CN114075552B - Hybridoma cell strain secreting anti-FGL 1 monoclonal antibody and application thereof - Google Patents

Hybridoma cell strain secreting anti-FGL 1 monoclonal antibody and application thereof Download PDF

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CN114075552B
CN114075552B CN202210057592.5A CN202210057592A CN114075552B CN 114075552 B CN114075552 B CN 114075552B CN 202210057592 A CN202210057592 A CN 202210057592A CN 114075552 B CN114075552 B CN 114075552B
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fgl1
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gly
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姜威
于永娟
张亚飞
谭佳佳
陆雯
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Meijie Transformation Medical Research Suzhou Co ltd
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Abstract

The invention provides a hybridoma cell strain secreting an anti-FGL 1 monoclonal antibody and application thereof, wherein the hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody is named as a mouse anti-human FGL1 monoclonal antibody hybridoma cell line MJ9-1 and a mouse anti-human FGL1 monoclonal antibody hybridoma cell line MJ9-4, and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation numbers are CGMCC No.23015 and CGMCC No.23016 respectively, and the preservation date is 2021 year, 7 month and 19 days. The anti-FGL 1 monoclonal antibody secreted by the hybridoma cell strain has good sensitivity and specificity, has good affinity with prokaryotic and eukaryotic FGL1 proteins, can be prepared into ELISA detection products, protein immunoblotting detection products and chemiluminescence detection products, and has practical application value.

Description

Hybridoma cell strain secreting anti-FGL 1 monoclonal antibody and application thereof
Technical Field
The invention belongs to the technical field of immunochemistry, tumor biology and cell biology, and particularly relates to a hybridoma cell strain secreting an anti-FGL 1 monoclonal antibody and application thereof.
Background
The up-regulated expression of Inhibitory Receptors (IRs) is important to balance co-stimulatory receptor activity and limit T cell activation, and may prevent autoimmunity, autoinflammation, and tissue damage. However, tumors can hijack the role of these immune checkpoints, producing a protective effect on the anti-tumor immune response induced by CD4+ and CD8+ T cells. Recent cancer immunotherapy approaches overcome immune tolerance by targeting inhibitory receptors and reactivate cytotoxic T cell responses to tumors, thereby reversing this failure.
Hepasocin, also called hepatocyte-derived fibrinogen-related protein 1 (HFREP-1) and fibrinogen-like protein 1 (FGL 1), is a liver-specific secreted protein, is a member of the EREP protein family, is mainly expressed in the liver, and is cloned from human hepatocellular carcinoma for the first time. The human fibrinogen Family (FREP) signaling pathway plays a key role in the development of cancer and in regulating immune cell function.
The human FGL1 protein is a secreted protein, and contains 312 amino acids in total, including a 22 amino acid signal peptide and a 290 amino acid mature protein. Lymphocyte activation gene 3 (LAG 3) Is an immunosuppressive receptor whose Ligand Is Major histocompatibility complex type II (MHC-II), and FGL1 Is independent of MHC-II and Is a Major functional Ligand of LAG3 (Wang et al, fibre-like Protein 1 Is a Major Immune inhibitor Ligand of LAG-3, Cell (2019), https:// doi. org/10.1016/j. cell.2018.11.010).
FGL1 is capable of inhibiting antigen-specific T cell activation, whose expression in the plasma of cancer patients is elevated indicating a poor prognosis of the treatment effect. Besides the role in cancer progression, FGL1 has a restoring function on hepatocytes, increases the expression level after partial hepatectomy and during liver regeneration, stimulates primary hepatocytes to take up 3H-thymidine, promotes hepatocyte proliferation, and has a protective effect on the liver damaged by rats with explosive liver failure (Yu, HT).et al(2009) j. biol. chem.284: 13335.). The above studies indicate that FGL1 plays an important role in liver regeneration and liver protection. Therefore, the preparation of the FGL1 antibody with high specificity and sensitivity and the FGL1 detection kit have important significance in clinical diagnosis.
At present, few antibodies and related kits are available on the market that can specifically and highly sensitively detect FGL 1. The provision of an anti-FGL 1 monoclonal antibody and a detection kit with strong specificity, high sensitivity and wide application range has become a problem to be solved urgently.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody and application thereof, wherein the hybridoma cell strain can secrete the monoclonal antibody with high sensitivity and strong specificity, has good affinity with FGL1 protein, and has important application value in related detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a hybridoma cell strain secreting an anti-FGL 1 monoclonal antibody, wherein the hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody is named as a mouse anti-human FGL1 monoclonal antibody hybridoma cell line MJ9-1, is preserved in the China general microbiological culture Collection center, has a preservation number of CGMCC No.23015 and a preservation date of 2021, 7 months and 19 days.
The invention provides a hybridoma cell strain secreting an anti-FGL 1 monoclonal antibody, which is named as a mouse anti-human FGL1 monoclonal antibody hybridoma cell line MJ9-4 and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.23016 and the preservation date of 2021, 7 months and 19 days.
In the invention, the hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody is obtained by taking human FGL1 protein as immunogen to immunize a mouse, fusing splenocytes of the immunized mouse with myeloma cells to form hybridoma cells and screening the hybridoma cells, and can secrete the monoclonal antibody with high sensitivity and good specificity.
In the invention, the immunogen is human FGL1 protein, and the amino acid sequence of the immunogen is shown in SEQ ID No. 1.
SEQ ID No: 1：
LEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVIHHHHHH。
In the invention, the screening method of the hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody comprises the following steps:
(1) constructing a eukaryotic expression vector, transiently expressing the eukaryotic expression vector in a 293T cell, transfecting the eukaryotic expression vector into a 293F cell to express after verifying that the protein can be expressed, collecting a culture and purifying to obtain FGL1 recombinant protein;
(2) and immunizing a mouse by using the obtained FGL1 recombinant protein, and taking spleen cells to fuse with myeloma cells after the serum titer is detected to be qualified. After culturing and screening, the hybridoma cell strain capable of stably secreting the anti-FGL 1 monoclonal antibody is obtained.
In a second aspect, the invention provides an anti-FGL 1 monoclonal antibody, wherein the anti-FGL 1 monoclonal antibody is secreted from the hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody of the first aspect.
In the invention, the subtype of the anti-FGL 1 monoclonal antibody is murine IgG 1.
In the invention, the monoclonal antibody secreted by the hybridoma cell strain with the preservation number of CGMCC No.23015 is named as MJ9-1, the sequences of the heavy chain CDR1, CDR2 and CDR3 are respectively shown as SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, the sequence of the heavy chain variable region is shown as SEQ ID No. 6, and the sequence of the heavy chain full length is shown as SEQ ID No. 7; the light chain CDR1, CDR2 and CDR3 have the sequences shown in SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10, respectively, the light chain variable region has the sequence shown in SEQ ID No. 11, and the light chain full-length sequence is shown in SEQ ID No. 12.
