under the same staining condition, the anti-human DNAM-1 antibody hardly bound to Jurkat-WT cells, showed high affinity with J-DNAM-1- #2, and detected high fluorescence values, while neither NKG2D nor NKP46 bound to both types of cells, and hardly detected fluorescence signals of the corresponding channels.
2. Functional specificity verification is reported.
Take 1X 105Marking the Jurkat cells as (i) and taking 4X 105J-DNAM-1- #2 cells, which were evenly divided into four, labeled: ② ③, fourthly, fifthly. The above 5 groups of cells were resuspended in 500. mu.l of 1640medium (10% fetal bovine serum) and transferred to a 24-well plate for culture, and then 1. mu.g of Biotin anti-human CD226(DNAM-1) Clone was added to the well (r): 11A8 antibody, wells # 2 No. 1. mu.g Biotin antiHuman CD226(DNAM-1) Clone:11A8 antibody, 1. mu.g of Biotin anti-human CD314(NKG2D) Clone: 1D11 antibody, 1. mu.g of Biotin anti-human CD335(NKp46) Clone: 9E2 antibody, 37 ℃ for 4h, after the incubation is finished, the cell suspension is collected, centrifuged and the supernatant is removed (10. mu.l volume can be left to avoid cell loss), 100. mu.l of ONE-Lite substrate and 1: 1 mix and transfer the cell pellet to a 96-well plate. The intensity of the luminescence signal after activation by the antibody was detected by a microplate reader (MD SpectraMax i3X) (absorbance, LM 1: 450, LM 2: 630, plate: 96-well standard well plate, reading height: 14.6 mm). As shown in FIG. 10, under the conditions of consistent antibody concentration and incubation time, the 11A8 antibody can activate the J-DNAM-1- #2 cell to express a higher luminescence signal window, and the 1D11 antibody can also activate the J-DNAM-1- #2 cell to express a luminescence signal window, but the luminescence signal window is far lower than that of the 11A8 antibody, and the 9E2 antibody has no activation function, compared with the control group (group II) incubated without any antibody.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Beijing ancient cooking peptide source Biotechnology Ltd
<120> recombinant plasmid and recombinant cell for coexpression of NFAT and human DNAM-1 protein, and construction method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2334
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
actagtgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 60
aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 120
ttatcatgtc tgctcgaagc ggccggccgc cccgactcta gagtcgcggc cttagacgtt 180
gatcctggcg ctggcgcaag cagcagggtg tctatccatg ccgctctcct gggcgcagct 240
catgggcagg gtgccggcgg cctgctcctc cacctcggga gggaagccgt gagaattcac 300
ggcgatcttg ccgcccttct tggccttaat gagaatctcg cggatcttgc gggcgtccaa 360
cttgccggtc agtcctttag gcacctcgtc cacgaacaca acaccaccgc gcagcttctt 420
ggcggttgta acctggctgg ccacatagtc cacgatctcc ttctcggtca tggttttacc 480
gtgttccagc acgacgactg cggcgggcag ctcgccggca tcgtcgtcgg gcaggccggc 540
gaccccggcg tcgaagatgt tggggtgttg cagcaggatg ctctccagtt cggctggggc 600
tacctggtag cccttgtatt tgatcaggct cttcagccgg tccacgatga agaagtgctc 660
gtcctcgtcc cagtaggcga tgtcgccgct gtgcagccag ccgtccttgt cgatgagagc 720
gtttgtagcc tcggggttgt taacgtagcc gctcatgatc atggggccac ggacgcacag 780
ctcgccgcgc tggttcacac ccagtgtctt accggtgtcc aagtccacca ccttagcctc 840
gaagaagggc accaccttgc ctactgcgcc aggcttgtcg tccccttcgg gggtgatcag 900
aatggcgctg gttgtttctg tcaggccgta gccctggcgg atgcctggta ggtggaagcg 960
tttggccacg gcctcaccta cctccttgct gagcggcgcc ccgccgctgg cgatctcgtg 1020
caagttgctt aggtcgtact tgtcgatgag agtgctctta gcgaagaagc taaatagtgt 1080
gggcaccagc agggcagatt gaatcttata gtcttgcaag ctgcgcaaga atagctcctc 1140
ctcgaagcgg tacatgagca cgacccgaaa gccgcagatc aagtagccca gcgtggtgaa 1200
catgccgaag ccgtggtgaa atggcaccac gctgaggata gcggtgtcgg ggatgatctg 1260
gttgccgaag atggggtcgc gggcatgact gaatcggaca caagcggtgc ggtgcggtag 1320
ggctacgccc ttgggcaatc cggtactgcc actactgttc atgatcaggg cgatggtttt 1380
gtcccggtcg aagctctcgg gcacgaagtc gtactcgttg aagccgggtg gcaaatggga 1440
agtcacgaag gtgtacatgc tttggaagcc ctggtagtcg gtcttgctat ccatgatgat 1500
gatcttttgt atgatcggta gcttcttttg cacgttgagg atcttttgca gccctttctt 1560
gctcacgaat acgacggtgg gctggctgat gcccatgctg ttcagcagct cgcgctcgtt 1620
gtagatgtcg ttagctgggg ccacagccac accgatgaac agggcaccca acacgggcat 1680
gaagaactgc aagctattct cgctgcacac cacgatccga tggtttgtat tcagcccata 1740
gcgcttcata gcttctgcca gccgaacgct catctcgaag tactcggcgt aggtaatgtc 1800
cacctcgata tgtgcgtcgg taaaggcgat ggtgccgggc accagggcgt agcgcttcat 1860
ggctttgtgc agctgctcgc cggcggtccc gtcttcgagt gggtagaatg gcgctgggcc 1920
cttcttaatg tttttggcat cttccatggt ggctttacca acagtaccgg attgccaagc 1980
tttttaccaa cagtaccgga atgccaagct ggaattcgag cttccattat ataccctcta 2040
