CN115786274B - Tilapia Ig lambda monoclonal antibody and application thereof - Google Patents
Tilapia Ig lambda monoclonal antibody and application thereof Download PDFInfo
- Publication number
- CN115786274B CN115786274B CN202211340017.2A CN202211340017A CN115786274B CN 115786274 B CN115786274 B CN 115786274B CN 202211340017 A CN202211340017 A CN 202211340017A CN 115786274 B CN115786274 B CN 115786274B
- Authority
- CN
- China
- Prior art keywords
- tilapia
- lambda
- antibody
- monoclonal antibody
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000276707 Tilapia Species 0.000 title claims abstract description 59
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 26
- 210000003719 b-lymphocyte Anatomy 0.000 claims abstract description 18
- 230000003053 immunization Effects 0.000 claims abstract description 5
- 238000002649 immunization Methods 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims description 8
- 108060003951 Immunoglobulin Proteins 0.000 claims description 6
- 102000018358 immunoglobulin Human genes 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000011835 investigation Methods 0.000 claims 1
- 210000002966 serum Anatomy 0.000 abstract description 17
- 229960005486 vaccine Drugs 0.000 abstract description 11
- 241000193985 Streptococcus agalactiae Species 0.000 abstract description 10
- 230000001900 immune effect Effects 0.000 abstract description 6
- 102000036639 antigens Human genes 0.000 abstract description 5
- 108091007433 antigens Proteins 0.000 abstract description 5
- 239000000427 antigen Substances 0.000 abstract description 4
- 238000011156 evaluation Methods 0.000 abstract description 4
- 238000012827 research and development Methods 0.000 abstract description 2
- 208000015181 infectious disease Diseases 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 29
- 239000000243 solution Substances 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 24
- 241001529936 Murinae Species 0.000 description 22
- 102000004169 proteins and genes Human genes 0.000 description 15
- 239000012528 membrane Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 13
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 241000251468 Actinopterygii Species 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 239000002033 PVDF binder Substances 0.000 description 10
- 239000006180 TBST buffer Substances 0.000 description 10
- 210000003734 kidney Anatomy 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 238000012546 transfer Methods 0.000 description 9
- 241000283707 Capra Species 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 8
- 238000001962 electrophoresis Methods 0.000 description 8
- 238000001976 enzyme digestion Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 235000013861 fat-free Nutrition 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 235000013336 milk Nutrition 0.000 description 7
- 239000008267 milk Substances 0.000 description 7
- 210000004080 milk Anatomy 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 210000000265 leukocyte Anatomy 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 210000005259 peripheral blood Anatomy 0.000 description 5
- 239000011886 peripheral blood Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 4
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000012160 loading buffer Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 3
- 238000011725 BALB/c mouse Methods 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 241000276701 Oreochromis mossambicus Species 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 241000252498 Ictalurus punctatus Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 208000010824 fish disease Diseases 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- -1 pH 9.6) Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of bioengineering, and relates to a tilapia Ig lambda monoclonal antibody and application thereof, wherein the monoclonal antibody is secreted by hybridoma cell strain (CCTCC NO: C202137), and the antibody can specifically recognize Ig lambda chain in serum and Ig lambda in tissues + B cells, further using mouse anti Iglambda monoclonal antibodies to detect the antibody level of the streptococcus agalactiae surface antigen Sip in tilapia serum after streptococcus agalactiae infection, and the result shows that the antibody level of the streptococcus agalactiae serum for recognizing Sip is obviously increased, which indicates that the antibody can be used for detecting the antibody level of tilapia after immunization, and lays a foundation for the research and development of tilapia vaccines and the evaluation of immune effects.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a tilapia Ig lambda monoclonal antibody and application thereof.