SEQ ID No: 3：GFTFSDGY；
SEQ ID No: 4：ISDGGSGT；
SEQ ID No: 5：AR；
SEQ ID No: 6：
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDGYMYWVRQTPEKRLEWVATISDGGSGTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARAATVTRWFAYWGQGTLVTVS；
SEQ ID No: 7：
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDGYMYWVRQTPEKRLEWVATISDGGSGTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARAATVTRWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTL GCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRD CGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTF RSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPED ITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK；
SEQ ID No: 8：YNINVW；
SEQ ID No: 9：KAS；
SEQ ID No: 10：QQGQSYP；
SEQ ID No: 11：
DIQMNQSPSSLSASLGDTITITCHASYNINVWLTWYQQKPGNIPKLLIYKASKLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGGGTKLEIK；
SEQ ID No: 12：
DIQMNQSPSSLSASLGDTITITCHASYNINVWLTWYQQKPGNIPKLLIYKASKLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
In the invention, the monoclonal antibody secreted by the hybridoma cell strain with the preservation number of CGMCC No.23016 is named as MJ9-4, the sequences of the heavy chain CDR1, CDR2 and CDR3 are respectively shown as SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15, the sequence of the heavy chain variable region is shown as SEQ ID No. 16, and the sequence of the heavy chain full length is shown as SEQ ID No. 17; the light chain CDR1, CDR2 and CDR3 have the sequences as shown in SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20, respectively, the light chain variable region has the sequence as shown in SEQ ID No. 21, and the light chain full-length sequence is shown in SEQ ID No. 22.
SEQ ID No: 13：GYIFTGGW；
SEQ ID No: 14：INPSTDGT；
SEQ ID No: 15：AR；
SEQ ID No: 16：
EVKLQQSGAELAKPGASVKMSCKASGYIFTGGWMHWVKQRPGQGLEWIGYINPSTDGTDYNQKFEDKATLTADKSSSTAYMQLSSLTSEDSAVYYCAREGYYGSSPYFDYWGQGTTLTVSS；
SEQ ID No: 17：
EVKLQQSGAELAKPGASVKMSCKASGYIFTGGWMHWVKQRPGQGLEWIGYINPSTDGTDYNQKFEDKATLTADKSSSTAYMQLSSLTSEDSAVYYCAREGYYGSSPYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMV TLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNS TFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFP EDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK；
SEQ ID No: 18：QSVSNN；
SEQ ID No: 19：YVS；
SEQ ID No: 20：QQSNSWP；
SEQ ID No: 21：
DIVITQTPATLSVTPGDSVSLSCRASQSVSNNLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLYTFGGGTKLEIK；
SEQ ID No: 22：
DIVITQTPATLSVTPGDSVSLSCRASQSVSNNLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
In a third aspect, the present invention provides a method for preparing the anti-FGL 1 monoclonal antibody of the second aspect, the method comprising:
culturing the hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody of the first aspect, and purifying to obtain the anti-FGL 1 monoclonal antibody.
In the invention, the preparation method of the monoclonal antibody comprises the following steps:
injecting the hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody into an abdominal cavity of a mouse, collecting ascites, purifying to obtain the anti-FGL 1 monoclonal antibody, and detecting.
In a fourth aspect, the invention provides a hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody of the first aspect and/or an application of the anti-FGL 1 monoclonal antibody of the second aspect in preparation of FGL1 expression detection products.
In a fifth aspect, the invention provides a product for detecting FGL1 expression, wherein the product for detecting FGL1 expression contains the anti-FGL 1 monoclonal antibody of the second aspect.
Preferably, the product comprises any one of a reagent, a reagent strip or a kit.
In the present invention, the anti-FGL 1 monoclonal antibody may be linked to a conjugate by chemical bond coupling, electrostatic adsorption or hydrophilic-hydrophobic adsorption, the conjugate including any one or a combination of at least two of horseradish peroxidase (HRP), Alkaline Phosphatase (AP), luciferase, Biotin (Biotin) or β -galactosidase.
In the present invention, the anti-FGL 1 monoclonal antibody can be used as a coating antibody or a detection antibody in the above products.
In a sixth aspect, the invention provides an enzyme-linked immunosorbent assay kit of FGL1, wherein the enzyme-linked immunosorbent assay kit of FGL1 contains the anti-FGL 1 monoclonal antibody of the second aspect.
Preferably, the FGL1 enzyme-linked immunosorbent assay kit detects the expression of FGL1 protein in a sample by a sandwich method or an indirect method by using an anti-FGL 1 monoclonal antibody secreted by a hybridoma cell line with the preservation number of CGMCC No.23015 as a capture antibody and an anti-FGL 1 monoclonal antibody secreted by a hybridoma cell line with the preservation number of CGMCC No.23016 coupled with biotin as a detection antibody.
In the invention, the anti-FGL 1 monoclonal antibody in the FGL1 enzyme-linked immunosorbent assay kit can form an antibody pair with other antibodies, and the FGL1 expression condition of a sample is qualitatively or quantitatively detected by a sandwich method.
In a preferred embodiment of the invention, when the monoclonal antibody secreted by the hybridoma with the clone number MJ9-1 is used as a capture antibody, and the monoclonal antibody secreted by the hybridoma with the clone number MJ9-4 coupled with biotin is used as a detection antibody, the amount of the target protein to be detected and the OD value of the reaction show good linear relation, and the sensitivity is high.
In a seventh aspect, the invention provides an FGL1 immunoblot detection kit, wherein the FGL1 immunoblot detection kit contains the anti-FGL 1 monoclonal antibody of the second aspect.
Preferably, the FGL1 immunoblot detection kit further comprises any one or at least two of SDS-PAGE gel, nitrocellulose membrane, blocking solution or enzyme-labeled secondary antibody.
In the invention, the anti-FGL 1 monoclonal antibody can detect eukaryotic and prokaryotic expressed FGL1 protein, and can be applied to immunoblot detection; the FGL1 immunoblotting detection kit can carry out qualitative or quantitative detection on a sample, and has clear bands, accurate results and good specificity.
In an eighth aspect, the present invention provides a FGL1 chemiluminescence detection kit, wherein the FGL1 chemiluminescence detection kit comprises the anti-FGL 1 monoclonal antibody of the second aspect.
In the invention, the anti-FGL 1 monoclonal antibody can be matched with antigen, and combined with chemiluminescence technology, qualitative or quantitative detection can be carried out on FGL1 protein in a sample.