gagtctagat ctacgccttc tgtatgaaac agtttttcct ccacgccttc tgtatgaaac 2100
agtttttcct ccacgccttc tgtatgaaac agtttttcct ccgctagcga gctcaggtac 2160
cggccagtta ggccagagaa atgttctggc acctgcactt gcactgggga cagcctattt 2220
tgctagtttg ttttgtttcg ttttgttttg atggagagcg tatgttagta ctatcgattc 2280
acacaaaaaa ccaacacaca gatgtaatga aaataaagat attttatttt cgaa 2334
<210> 2
<211> 1289
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
cctgaggccg ccatccacgc cggttgagtc gcgttctgcc gcctcccgcc tgtggtgcct 60
cctgaactgc gtccgccgtc taggtaagtt taaagctcag gtcgagaccg ggcctttgtc 120
cggcgctccc ttggagccta cctagactca gccggctctc cacgctttgc ctgaccctgc 180
ttgctcaact ctacgtcttt gtttcgtttt ctgttctgcg ccgttacaga tccaagctgt 240
gaccggcgcc tacgctagac gccaccatgg tcgacatgga ctaccccacc ctgctgctgg 300
ccctgctgca tgtgtacaga gccctgtgtg aagaggtgct gtggcacacc tctgtgccct 360
ttgctgagaa catgagcctg gagtgtgtgt accctagcat gggcatcctg acccaagtgg 420
agtggttcaa gattggcaca cagcaagaca gcattgccat cttcagcccc acccatggca 480
tggtgatcag aaagccctat gctgagagag tgtacttcct caacagcacc atggctagca 540
acaacatgac cctgttcttc agaaatgcct ctgaggatga tgtgggctac tacagctgca 600
gcctgtacac ctacccccaa ggcacctggc agaaggtgat ccaagtggtg cagtctgaca 660
gctttgaggc tgctgtgcct agcaacagcc acattgtgtc tgagcctggc aagaatgtga 720
ccctgacctg tcagcctcag atgacctggc ctgtgcaagc tgtgagatgg gagaagattc 780
agcctagaca gattgacctg ctgacctact gcaacctggt gcatggcaga aacttcacaa 840
gcaagttccc tagacagatt gtgagcaact gcagccatgg cagatggtct gtgattgtga 900
tccctgatgt gacagtgtct gactctggcc tgtacagatg ctacctgcaa gcctctgctg 960
gggagaatga gacctttgtg atgagactga cagtggctga gggcaagaca gacaatcagt 1020
acaccctgtt tgtggctggg ggcacagtgc tgctgctgct gtttgtgatc agcatcacca 1080
ccatcattgt gatcttcctg aacagaagaa gaagaagaga gagaagagac ctgttcacag 1140
agagctggga cacacagaag gcccccaaca actacagaag ccccatcagc acatctcagc 1200
ccaccaatca gagcatggat gacacaagag aggacatcta tgtgaactac cccaccttca 1260
gcagaagacc caagacaaga gtgcatatg 1289
<210> 3
<211> 119
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 3
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Tyr Trp Arg Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 4
<211> 107
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 4
Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp
1 5 10 15
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val
20 25 30
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
35 40 45
Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser
50 55 60
Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala Glu
65 70 75 80
Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
Claims (6)
1. A construction method of a recombinant plasmid for co-expressing NFAT and human DNAM-1 protein comprises the following steps:
(1) inserting the gene sequence encoding NFAT-reverse into the vector PB-713B-1SpeI andBstBcarrying out small extraction preparation of endotoxin-free plasmids between the I enzyme cutting sites to obtain PB-NFAT-Luc-713 plasmids; the sequence of the NFAT-reverse coding gene is shown in SEQ ID NO. 1;
(2) after codon optimization is carried out on the gene sequence of human DNAM-1, the gene sequence is inserted into the PB-NFAT-Luc-713 plasmidBsu36I andNdecarrying out large-scale extraction preparation of endotoxin-free plasmids between the I enzyme digestion sites to obtain recombinant plasmids PB-NFAT-Luc-CD 226; the gene sequence of the codon-optimized human DNAM-1 is shown in SEQ ID NO. 2.
2. The recombinant plasmid PB-NFAT-Luc-CD226 constructed by the method of claim 1.
3. A recombinant cell Jurkat-NFAT-Luc-CD226 comprising the recombinant plasmid PB-NFAT-Luc-CD226 of claim 2.
4. The use of the recombinant cell Jurkat-NFAT-Luc-CD226 of claim 3 for the detection and/or screening of anti-human DNAM-1 antibodies for non-disease diagnostic and therapeutic purposes.
5. A method for detecting and/or screening anti-human DNAM-1 antibodies for non-disease diagnosis and treatment purposes, comprising the steps of: the recombinant cell Jurkat-NFAT-Luc-CD226 of claim 3 and the anti-human DNAM-1 antibody are mixed and incubated for 4h, the cell suspension is collected, the supernatant is centrifuged, and the mixture is mixed with the ONE-Lite substrate and 1: 1, mixing the mixed solution, measuring a fluorescence signal on an enzyme labeling instrument, and detecting and/or screening the anti-human DNAM-1 antibody according to the intensity of the fluorescence signal.
6. The anti-human DNAM-1 antibody obtained by screening according to the method of claim 5, wherein the amino acid sequence of the heavy chain variable region of the anti-human DNAM-1 antibody is shown as SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 4.
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