Background
Tilapia (Oreochromis mossambicus) is a worldwide economic fish, and has the advantages of fast growth, good meat quality, strong reproductive capacity, wide adaptability and the like. But in recent years, diseases frequently occur, and streptococcosis caused by streptococcus agalactiae (Streptococcus agalactiae) is particularly prominent, and causes great economic loss to farmers. Because of the continuous use of antibiotics, the problem of antibiotic drug resistance is increasingly serious, and the sustainable development of aquaculture industry is seriously affected, and the vaccine can activate the immune system of fish, improve the disease resistance of organisms against specific pathogens, is an important immunoprophylaxis means of streptococcicosis of tilapia, and is also the main flow direction of prevention and control of fish diseases in the future. However, the method for evaluating the immune effect of the vaccine is often complicated in process, takes long time and lacks an evaluation standard, and based on monoclonal antibodies of fish immunoglobulin (Ig), the method for evaluating the immune effect of the vaccine by analyzing the level change rule of antibodies (antigen-specific Ig secreted into body fluid) in blood and mucus of vaccinated fish is provided with a powerful tool for establishing an evaluation standard system of the immune effect of the tilapia vaccine. Therefore, the research on the Ig monoclonal antibody of the tilapia is helpful for in-depth understanding of the humoral immune response rule of the tilapia, and can help to establish a tilapia antibody analysis and detection method, and then establish a method for evaluating the immune effect of the vaccine based on the tilapia antibody level, so as to promote the development of epidemic disease prevention and control technologies such as tilapia immune diagnosis, vaccination and the like.
Teleosts contain 3 immunoglobulins, namely: igM, igT and IgD, of which IgM and IgT are the main functions of antibodies. The existing research shows that teleost IgM mainly plays a role in systemic immunity, while IgT mainly plays a role in mucosal immunity. Many vaccines, particularly oral and infusion vaccines, are capable of activating both systemic and mucosal immunity of the body, and therefore, to accurately evaluate the vaccination efficacy of such vaccines, it is necessary to detect the antibody titres of IgM and IgT simultaneously, which requires antibodies to both classes of Ig simultaneously, and both preparation and detection are time-consuming and laborious.
Fish Ig light chains include three subtypes igλ, igσ, and igκ, and monoclonal antibodies against fish Ig light chains are more difficult to obtain than monoclonal antibodies against heavy chains because the constant region of fish Ig light chains has only one Ig domain, while the heavy chain constant region contains at least 4 Ig domains, and thus fewer hybridoma cells against light chains are involved in the monoclonal antibody screening process. Up to now, only monoclonal antibodies against channel catfish igσ and igκ have been reported to recognize fish Ig light chains, and no monoclonal antibodies against igλ have been reported.
Disclosure of Invention
Aiming at the problem of tilapia Ig lambda monoclonal antibody deletion in the prior art, the invention provides a hybridoma cell secreting a mouse anti-tilapia Ig lambda monoclonal antibody, wherein the preservation number of the hybridoma cell strain is as follows: CCTCC NO: C202137.
Another object of the present invention is to provide a tilapia Ig lambda monoclonal antibody, which is prepared from the following materials in storage number: the CCTCC NO is secreted by the hybridoma cell of C202137.
The final object of the invention is to provide the application of the monoclonal antibody secreted by the hybridoma cells in detecting tilapia immunoglobulin Ig lambda.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the applicant uses purified natural tilapia IgM as antigen to immunize mice, and performs screening of positive B cell, WB, monoclonal antibody output and other conditions, finally a hybridoma cell strain is obtained, monoclonal antibodies secreted by the hybridoma cell strain specifically recognize Ig lambda, the monoclonal antibodies have high yield and high titer, and the hybridoma cells are preserved in China center for type culture collection (China) for 01 month 27 of 2021, and are classified and named: hybridoma cell lines 4-6, accession numbers: CCTCC NO: C202137, address: university of martial arts in chinese.
The protection scope of the invention also comprises: the preservation number is as follows: the hybridoma cell of CCTCC NO: C202137 secretes the obtained monoclonal antibody.
The application of the monoclonal antibody secreted by the hybridoma cells in detecting tilapia immunoglobulin Ig lambda comprises that the monoclonal antibody can be used for detecting tilapia Ig lambda + Sorting B cells or analyzing Ig lambda of tilapia + B cell ratio, or antibody levels after immunization of tilapia.