In a ninth aspect, the present invention provides a method for detecting FGL1 expression, the method for detecting FGL1 expression comprises:
detecting a sample to be detected by using any one or a combination of at least two of the anti-FGL 1 monoclonal antibody of the second aspect, the FGL1 expression detection product of the fifth aspect, the FGL1 enzyme-linked immunosorbent assay kit of the sixth aspect, the FGL1 immunoblot detection kit of the seventh aspect or the FGL1 chemiluminescence detection kit of the eighth aspect.
In a tenth aspect, the invention provides the use of the anti-FGL 1 monoclonal antibody of the second aspect, the FGL1 expression detection product of the fifth aspect, the FGL1 enzyme-linked immunosorbent assay kit of the sixth aspect, the FGL1 immunoblotting detection kit of the seventh aspect, the FGL1 chemiluminescence detection kit of the eighth aspect, or the method for detecting FGL1 expression of the ninth aspect, in FGL1 protein expression detection.
Compared with the prior art, the invention has the following beneficial effects:
(1) the screened hybridoma cell strain MJ 9-1-MJ 9-5 secreting the anti-FGL 1 monoclonal antibody and the monoclonal antibody secreted by MJ 9-7-MJ 9-15 secreting the eukaryotic and prokaryotic recombinant expression can specifically recognize FGL1 protein obtained by eukaryotic and prokaryotic recombinant expression, and has strong specificity and high sensitivity, wherein the monoclonal antibody secreted by the hybridoma cell with the clone number MJ9-1 has optimal sensitivity and wide application;
(2) the anti-FGL 1 monoclonal antibody obtained by the invention can specifically identify FGL1 protein expressed by prokaryotic and eukaryotic expression systems by an indirect method in enzyme-linked immunosorbent assay (ELISA); the anti-FGL 1 monoclonal antibodies can be paired and used as capture antibodies or detection antibodies, the content of FGL1 protein can be detected in enzyme-linked immunosorbent assay and chemiluminescence assay, the specificity is strong, the sensitivity is high, the effect is good, when the monoclonal antibody secreted by the hybridoma with the clone number of MJ9-1 is used as the capture antibody and the monoclonal antibody secreted by the hybridoma with the clone number of MJ9-4 coupled with biotin is used as the detection antibody in ELISA assay, the optimal linear relation is presented between the amount of target protein to be detected and the OD value of the reaction, the sensitivity is high, and the detection limit of the eukaryotic expression FGL1 protein is as low as 1.95 ng/mL;
(3) the anti-FGL 1 monoclonal antibody obtained by the invention can specifically identify FGL1 protein expressed by a prokaryotic and eukaryotic expression system in Western Blot (Western Blot) detection, has clear bands, no non-specific background, strong specificity and high sensitivity;
(4) the monoclonal antibody resisting FGL1 obtained by the invention can be connected with conjugates such as horseradish peroxidase (HRP), Alkaline Phosphatase (AP), Luciferase (Luciferase), Biotin (Biotin) and beta-galactosidase, etc., so as to detect samples, thereby further expanding the application range.
Drawings
FIG. 1 is a photograph showing the SDS-PAGE electrophoresis result of purified eukaryotic expressed human FGL1 recombinant protein, wherein, lane M-standard protein molecular weight Marker, lane BSA-bovine serum albumin, lane R-human FGL1 protein under reduced state, and lane N-human FGL1 protein under non-reduced state;
FIG. 2 is a photograph showing the SDS-PAGE results of the anti-FGL 1 monoclonal antibody secreted from the hybridoma cell with clone number MJ9-1, wherein the lane M shows a standard protein molecular weight Marker, the lane BSA shows bovine serum albumin, the lane R shows an anti-FGL 1 monoclonal antibody in a reduced state, and the lane N shows an anti-FGL 1 monoclonal antibody in a non-reduced state;
FIG. 3 is a picture of indirect ELISA detection results of anti-FGL 1 monoclonal antibodies secreted by hybridoma cell strain clones MJ 9-1-MJ 9-5, MJ 9-7-MJ 9-10 and MJ 9-12-MJ 9-15;
FIG. 4 is a scattergram showing the results of detecting FGL1 protein with gradient concentration by a double antibody sandwich ELISA method using purified MJ9-1 secreted monoclonal antibody and biotin-labeled MJ9-4 secreted monoclonal antibody;
FIG. 5 is a picture of the detection results of purified MJ9-1 and MJ9-4 secreted monoclonal antibodies on FGL1 proteins from different expression sources, wherein, the lane M-standard protein molecular weight Marker, the lane 2-eukaryotic expression FGL1 protein, the lane 3-negative control BSA, and the lane 4-prokaryotic expression FGL1 protein; lane 6-eukaryotic FGL1 protein, lane 7-negative control BSA, lane 8-prokaryotic FGL1 protein, wherein lanes 2-4 were detected using MJ9-4 secreted monoclonal antibody, and lanes 6-8 were detected using MJ9-1 secreted monoclonal antibody.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1
This example prepares eukaryotic expressed human FGL1 antigen by the following steps:
the amino acid sequence of the extracellular region (UNIPROT ID: Q08830, Leu23-Ile 312) of the human FGL1 protein is obtained, 6 histidine tags are added at the C end of the protein, and a signal peptide suitable for the secretion of eukaryotic protein is added at the N end of the protein to form a recombinant protein, and the sequence is shown as SEQ ID No: 1. The target gene of the coding recombinant protein is connected with an expression vector PTT5 by utilizing a homologous recombination method to construct an expression plasmid PTT5-FGL 1.
SEQ ID No: 1：
LEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVIHHHHHH。
Transient expression of the constructed eukaryotic expression plasmid in 293T cells, collecting cell expression supernatant after 48h, verifying the expression condition of the protein by ELISA, and confirming the successful expression of the protein by experimental results.
The glycerol strain containing the target plasmid is inoculated in 200mL LB culture medium, shake culture is carried out for 16 h at 37 ℃ and 200rpm, plasmid extraction and sequencing are carried out, and the plasmid sequence is ensured to be correct. Plasmid DNA was transiently transfected into 293F cells by protein expression in 293F cells according to the instructions of Expi293 cell expression System kit (cat. No.: A14635) of Thermofish, and cell culture was harvested at 96h, centrifuged at 10000rpm for 30min, and the liquid was filtered through a 0.45 μm disposable sterile filter to obtain a culture medium supernatant.
Human FGL1 protein was affinity purified by AKTA protein purification instrument, the purified protein was replaced by buffer solution and then detected by SDS-PAGE electrophoresis, the molecular weight and purity of the purified protein were determined, the loading amount was 1. mu.g, the results are shown in FIG. 1.
The theoretical molecular total amount of human FGL1 protein expressed by eukaryotic cells is 35kD, and as can be seen from the figure, the size of the band of the purified FGL1 protein is consistent with the theoretical molecular weight of the protein under the condition of reducing buffer solution, the position of the band is correct, and the purity is more than 95%; in the non-reducing buffer, the protein is in a dimer state due to the cysteine and disulfide bonds contained in the protein, and the molecular weight is about 70 kD.