In the above applications, the hybridoma cells or monoclonal antibodies secreted by the hybridoma cells can be prepared into related detection kits. Compared with the prior art, the invention has the beneficial effects that:
the invention aims at the deficiency of the Ig lambda monoclonal antibody of tilapia mossambica in the prior artThe natural tilapia IgM is used as an antigen to immunize a mouse and a hybridoma cell strain 4-6 capable of specifically secreting mouse anti-tilapia Ig lambda monoclonal antibody is obtained by screening, the monoclonal antibody secreted by the hybridoma cell strain 4-6 can specifically identify tilapia Ig lambda and does not react with other proteins (including H chains of IgM) of the tilapia, the titer is high, and the titer after purification is 128000, so that the method can be used for specifically detecting the tilapia Ig lambda and the antibody level after the tilapia is immunized, has high specificity and sensitive response, and lays a foundation for research and development of tilapia vaccines and evaluation of immune effects. In addition, the mouse anti-tilapia Ig lambda monoclonal antibody can be used for specifically detecting tilapia Ig lambda + B cell for realizing Ig lambda of tilapia + Isolation and functional study of B cells.
The monoclonal antibody of Iglambda shared by IgM and IgT can be used for accurately evaluating the vaccination effect of the vaccine, and the time and labor are saved and the result is accurate and reliable.
Drawings
FIG. 1 is a graph showing the results of SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions of analysis of purified IgM heavy (Igμ) and light (Igk, igλ and/or Igσ) chains in tilapia serum, followed by coomassie brilliant blue staining in example 1 according to the present invention;
FIG. 2 is a graph showing the results of detection of purified L chain and multimers of tilapia IgM by immunoblotting (Western blot) under reducing and non-reducing conditions, respectively, using murine anti-tilapia Ig lambda monoclonal antibodies in example 3 of the present invention;
FIG. 3 shows the result of Coomassie brilliant blue staining of light chain Igkappa and Iglambda constant region proteins of tilapia prokaryotic expression in example 4 of the present invention, followed by recognition of Iglambda only using murine anti-tilapia Iglambda monoclonal antibodies;
FIG. 4 is a graph showing the results of sorting tilapia head and kidney leukocytes using murine anti-tilapia Ig lambda monoclonal antibody in example 5, and classifying lymphocytes into Ig lambda Positive (Positive) and Negative (Negative) populations;
FIG. 5 is a graph showing the results of quantitative analysis of CD4, CD8 and IgT, igM1, igM 2H chain gene and L chain gene (Igkappa, iglambda) expression in Iglambda Positive (Positive) cells and Negative (Negative) cells of the head and kidney of the selected tilapia according to example 5 of the present invention, wherein the values shown are mean.+ -. Standard deviation of four fish with beta-actin as an internal reference, and ". Times." indicates significant difference (p < 0.05).
FIG. 6 shows the flow cytometry detection of leukocytes in tilapia head kidney, spleen and peripheral blood in example 6 of the present invention. A: results of co-staining of cephalic and renal leukocytes with murine anti-tilapia IgM heavy chain monoclonal antibody (purchased from Aquatic Diagnostics company) and murine anti-tilapia iglambda monoclonal antibody; b: the result of co-staining spleen leucocytes with a murine anti-tilapia IgM heavy chain monoclonal antibody and a murine anti-tilapia Ig lambda monoclonal antibody; c: the result of co-staining peripheral blood leucocytes with a murine anti-tilapia IgM heavy chain monoclonal antibody and a murine anti-tilapia Ig lambda monoclonal antibody; d: igM in head kidney, spleen and peripheral blood + Igλ + B cells account for total IgM + Proportion of B cells and Ig lambda + IgM - B cells account for total iglambda + Proportion of B cells. The values shown are mean ± standard deviation of four fish, "×" indicates that the difference is significant (p<0.05)。
FIG. 7 shows ELISA detection results of specific antibodies against the surface immunogenic protein Sip of Streptococcus agalactiae in serum of control (PBS) and infected (S.agalactiae) tilapia in example 7 according to the present invention.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention. The technical scheme of the invention is conventional in the field unless specifically stated otherwise, and the reagents or materials are commercially available unless specifically stated otherwise.