Example 2
This example provides a prokaryotic expression of human FGL1 protein antigen, which is prepared as follows:
the sequence of the extracellular domain (UNIPROT ID: Q08830, Leu23-Ile 312) of the human FGL1 protein is connected with an expression vector pCold TF (Takara, Code No: 3365) by a homologous recombination method to construct an expression cloning vector Pold TF-FGL1, so as to form a complete TF-FGL1 fusion protein, wherein the amino acid sequence of the complete fusion protein is shown as SEQ ID No: 2.
SEQ ID No:2：
MNHKVHHHHHHMQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAGLEVLFQGPSAGLVPRGSGGIEGRHMLEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVI。
Prokaryotic expression plasmids were constructed, and after correct sequencing, BL21 (DE 3) strain was transformed, coated with ampicillin resistant plates, and cultured for 12 h at 37 ℃ in an inverted manner. Selecting a single colony, carrying out shake culture, inducing by adopting IPTG (isopropyl thiogalactoside) with the final concentration of 0.1 mM, crushing thalli, collecting supernatant, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis, and verifying whether protein is expressed and molecular weight, wherein the protein is soluble expressed in the supernatant after the thalli are crushed and the molecular size is also in accordance with the expectation.
Inoculating the strain into LB culture medium containing ampicillin, culturing at 37 deg.C and 200rpm, raising OD value of the strain to 0.5, performing low temperature induced expression with 0.1 mM IPTG at 16 deg.C for 18 h, and centrifuging at 10000rpm to collect thallus after the induced expression. After the bacteria breaking treatment is carried out by an ultrasonic crusher, centrifugation is carried out for 30min at 10000rpm, and the bacteria breaking supernatant is filtered by a filter with the diameter of 0.45 mu m to obtain a sample to be purified. And (3) carrying out affinity chromatography purification by using a Ni column (NI sepharose High performance, GE, cat No. 17-5268-01) to obtain prokaryotic expression FGL1 human antigen, and verifying the activity of the protein by ELISA.
Example 3
This example prepares hybridoma cell lines secreting anti-FGL 1 monoclonal antibodies, which includes the following steps:
(1) immunization
A4-6-week-old female Balb/c mouse (Cavens hundred-grid (Suzhou) model animal research Co., Ltd.) is immunized by eukaryotic expression human FGL1 recombinant protein, 20 muL of blood is collected in an orbit before immunization, and serum is obtained after centrifugation to serve as a negative control.
The first immunization dose is that 50 mug of human FGL1 recombinant protein is injected into each mouse, the human FGL1 recombinant protein is diluted to 200 mug by physiological saline, the diluted human FGL recombinant protein is mixed with equal volume of Freund's complete adjuvant, and abdominal subcutaneous multi-point injection is carried out after full emulsification. The subcutaneous boosting immunization is carried out once every 14 days, the dose is 25 mug of human FGL1 recombinant protein injected into each mouse, the human FGL1 recombinant protein is diluted to 200 mug by physiological saline, the diluted human FGL1 recombinant protein is mixed with equal volume of Freund incomplete adjuvant, and the mixture is injected after full emulsification. And (3) taking blood from the orbit 7 days after the 3 rd boosting immunization, detecting the titer of serum antibodies by using ELISA, performing impact immunization on the mice meeting the requirement, injecting 50 micrograms of human FGL1 recombinant protein into each mouse at a dosage, diluting the recombinant protein to a volume of 100 micrograms by using physiological saline, performing intraperitoneal injection, and fusing after 3 days.
(2) Fusion
Freshly picked mouse spleens were crushed on a cell sieve, filtered, mixed with SP2/0 cells at a ratio of 3:1, centrifuged at 1000rpm for 5min at room temperature, and the medium was discarded. The centrifuge tube containing the centrifuged, mixed cells was placed in a 37 ℃ warm water bath, and 1mL of polyethylene glycol solution (Sigma, P7181-5X 5 mL) was added slowly while agitating the cells. After standing in a water bath for 1min, 10mL of serum-free DMEM medium (Hyclone, SH30243.01B) was slowly added thereto, and the mixture was centrifuged at 1000rpm for 5min, and the supernatant was discarded.
Serum-containing DMEM medium was added according to the total number of cells, and the cells were seeded into 96-well cell culture plates to have a cell density of 1X 105Inoculating 150 muL of the strain per well, placing the strain at 37 ℃ and 5% CO2Culturing in an incubator. After 24 h 50 μ L of HAT (Gibco, 21060017) -containing medium was added. And then changing the liquid every 3-5 days.
(3) Screening
And when the cell density reaches 50%, taking the supernatant, and screening the hybridoma cell strain secreting the anti-FGL 1 antibody by using an indirect ELISA method. The indirect ELISA method was performed as follows:
the detection plate is coated with 100 muL of human FGL1 recombinant protein with the concentration of 0.5 mug/mL, and the mixture is kept stand for 1h at 37 ℃. After washing 4 times with PBST wash, 100. mu.L of culture supernatant of fused cells was added to each ELISA well, and immune mouse serum (diluted 1: 10000) was used as a positive control, and SP2/0 culture supernatant was used as a negative control, and the mixture was allowed to stand at 37 ℃ for 1 hour. After 4 washes with PBST, 100. mu.L of horseradish peroxidase HRP-labeled affinity purified goat anti-mouse IgG (H + L) F (ab')2 fragment (brand: Jackson, cat # 115-036-plus 003, diluted 1:5000 using 1% BSA) was added to each well and incubated at 37 ℃ for 1H. After washing 4 times with PBST, TMB (Solarbio, PR 1210-2X 250 mL) was added for color development, and the OD at 450nm was measured. OD450If the value is greater than 0.5, the clone is determined to be positive.
And screening subclones of the obtained positive cell clone strain by adopting a limiting dilution method.
(4) Establishment of hybridoma cell strain
After 3 times of subcloning and indirect ELISA screening, 14 hybridoma cell strains which specifically identify human FGL1 protein and stably secrete monoclonal antibodies are obtained, and the clone numbers are MJ9-1, MJ9-2, MJ9-3, MJ9-4, MJ9-5, MJ9-7, MJ9-8, MJ9-9, MJ9-10, MJ9-11, MJ9-12, MJ9-13, MJ9-14 and MJ9-15 in sequence.
Example 4
This example prepares an anti-FGL 1 monoclonal antibody and performs subtype identification by the following steps:
(1) preparation of ascites
Taking hybridoma cell strain secreting mouse anti-human FGL1 monoclonal antibody in logarithmic growth phase, washing with serum-free culture medium, resuspending, and adjusting density to 5 × 105one/mL.