Example 1:
purification of natural tilapia IgM
(1) Separation and purification of IgM in tilapia serum
1) Preparation of an affinity column: 5mg Protein A+G Agarose (from Biyun biotechnology Co., ltd.) was suspended in 5mL 10mM PBS-20mM EDTA (pH 7.4), then taken into a PD-10 purification column, and passed through the equilibrated column with 5 column volumes of 10mM PBS-20mM EDTA (pH 7.4);
2) Co-incubation: the well-balanced column material is resuspended by 5mL of 10mM PBS-20mM EDTA (pH 7.4), then added into tilapia serum diluted by PBS according to the proportion of 1:1, and incubated at 4 ℃ in a co-rotating way for overnight, so that antibodies in the serum are fully combined with Protein A+G, and the mixture is transferred into a PD-10 purification column for flow through after incubation;
3) Neutral elution: washing with 10 times of column volume of 10mM PBS-20mM EDTA (pH 7.4) to remove nonspecifically adsorbed impurity proteins;
4) Acid elution: 500. Mu.L per tube of eluted IgM was collected using 100mM glycine (pH 2.5) as eluent, and immediately pH was adjusted to neutral with 1M Tris;
5) Desalting: desalting the eluted IgM on PD-10 desalting column, replacing IgM in PBS solution, and temporarily storing at 4deg.C.
(3) Detection of purified IgM of tilapia
SDS-PAGE was used to measure the purification effect of IgM in serum. Preparing 12% Tris-glycine polyacrylamide lower layer separating gel and 5% upper layer concentrating gel. Taking a collected sample and a reduced protein loading buffer solution (containing beta-mercaptoethanol) according to a proportion, heating the sample in a metal bath at a constant temperature of 100 ℃ for 10min, carrying out instantaneous centrifugation, loading the sample for electrophoresis, wherein the electrophoresis voltage is 120V, the electrophoresis time is 1h, and after electrophoresis, staining the sample by using coomassie brilliant blue staining solution, decolorizing the sample by using boiling water, and then imaging the sample.
The results of the purification of IgM from tilapia are shown in FIG. 1, and the IgM is separated into two main bands, namely IgH chain and IgL chain under the reducing condition, wherein the heavy chain is about 75kD, and the light chain is about 27kD, and the sizes are consistent with the molecular weight ranges of H chain and L chain of other teleosts IgM reported before.
Example 2:
preparation of murine anti-tilapia Ig lambda monoclonal antibody
(1) Immunization of mice
Healthy female BALB/c mice of 7-8 weeks old are immunized 3 times with affinity purified tilapia IgM, each time is separated by 2 weeks, freund's complete adjuvant is added for the first time, freund's incomplete adjuvant is added for the second and third times, and blood collection is started after the second time to detect the antibody production.
(2) Cell fusion
Feeder cells are prepared 3-5 days before fusion, and BALB/c mouse peritoneal macrophages are taken according to a conventional method and spread in a 96-well cell culture plate for later use. The antibody positive mice were sacrificed by cervical scission, spleen cells were aseptically taken, the spleen cells and SP2/0 myeloma cells were fused with PEG-4000 at a ratio of 5:1, and the fused cells were plated on prepared feeder cells.
(3) Screening and cloning of positive hybridoma cell strains
Coating polystyrene plates with purified tilapia IgM, collecting cell culture supernatants of 10 th to 15 th days after fusion, and performing indirect ELISA detection; and (3) performing amplification culture on the detected positive hybridoma cells, cloning the positive hybridoma cells by a limiting dilution method, cloning for at least 3 rounds, and timely freezing the cloned positive hybridoma cells.
The applicant has sent the hybridoma cells screened by the invention to China center for type culture collection (China, accession number: CCTCC NO: C202137, classified naming: hybridoma cell lines 4-6, addressed: chinese, university of martial arts, martial arts.
(4) Mass production of monoclonal antibodies
The liquid paraffin was injected intraperitoneally into healthy BALB/c mice of about 10 weeks of age, 0.5 mL/mouse, and 5X 10 per pretreated mice after 1 week 7 And (3) extracting ascites when the abdomen of the mouse is extremely swelled after 10-14 days, extracting 1 time at intervals of 2d, centrifuging the extracted ascites at 10000r/min for 10min, taking the supernatant, and purifying by affinity chromatography to obtain the mouse anti-tilapia Ig lambda monoclonal antibody, wherein the titer of the monoclonal antibody after purification is 1:128000.
Example 3:
identification of murine anti-tilapia Ig lambda monoclonal antibodies
1 antibody subclass
Coating purified tilapia IgM into ELISA plate, 4Incubating overnight at a temperature; washing 3 times with washing solution, adding 100 mu L of mouse anti-tilapia Ig lambda monoclonal antibody into each hole, and incubating for 1h at 37 ℃; after 3 washes, diluted HRP-labeled goat anti-mouse IgG1, igG2a, igG2b, igG3, igA, and IgM monoclonal antibodies were added, respectively, and 100 μl per well was incubated for 1h at 37 ℃; after 3 washes, 100 μl of TMB color development solution (available from bi yun biotechnology limited) was added to each well and incubated at 37 ℃ for 30min in the absence of light; then 50. Mu.L of stop solution (2M H) was added to each well 2 SO 4 ) The reaction was terminated. The absorbance was measured with an enzyme-labeled instrument at 450nm wavelength, and the HRP-labeled goat anti-mouse Ig subclass used for the positive wells was used as the antibody class to be measured. The identification result shows that the subclass of the Ig lambda monoclonal antibody of the mouse anti-tilapia is IgG2a.
2Western blot detection
(1) Preparation of serum samples
Serum of healthy tilapia mossambica is prepared, diluted by PBS for 20 times, and 1/4 volume of 5 Xreduction type SDS-PAGE loading buffer solution and Non-reduction type SDS-PAGE loading buffer solution are respectively added, the mixture is uniformly mixed by vortex, and the mixture is subjected to metal bath at 100 ℃ for 10min and is placed on ice for standby or frozen at-80 ℃.
(2)SDS-PAGE
Preparing 12% and 8% Tris-glycine polyacrylamide lower layer separating gel and 5% upper layer concentrating gel respectively. Loading samples for electrophoresis, wherein the electrophoresis voltage is 80V and 0.5h;120V,1h.
(3) Transfer film
SDS-PAGE gel was removed from the gel plate, soaked in pre-prepared transfer buffer (25 mM Tris,192mM glycine, 15% methanol), and PVDF membrane of 0.22 μm was cut to the appropriate size, soaked in methanol for 30s, and transferred into transfer buffer. The filter paper-PVDF membrane-glue-filter paper is arranged from bottom to top in the order of filter paper-PVDF membrane-glue-filter paper, so that each layer cannot have bubbles, and the protein on the SDS-PAGE gel is transferred onto the PVDF membrane after 300mA semi-drying transfer for 60 min.
(4) Closure
After the transfer was completed, PVDF membrane was transferred into TBST (25mM Tris,150mM NaCl,0.1% Tween-20) solution containing 5% skimmed milk powder, and incubated at room temperature for 2h.
(5) Incubation with primary antibody
The blocking solution was discarded, and the murine anti-tilapia Ig lambda monoclonal antibody was diluted to 2. Mu.g/mL with a TBST solution containing 5% nonfat milk powder and incubated overnight at 4 ℃.
(6) Second antibody incubation
The primary antibody solution was removed, the membrane was washed 3 times with TBST for about 10min each, and HRP-labeled goat anti-mouse IgG (H+L) antibody (purchased from Thermo Fisher Scientific company) was diluted 1:5000 with TBST solution containing 5% nonfat milk powder and incubated at room temperature for 1H.
(7) ECL color development
The secondary antibody solution was removed, the membrane was washed 3 times with TBST for 10min each, an appropriate amount of ECL color developing solution (purchased from Biosharp Co.) was prepared at 1:1, and the color developing solution was applied dropwise to the membrane, and after sufficient contact, analyzed using a chemiluminescent imager and photographed.
The recognition of IgM by murine anti-tilapia Ig lambda monoclonal antibody is shown in FIG. 2, and the monoclonal antibody recognizes L chain of IgM of tilapia with the size of about 27kD under the reducing condition. Under non-reducing conditions, the monoclonal antibodies can recognize both monomeric and multimeric forms of IgM. Example 4: murine anti-tilapia Ig lambda monoclonal antibodies recognize Ig lambda only
1. Prokaryotic expression
(1) Expression vector construction
The cDNA of the head and kidney of tilapia is used as a template, ecoR I and Not I restriction enzyme sites (Ig kappa F: ATA) are respectively added at the 5' ends of the forward and reverse primersGAATTCAGTGATACCCGTCCCATCCT，IgκR：ATAGCGGCCGCTCAGGACTGGGAACACTGGA；IgλF：ATAGAATTCTCCAGCCTCCCTCCTCCTG，IgλR：
ATAGCGGCCGCTTCAGTGGAACATTCTGACTTC), the constant regions Igkappa and Iglambda of tilapia are PCR amplified. The pET-32a plasmid and the PCR product were digested with EcoR I and Not I at 37℃for 1h. And (3) recovering the product after enzyme digestion, and connecting the vector after enzyme digestion with a gene sequence at 16 ℃ for 12 hours. And (3) recovering the product after enzyme digestion, and connecting the vector after enzyme digestion with a gene sequence at 16 ℃ for 12 hours. And (3) recovering the product after enzyme digestion, and connecting the vector after enzyme digestion with a gene sequence at 16 ℃ for 12 hours. And (3) recovering the product after enzyme digestion, and connecting the vector after enzyme digestion with a gene sequence at 16 ℃ for 12 hours.