Mice were sensitized with paraffin oil, then the suspended cells were injected intraperitoneally, and ascites were collected 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites in the middle layer was carefully aspirated, collected in a centrifuge tube, stored at 4 ℃ and used for antibody purification.
(2) Purification of monoclonal antibodies
Monoclonal antibodies were purified from ascites fluid using Hi Trap protein G FF (GE Healthcare, 17061802) affinity chromatography packing and AKTA protein purification apparatus, according to the instructions.
The equilibration solution was 20mM phosphate buffer (pH 7.0), the eluent was 100mM glycine solution (pH 2.7), and the neutralization solution was 1M Tris-HCl solution (pH 8.5). The purification method comprises the following specific steps:
a sample was loaded by washing 1mL of the purification column with 10mL of deionized water and then with 10mL of the equilibration solution. The column was washed with 10mL of the equilibration solution, and finally the elution of the antibody was performed with 10mL of the eluent, and the neutralization solution was immediately added to bring the pH to 7.0. The liquid flow rate for all steps was 1 mL/min.
The antibody was subjected to SDS-PAGE to determine the purity of the antibody, and the concentration was determined by a Nanodrop protein concentration meter. The loading amount of SDS-PAGE was 1. mu.g, and the results of detection of the anti-FGL 1 monoclonal antibody secreted from the hybridoma cell with clone number MJ9-1 are shown in FIG. 2. As can be seen, the purified anti-FGL 1 monoclonal antibody had a band size consistent with that expected, a purity of > 95%, and was amenable to subsequent experiments.
The detection results of monoclonal antibodies secreted by other clone-numbered hybridoma cells are similar to MJ9-1, and are not described herein for brevity.
(3) Monoclonal antibody subtype identification
Diluting goat anti-mouse IgG (SL 200101, Solai Bo Ou Biotechnology Co., Ltd. in Hangzhou) antibody with PBS (pH7.4) to a concentration of 1 mug/mL, adding 100 mug L to each well, and incubating overnight at 4 ℃;
emptying the liquid, washing the liquid for 5 times by using PBST containing 0.05% Tween-20, adding 300 mu L of sealing liquid into each hole, sealing the hole for 1h at 37 ℃, emptying the liquid, and washing the liquid for 5 times by using PBST;
adding 100 mu L of hybridoma cell culture supernatant into each well, incubating for 1h at 37 ℃, emptying liquid, and washing for 5 times by PBST;
100 μ of LHRP-labeled goat anti-mouse IgM (Jackson, 115-;
adding 100 mu L of substrate TMB solution into each hole, developing for 5min at room temperature, and measuring OD value at the wavelength of 450 nm.
The results show that the 14 hybridoma cells secrete anti-FGL 1 monoclonal antibody subtypes which are MouseIgG 1.
Example 5
In this embodiment, the specificity of the purified anti-FGL 1 monoclonal antibody is detected by indirect ELISA, and the used coating antigen is human FGL1 protein expressed by eukaryotic expression system and prokaryotic expression system, and the steps are as follows:
respectively coating a detection plate with 100 muL of human FGL1 recombinant protein (eukaryotic expression protein and prokaryotic expression protein) with the concentration of 0.5 mug/mL, standing for 1h at 37 ℃, washing for 4 times with PBST washing liquid, then sealing the ELISA detection plate with 5% BSA, standing for 1h at 37 ℃, and washing for 4 times with PBST;
diluting the antibody with 1% BSA to a final concentration of 0.5 mug/mL, respectively taking 100 mug L diluted antibodies (hybridoma MJ 9-1-MJ 9-5, MJ 9-7-MJ 9-10 and MJ 9-12-MJ 9-15 secreted anti-FGL 1 monoclonal antibodies in the invention) to be added into detection holes, taking immune mouse serum (diluted according to a ratio of 1: 10000) as a positive control (P), taking 1% BSA as a negative control (N), and standing for 1h at 37 ℃;
after washing 4 times with PBST, 100 μ LHRP goat anti-mouse IgG antibody (1% BSA diluted 1: 5000) was added to each well, incubated at 37 ℃ for 1h, washed 4 times with PBST, and then TMB was added for color development to determine the OD value at 450 nm.
The results are shown in fig. 3, and the results show that the monoclonal antibodies secreted by the remaining hybridoma cells show strong positive and good antibody effects in detecting the human FGL1 proteins from different expression sources, except that the OD value of the monoclonal antibody secreted by MJ9-10 is low and the ability to recognize FGL1 protein is poor.
Based on the results of the tests, the monoclonal antibodies secreted by MJ9-1, MJ9-4, MJ9-5, MJ9-8 and MJ9-12 were further selected to be compared with the commercial monoclonal antibody Abnova monoclonal antibody 2A4 (cat No. H00002267-M01), Abnova monoclonal antibody 2C7 (cat No. H00002267-M06) and Santa Cruz monoclonal antibody A-8 (cat No. sc-514057) in ELISA experiments. Eukaryotic expression human FGL1 recombinant protein is coated according to the concentration of 0.5 mug/mL, and the concentration of a primary antibody is 500ng/mL, 250ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 10ng/mL, 5ng/mL and 0 in sequence. The dilution ratio of the secondary antibody HRP goat anti-mouse IgG is 1:5000, and the rest experimental steps are the same as above. The results are shown in Table 1.
TABLE 1
Figure 651867DEST_PATH_IMAGE001
As shown in Table 1, the monoclonal antibodies secreted by MJ9-1, MJ9-4, MJ9-5, MJ9-8 and MJ9-12 can bind to human FGL1 protein expressed in eukaryotic cells, and the A-8 antibody can specifically recognize human FGL1 protein. MJ9-1 and MJ9-5 secreted monoclonal antibodies with a sensitivity superior to the A-8 antibody at a primary antibody concentration of 5 ng/mL.
According to the detection results of this example, the commercial antibodies 2a4 and 2C7 did not bind to the eukaryotic expressed human FGL1 protein in the ELISA assay. However, according to the relevant descriptions in the commercial specifications, 2a4 and 2C7 both can specifically bind to GST-FGL1 protein, and the sensitivity is very high, and the reasons why the results of this example are inconsistent with the description are presumed to be as follows:
in the embodiment, an indirect ELISA method is adopted for detection, the antigen protein is directly coated on a detection plate, and the antigen coating may coat the epitopes recognized by 2A4 and 2C7 on the inner side, so that the epitopes cannot be exposed and cannot be combined with antibodies; 2A4 and 2C7 are detected by a double-antibody sandwich ELISA method, antibody of GST-FGL1 protein is coated on the detection plate, the GST-FGL1 protein is not directly contacted with the detection plate, more antigenic epitope is exposed, and therefore the corresponding antigenic epitope can be combined with 2A4 and 2C7 and successfully develops color;
secondly, the eukaryotic FGL1 protein in the embodiment is added with a histidine tag, so that the molecular weight is small and the influence on the structure of the antigen is small; 2A4 and 2C7 are GST-FGL1 fusion proteins, and the GST tag is large (26 kD), so that the GST tag may affect the structure of the antigen itself. The FGL1 protein used in the detection of this example does not carry a GST tag, and it may have a difference in spatial structure from the FGL1 protein used in the specifications of 2a4 and 2C7, and further, the epitope may be changed, resulting in a difference in affinity of antibodies recognizing different epitopes to the antigen.