(2) Transformation
The pET32 a-Igkappa and pET32 a-Iglambda recombinant plasmids (5 mu L) which are connected are transferred into DH5 alpha (50 mu L) competence, incubated on ice for 30min, thermally shocked for 90s at 42 ℃, immediately cooled on ice for 3min, 400 mu L sterilized LB culture medium is added, the mixture is placed in a shaking table at 37 ℃ for shaking for 45min at 100rpm, 150 mu L of bacterial liquid is sucked and coated on an LB plate containing Amp (100 mu g/mL), and after the bacterial liquid is dried, the bacterial liquid is placed in a constant temperature incubator at 37 ℃ in an inverted mode for 14h of culture.
(3) Positive plasmid selection
Single colonies on the plates were picked up to 400. Mu.L of LB liquid medium containing Amp (100. Mu.g/mL) with a sterile gun head, shake-cultured in a shaker at 37℃for 4h, 1. Mu.L of bacterial liquid was used as a template for PCR verification, and the resulting positive bacterial liquid was sent to the Programme (Wuhan) Biotechnology Co., ltd.
(4) Plasmid extraction
The bacterial liquid with correct sequencing result is expanded and cultured to 50mL LB liquid medium containing Amp (100 mug/mL), a constant temperature shaking table is used for culturing for 9h at 37 ℃ and 200rpm, bacterial cells are collected, and plasmids are extracted by using HiPure Plasmid Micro Kit rapid plasmid small extraction kit (purchased from Magen).
(5) Induction of expression
The extracted plasmid (5. Mu.L) was transferred into the expression strain BL21 (DE 3), for specific procedures reference (4). Positive clones were inoculated into 500mL LB liquid medium containing Amp (100. Mu.g/mL), incubated at 37℃with shaking table at 180rpm until the bacterial liquid OD600 = 0.6-0.8, IPTG was added to a final concentration of 1mM, protein expression was induced, shaking table at 37℃for 4h, and cells were collected.
(6) Protein purification
The collected cells were resuspended in PBS, and the cells were broken by a high-pressure cell breaker, and the broken cell suspension was centrifuged at 12000rcf for 30min at 4℃to collect the supernatant. The supernatant was incubated with Ni filler (available from Huisha Biotechnology Co., ltd.) at room temperature for 1h, washed with 120mL of PBS containing 50mM imidazole (pH=7.5), and the protein was eluted with PBS containing 300mM imidazole (pH=7.5), and subjected to SDS-PAGE gel electrophoresis to examine the purity of the protein. Selecting high-purity protein solution, centrifuging at 12000rcf and 4 ℃ for 15min, sucking the supernatant into a 2mL sterile syringe, ejecting bubbles, pushing the supernatant into a sample inlet of an AKTA protein purifier, purifying the protein by using a molecular sieve principle, and detecting the purity of the purified pET32 a-Igkappa and pET32 a-Iglambda recombinant proteins by SDS-PAGE (figure 3).
Western blot detection
(1) Preparation of protein samples
Purified pET32 a-Igkappa and pET32 a-Iglambda recombinant proteins are diluted to the same concentration, added with 5X reduction (Reducing) SDS-PAGE loading buffer, mixed uniformly by vortex, subjected to metal bath at 100 ℃ for 10min, and placed on ice for standby or frozen at-80 ℃.
(2)SDS-PAGE
Preparing 12% Tris-glycine polyacrylamide lower layer separating gel and 5% upper layer concentrating gel. Loading samples for electrophoresis, wherein the electrophoresis voltage is 80V and 0.5h;120V,1h.