Example 6
This example performs an ELISA antibody pairing experiment between anti-FGL 1 monoclonal antibodies, as follows:
and (3) monoclonal antibody pairing screening: for 14 kinds of monoclonal antibodies, 14X 14 groups of monoclonal antibodies are obtained. The optimal pairing monoclonal antibody pair is obtained by primary pairing screening through a chemiluminescence detection method, and the pairing detection optimization is carried out through an ELISA experiment on the monoclonal antibodies secreted by hybridoma MJ9-1 and MJ 9-4.
The experimental steps are as follows:
(1) coating: diluting the capture antibody with PBS (pH7.4) to a final concentration of 2 mug/mL, adding 100 mug L to each well, incubating for 1h at 37 ℃, discarding the liquid, and washing 5 times with PBST;
(2) and (3) sealing: preparing 5% BSA by using PBST, adding 300 muL into each hole, incubating for 1h at 37 ℃, discarding liquid, and washing for 5 times by using PBST;
(3) adding an antigen: diluting eukaryotic expression human FGL1 protein with 1% BSA to gradient concentration, adding 100 muL into each well, incubating for 1h at 37 ℃, discarding liquid, and washing with PBST for 5 times;
(4) primary antibody incubation: diluting a biotin-coupled detection antibody with 1% BSA to a final concentration of 0.25 mug/mL, adding 100 mug L to each well, incubating at 37 ℃ for 1h, discarding the liquid, and washing 5 times with PBST;
(5) adding 100 mu LHRP marked streptavidin into each hole, and incubating for 1h at 37 ℃; discard the liquid and wash 5 times with PBST; 100 mu LTMB (Solarbio, PR 1210-2X 250 mL) is added into each well for color development, the mixture is incubated for 5min in a dark place at room temperature, 50 mu L of 1mol/L sulfuric acid solution is added into each well to stop the reaction, and the OD value at 450nm is measured on a microplate reader.
Through optimization, the monoclonal antibody secreted by the monoclonal antibody with the concentration of 8 mug/mL and the clone number of MJ9-1 is determined to be used as a capture antibody, and the monoclonal antibody coupled biotin secreted by the monoclonal antibody with the concentration of 0.2 mug/mL and the clone number of MJ9-4 is used as a detection antibody, so that the detection mode of optimal antibody pairing is realized, and the optimal linear relation is presented between the amount of the target protein to be detected and the OD value of the reaction.
Evaluation of the sensitivity of the partner antibody:
the antibody secreted by MJ9-1 and MJ9-4 is used as a pairing antibody, the coating concentration of the first monoclonal antibody (MJ 9-1 secretion) is 8 mug/mL, the detection concentration of the biotin-labeled second monoclonal antibody (MJ 9-4 secretion) is 0.2 mug/mL, the FGL1 protein solution of eukaryotic expression with gradient concentration is detected, and the detection result is shown in Table 2. The table data is collated, and the result is shown in fig. 4. As shown in Table 2 and FIG. 4, the detection sensitivity of the paired antibodies MJ9-1 and MJ9-4 to the eukaryotic FGL1 protein is 1.95ng/mL, and the protein concentration and the OD value detected by ELISA are in a linear relationship within the range of 1.95-31.15 ng/mL.
TABLE 2
Figure 466239DEST_PATH_IMAGE002
Example 7
The embodiment provides an FGL1 enzyme-linked immunosorbent assay kit, wherein the FGL1 enzyme-linked immunosorbent assay kit contains anti-FGL 1 monoclonal antibodies secreted by clone numbers MJ9-1 and MJ 9-4;
the FGL1 enzyme-linked immunosorbent assay kit takes antibodies secreted by MJ9-1 and MJ9-4 as paired antibodies, wherein the monoclonal antibody secreted by MJ9-1 is taken as a capture antibody, the monoclonal antibody secreted by MJ9-4 coupled with biotin is taken as a detection antibody, and the expression of FGL1 protein in a sample is detected by a sandwich method.
Example 8
The embodiment provides an FGL1 immunoblotting detection kit, and the FGL1 immunoblotting detection kit contains the anti-FGL 1 monoclonal antibody, SDS-PAGE gel, nitrocellulose membrane, a confining liquid and an enzyme-labeled secondary antibody;
wherein, the anti-FGL 1 monoclonal antibody is a monoclonal antibody secreted by hybridoma MJ9-1 or MJ 9-4.
Example 9
This example used the FGL1 immunoblot detection kit prepared in example 8 for detection with the following steps:
taking eukaryotic expression FGL1 protein (molecular weight: 35 kD) and prokaryotic expression TF-FGL1 protein (molecular weight: 86.3 kD) as samples to be detected, adding reducing buffer solution, and boiling for 10 min;
preparing 12% SDS-PAGE gel, loading, and performing electrophoresis at 100V until the electrophoresis is finished;
preparing 1 Xmembrane transferring solution, and transferring the nitrocellulose membrane and the gel together under the voltage of 100V for 60 min;
putting the nitrocellulose membrane in PBST sealing solution containing 5% skimmed milk powder, sealing overnight at 4 ℃;
after washing with PBST, the anti-FGL 1 monoclonal antibody is diluted with 1% skimmed milk powder to a final concentration of 0.5. mu.g/mL, the nitrocellulose membrane is placed in an antibody diluent and incubated for 60min at room temperature, and the commercial FGL1 monoclonal antibody A-8 is used as a control and diluted to a final concentration of 0.5. mu.g/mL;
after being washed by PBST, the goat anti-mouse IgG (H + L) F (ab')2 fragment (brand: Jackson, cat # 115-;
exposure was performed using a ChemiDoc. TM.MP imaging system.
The loading amount, the antibody used and the detection procedure are shown in Table 3, wherein the total loading amount of the protein is shown in Table 3, and the loading volume is 10. mu.L.