(3) Transfer film
SDS-PAGE gel was removed from the gel plate, soaked in pre-prepared transfer buffer (25 mM Tris,192mM glycine, 15% methanol), and PVDF membrane of 0.22 μm was cut to the appropriate size, soaked in methanol for 30s, and transferred into transfer buffer. The proteins on the SDS-PAGE gel were transferred to the PVDF membrane by arranging the filter paper-PVDF membrane-gel-filter paper in the order from bottom to top, ensuring that each layer cannot have bubbles, and transferring the membrane to the PVDF membrane in 25V semi-dry mode for 15 min.
(4) Closure
After the transfer was completed, PVDF membrane was transferred into TBST (25mM Tris,150mM NaCl,0.1% Tween-20) solution containing 5% skimmed milk powder, and incubated at room temperature for 2h.
(5) Incubation with primary antibody
The blocking solution was discarded, and the murine anti-tilapia Ig lambda monoclonal antibody was diluted to 2. Mu.g/mL with a TBST solution containing 5% nonfat milk powder and incubated overnight at 4 ℃.
(6) Second antibody incubation
The primary antibody solution was removed, the membrane was washed 3 times with TBST for about 10min each, and HRP-labeled goat anti-mouse IgG (H+L) antibody (purchased from Thermo Fisher Scientific company) was diluted 1:5000 with TBST solution containing 5% nonfat milk powder and incubated at room temperature for 1H.
(7) ECL color development
The secondary antibody solution was removed, the membrane was washed 3 times with TBST for 10min each, an appropriate amount of ECL color developing solution (purchased from Biosharp Co.) was prepared at 1:1, and the color developing solution was applied dropwise to the membrane, and after sufficient contact, analyzed using a chemiluminescent imager and photographed. The results indicated that the murine anti-igλ monoclonal antibody recognizes only igλ, not igκ (fig. 3).
Example 5:
murine anti-tilapia Ig lambda monoclonal antibodies divide lymphocytes into positive and negative two populations
(1) Tilapia head and kidney leukocytes isolated by Percoll density gradient centrifugation were dispensed into flow tubes and resuspended in 2mL PBS containing 2% Fetal Bovine Serum (FBS) for about 10 8 A cell;
(2) Adding a mouse anti-tilapia Ig lambda monoclonal antibody, wherein the final concentration of the antibody is 2 mug/mL, incubating on ice for 60min, and uniformly mixing every 15min by vortex; (3) rinsing twice with 4mL PBS;
(4) Secondary antibody incubation, re-suspending with 1mL PBS containing 2% FBS, adding APC-labeled goat anti-mouse IgG (purchased from BioLegend company) at a final concentration of 2 μg/mL, incubating on ice for 30min in the absence of light, and vortex mixing once every 15 min;
(5) Wash twice with 4mL PBS in the dark;
(6) The cells were resuspended in 4mL PBS and sorted by flow cytometry after passing through the cell strainer;
(7) In the flow cell sorting process, preparing a 15mL centrifuge tube to collect sorted cells, and pre-filling about 5mL of PBS in the centrifuge tube;
(8) The sorted cells are collected by centrifugation, and the subsequent experimental operations such as RNA extraction, fluorescent quantitative PCR and the like are performed.
The results of flow cell sorting are shown in FIG. 4, and the murine anti-tilapia Ig lambda monoclonal antibody can divide tilapia head kidney lymphocytes into Ig lambda positive cells and negative cells. As shown in FIG. 5, the expression of the immune-related genes in these two cell populations was high in CD4 and CD8 genes in Ig lambda negative cells, while the expression level in positive cells was very low. In contrast, the H chain genes and L chain Igkappa and Iglambda genes of IgT, igM1, igM2 of immunoglobulins were expressed higher in positive cells than in negative cells, indicating that Iglambda antibodies recognized most IgM simultaneously + And IgT + B cells. Ig lambda expression in Ig lambda positive cellsThe levels were significantly higher than for iglambda negative cells.