TABLE 3
Figure 711538DEST_PATH_IMAGE003
The electrophoresis results are shown in FIG. 5. As can be seen from the figure, in the detection using MJ9-1 secretion antibody, the immunoblot band was clear when the loading amount of the protein for eukaryotic expression of FGL1 protein was 1. mu.g, the loading amount of the protein for prokaryotic expression of FGL1 protein was 1. mu.g, and the first antibody dilution concentration was 0.5. mu.g/mL. In the detection using MJ9-4 secretion antibody, the immunoblot band is clear when the loading amount of the protein for eukaryotic expression of FGL1 protein is 0.05. mu.g, the loading amount of the protein for prokaryotic expression of FGL1 protein is 0.5. mu.g, and the dilution concentration of the primary antibody is 0.5. mu.g/mL.
The experimental result shows that the monoclonal antibodies secreted by two hybridoma cells with clone numbers MJ9-1 and MJ9-4 have excellent effect in an immunoblotting experiment, can detect FGL1 proteins from different expression sources, and have correct strip size, good specificity and no non-specific background. Under the two optimized conditions, the brightness and the width of the strips are consistent, and the antibody sensitivity of MJ9-4 is slightly higher than that of MJ 9-1.
In this example, the primary antibody was used at a concentration of 0.5. mu.g/mL, and the monoclonal antibodies secreted by MJ9-1 and MJ9-4 could be detected accurately, whereas the lanes using the A-8 antibody had no bands, and the reason for this was presumed to be as follows: the primary antibody used in this application was used at a concentration of 0.5. mu.g/mL, which is lower than the working concentration of 200. mu.g/mL disclosed in the A-8 specification, resulting in less antigen-protein bound antibody on the nitrocellulose membrane to develop normally. The monoclonal antibody in the application can also be detected at the use concentration of 0.5 mu g/mL, and the sensitivity of the monoclonal antibody secreted by MJ9-1 and MJ9-4 in the application is further shown to be better than that of the control antibody A-8.
Example 10
In the embodiment, a hybridoma cell strain MJ9-1 secreting an anti-FGL 1 monoclonal antibody is preserved, named as a mouse anti-human FGL1 monoclonal antibody hybridoma cell line MJ9-1, in 2021, 7 months and 19 days, at China general microbiological culture Collection center (CGMCC) with the address of No. 3 institute No. 1 Xilu-Shi-1 of the morning-Yang district in Beijing, the postal code 100101 and the preservation number of CGMCC No. 23015.
Example 11
In the embodiment, a hybridoma cell strain MJ9-4 secreting an anti-FGL 1 monoclonal antibody is preserved, named as a mouse anti-human FGL1 monoclonal antibody hybridoma cell line MJ9-4, in 2021, 7 months and 19 days, at China general microbiological culture Collection center (CGMCC) with the address of No. 3 institute Xilu 1 of Xingyang district, Beijing, and the preservation number of the hybridoma cell line is CGMCC No. 23016.
In conclusion, the hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody provided by the invention has high sensitivity and good specificity, has good affinity with FGL1 proteins from different sources, can be applied to ELISA, protein immunoblotting, chemiluminescence and other related detection, and has wide application prospects.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> Mejie transformation medical research (Suzhou) Co., Ltd
<120> hybridoma cell strain secreting anti-FGL 1 monoclonal antibody and application thereof
<130> 2022
<160> 22
<170> PatentIn version 3.3
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Asp Lys Gly Asp Glu Asn Thr Val Ile Asp Leu Gly Ser Lys Arg Gln
515 520 525
Tyr Ala Asp Cys Ser Glu Ile Phe Asn Asp Gly Tyr Lys Leu Ser Gly
530 535 540
Phe Tyr Lys Ile Lys Pro Leu Gln Ser Pro Ala Glu Phe Ser Val Tyr
545 550 555 560
Cys Asp Met Ser Asp Gly Gly Gly Trp Thr Val Ile Gln Arg Arg Ser
565 570 575
Asp Gly Ser Glu Asn Phe Asn Arg Gly Trp Lys Asp Tyr Glu Asn Gly
580 585 590
Phe Gly Asn Phe Val Gln Lys His Gly Glu Tyr Trp Leu Gly Asn Lys
595 600 605
Asn Leu His Phe Leu Thr Thr Gln Glu Asp Tyr Thr Leu Lys Ile Asp
610 615 620
Leu Ala Asp Phe Glu Lys Asn Ser Arg Tyr Ala Gln Tyr Lys Asn Phe
625 630 635 640
Lys Val Gly Asp Glu Lys Asn Phe Tyr Glu Leu Asn Ile Gly Glu Tyr
645 650 655
Ser Gly Thr Ala Gly Asp Ser Leu Ala Gly Asn Phe His Pro Glu Val
660 665 670
Gln Trp Trp Ala Ser His Gln Arg Met Lys Phe Ser Thr Trp Asp Arg
675 680 685
Asp His Asp Asn Tyr Glu Gly Asn Cys Ala Glu Glu Asp Gln Ser Gly
690 695 700
Trp Trp Phe Asn Arg Cys His Ser Ala Asn Leu Asn Gly Val Tyr Tyr
705 710 715 720
Ser Gly Pro Tyr Thr Ala Lys Thr Asp Asn Gly Ile Val Trp Tyr Thr
725 730 735
Trp His Gly Trp Trp Tyr Ser Leu Lys Ser Val Val Met Lys Ile Arg
740 745 750
Pro Asn Asp Phe Ile Pro Asn Val Ile
755 760
<210> 3
<211> 8
<212> PRT
<213> Artificial sequence
<400> 3
Gly Phe Thr Phe Ser Asp Gly Tyr
1 5
<210> 4
<211> 8
<212> PRT
<213> Artificial sequence
<400> 4
Ile Ser Asp Gly Gly Ser Gly Thr
1 5
<210> 5
<211> 2
<212> PRT
<213> Artificial sequence
<400> 5
Ala Arg
1
<210> 6
<211> 118
<212> PRT
<213> Artificial sequence
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Gly
20 25 30
Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Gly Gly Ser Gly Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Ala Ala Thr Val Thr Arg Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser
115
<210> 7
<211> 443
<212> PRT
<213> Artificial sequence
<400> 7
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Gly
20 25 30
Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Gly Gly Ser Gly Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Ala Ala Thr Val Thr Arg Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr
115 120 125
Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu
130 135 140
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp
145 150 155 160
Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser
180 185 190
Pro Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys
210 215 220
Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr
245 250 255
Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
260 265 270
Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg
275 280 285
Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile
290 295 300
Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn
305 310 315 320
Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
325 330 335
Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu
340 345 350
Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe
355 360 365
Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala
370 375 380
Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr
385 390 395 400
Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly
405 410 415
Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His
420 425 430
Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440
<210> 8
<211> 6
<212> PRT
<213> Artificial sequence
<400> 8
Tyr Asn Ile Asn Val Trp
1 5
<210> 9
<211> 3
<212> PRT
<213> Artificial sequence
<400> 9
Lys Ala Ser
1
<210> 10
<211> 7
<212> PRT
<213> Artificial sequence
<400> 10
Gln Gln Gly Gln Ser Tyr Pro
1 5
<210> 11
<211> 107
<212> PRT
<213> Artificial sequence
<400> 11
Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Thr Ile Thr Ile Thr Cys His Ala Ser Tyr Asn Ile Asn Val Trp
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Lys Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 12
<211> 214
<212> PRT
<213> Artificial sequence
<400> 12
Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Thr Ile Thr Ile Thr Cys His Ala Ser Tyr Asn Ile Asn Val Trp
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Lys Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 13
<211> 8
<212> PRT
<213> Artificial sequence
<400> 13
Gly Tyr Ile Phe Thr Gly Gly Trp
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial sequence
<400> 14
Ile Asn Pro Ser Thr Asp Gly Thr
1 5
<210> 15
<211> 2
<212> PRT
<213> Artificial sequence
<400> 15
Ala Arg
1
<210> 16
<211> 121
<212> PRT
<213> Artificial sequence
<400> 16
Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Gly
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Thr Asp Gly Thr Asp Tyr Asn Gln Lys Phe
50 55 60
Glu Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Tyr Tyr Gly Ser Ser Pro Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 17
<211> 445
<212> PRT
<213> Artificial sequence
<400> 17
Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Gly
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Thr Asp Gly Thr Asp Tyr Asn Gln Lys Phe
50 55 60
Glu Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Tyr Tyr Gly Ser Ser Pro Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
115 120 125
Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro
180 185 190
Ser Ser Pro Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro
195 200 205
Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly
210 215 220
Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys
245 250 255
Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln
260 265 270
Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu
290 295 300
Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg
305 310 315 320
Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro
340 345 350
Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr
355 360 365
Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln
370 375 380
Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly
385 390 395 400
Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu
405 410 415
Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn
420 425 430
His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440 445
<210> 18
<211> 6
<212> PRT
<213> Artificial sequence
<400> 18
Gln Ser Val Ser Asn Asn
1 5
<210> 19
<211> 3
<212> PRT
<213> Artificial sequence
<400> 19
Tyr Val Ser
1
<210> 20
<211> 7
<212> PRT
<213> Artificial sequence
<400> 20
Gln Gln Ser Asn Ser Trp Pro
1 5
<210> 21
<211> 108
<212> PRT
<213> Artificial sequence
<400> 21
Asp Ile Val Ile Thr Gln Thr Pro Ala Thr Leu Ser Val Thr Pro Gly
1 5 10 15
Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asn Asn
20 25 30
Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Val Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr
65 70 75 80
Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu
85 90 95
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 22
<211> 215
<212> PRT
<213> Artificial sequence
<400> 22
Asp Ile Val Ile Thr Gln Thr Pro Ala Thr Leu Ser Val Thr Pro Gly
1 5 10 15
Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asn Asn
20 25 30
Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Val Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr
65 70 75 80
Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu
85 90 95
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala
100 105 110
Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser
115 120 125
Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp
130 135 140
Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val
145 150 155 160
Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met
165 170 175
Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser
180 185 190
Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys
195 200 205
Ser Phe Asn Arg Asn Glu Cys
210 215

Claims (11)

1. The hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody is named as a mouse anti-human FGL1 monoclonal antibody hybridoma cell line MJ9-1, is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23015 and has the preservation date of 2021, 7 months and 19 days.
2. The hybridoma cell strain secreting the anti-FGL 1 monoclonal antibody is named as a mouse anti-human FGL1 monoclonal antibody hybridoma cell line MJ9-4, is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23016 and the preservation date of 2021, 7 months and 19 days.
3. The anti-FGL 1 monoclonal antibody, wherein the anti-FGL 1 monoclonal antibody is secreted from the hybridoma cell line secreting anti-FGL 1 monoclonal antibody of claim 1 or 2.
4. A method for preparing an anti-FGL 1 monoclonal antibody according to claim 3, comprising:
culturing the hybridoma cell line secreting the anti-FGL 1 monoclonal antibody of claim 1 or 2, and purifying to obtain the anti-FGL 1 monoclonal antibody.
5. The hybridoma cell line secreting the anti-FGL 1 monoclonal antibody of claim 1 or 2 and/or the anti-FGL 1 monoclonal antibody of claim 3 for use in preparation of FGL1 expression detection products.
6. A FGL1 expression testing product, wherein the FGL1 expression testing product contains the anti-FGL 1 monoclonal antibody of claim 3;
the product comprises any one of a reagent, a reagent strip or a kit.
7. An enzyme-linked immunosorbent assay kit of FGL1, wherein the enzyme-linked immunosorbent assay kit of FGL1 contains the anti-FGL 1 monoclonal antibody of claim 3;
the FGL1 enzyme-linked immunosorbent assay kit takes an anti-FGL 1 monoclonal antibody secreted by a hybridoma cell strain with the preservation number of CGMCC No.23015 as a capture antibody and takes an anti-FGL 1 monoclonal antibody secreted by a hybridoma cell strain with the preservation number of CGMCC No.23016 coupled with biotin as a detection antibody, and the expression of FGL1 protein in a sample is detected by a sandwich method or an indirect method.
8. An FGL1 immunoblot detection kit, wherein the FGL1 immunoblot detection kit contains the anti-FGL 1 monoclonal antibody of claim 3;
the FGL1 immunoblot detection kit further comprises any one or at least two of SDS-PAGE gel, nitrocellulose membrane, confining liquid or enzyme-labeled secondary antibody.
9. An FGL1 chemiluminescence detection kit, which is characterized in that the FGL1 chemiluminescence detection kit contains the anti-FGL 1 monoclonal antibody of claim 3.
10. A method for detecting FGL1 expression for the purpose of non-disease diagnosis and/or treatment, wherein the method for detecting FGL1 expression for the purpose of non-disease diagnosis and/or treatment comprises:
detecting a sample to be detected by using any one or a combination of at least two of the anti-FGL 1 monoclonal antibody of claim 3, the FGL1 expression detection product of claim 6, the FGL1 enzyme-linked immunosorbent assay kit of claim 7, the FGL1 immunoblot detection kit of claim 8, or the FGL1 chemiluminescence detection kit of claim 9.
11. Use of the anti-FGL 1 monoclonal antibody of claim 3, the FGL1 expression detection product of claim 6, the FGL1 enzyme linked immunosorbent assay kit of claim 7, the FGL1 immunoblot assay kit of claim 8, the FGL1 chemiluminescence assay kit of claim 9, or the method of detecting FGL1 expression for non-disease diagnosis and/or treatment of claim 10 for detection of FGL1 protein expression for non-disease diagnosis and/or treatment.
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CN114560937B (en) * 2021-05-27 2022-07-05 山东博安生物技术股份有限公司 anti-FGL 1 antibody and application thereof
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