Example 6:
IgM analysis by murine anti-tilapia Ig lambda monoclonal antibody + 、Igλ + And IgM + Igλ + Proportion of B cells
(1) White blood cells of head kidney, spleen and peripheral blood of tilapia, which were separated by Percoll density gradient centrifugation, were packed in flow tubes, and cell concentration was adjusted to 4X 10 with PBS containing 2% FBS 6 cells/mL;
(2) Split charging the cell suspension into 1.5mL EP tubes, 200 mu L each tube, adding a murine anti-tilapia IgM heavy chain monoclonal antibody (IgG 1 subtype) and a murine anti-tilapia Ig lambda monoclonal antibody (IgG 2a subtype) respectively, wherein the final concentration of the antibodies is 2 mu g/mL, incubating on ice for 45min, and vortex mixing once every 15 min;
(3) After washing twice with PBS containing 2% FBS, the mixture was resuspended to 200. Mu.L, and an APC-labeled goat anti-mouse IgG1 antibody (purchased from Jackson corporation) and a FITC-labeled goat anti-mouse IgG2a antibody (purchased from Jackson corporation) were added, respectively, and the final concentration of the antibodies was 2. Mu.g/mL, incubated on ice for 30min in the absence of light, and vortexed and mixed uniformly every 15 min;
(4) After washing twice with PBS containing 2% FBS, the suspension was resuspended to 200. Mu.L, and the suspension was examined by flow cytometry after passing through a cell filter.
The detection results are shown in FIG. 6, and IgM in the head and kidney and peripheral blood is shown according to the double-dyeing results + Igλ + B cells account for total IgM + About 30% of B cells, whereas the proportion in spleen is about 45%; igM in head kidney and peripheral blood - Igλ + B cells occupy Iglambda + About 45% of B cells, about 25% in spleen.
Example 7: ELISA detection of serum antibody level of tilapia
Tilapia used in the experiment has a weight of 350+/-50 g, and is fed to a culture base of aquatic university of Huazhong agricultural university, and after temporary culture for 2 weeks, the main experiment is started. Tilapia is randomly divided into two groups, 200 mu L of sterile PBS is injected into the abdominal cavity of a control group, and 200 mu L of 1 multiplied by 10 is injected into the abdominal cavity of an infected group 8 cfu/mL Streptococcus agalactiae, 200. Mu.L of sterile PBS was injected intraperitoneally into the control group after 21d, and 200. Mu.L of 1X 10 was injected intraperitoneally into the infected group 7 cfu/mL Streptococcus agalactiae secondaryAfter immunization, 10 fish were randomly drawn from the control and infected groups, respectively, and blood serum was prepared for ELISA detection as follows: (1) Diluting antigen protein (recombinant streptococcus agalactiae surface immunogenic protein Sip) to 10 μg/mL with coating buffer (0.05M carbonate buffer, pH 9.6), coating 100 μl protein per well of the elisa plate, and incubating overnight at 4 ℃;
(2) After washing 5 times with PBST (10 mM PBS,0.05% Tween-20), 250. Mu.L of a 5% nonfat dry milk in PBST solution was added to each well, and incubated at 37℃for 2 hours;
(3) After washing 5 times with PBST, control group serum and immune group serum were diluted 50-fold respectively, 100 μl was added to each well, PBS was used as a blank control, and incubated for 3h at 28 ℃;
(4) After washing 5 times with PBST, the murine anti-tilapia Ig lambda monoclonal antibody was diluted to 2. Mu.g/mL with a 5% nonfat dry milk in PBST, 100. Mu.L was added to each well and incubated for 1h at 37 ℃;
(5) After washing 5 times with PBST, HRP-labeled goat anti-mouse IgG (H+L) antibody (available from Thermo Fisher Scientific company) was diluted 1:2000 with a 5% nonfat dry milk in PBST solution, 100. Mu.L was added to each well, and incubated at 37℃for 1H;
(6) After washing 5 times with PBST, 100 mu L of TMB developing solution is added into each hole, and the color is developed for 30min at 37 ℃ in a dark place;
(7) mu.L of stop solution (2M H) was added to each well 2 SO 4 ) Stopping the reaction for 10min, mixing uniformly, and detecting the light absorption value by using an enzyme-labeled instrument at the wavelength of 450 nm.
The results of the assay are shown in FIG. 7, where the level of Sip-specific antibodies in the serum of the infected group was significantly higher than that in the serum of the PBS control group.
Claims (6)
1. A hybridoma cell line, wherein the hybridoma cell line has a deposit number of: CCTCC NO: C202137.
2. The monoclonal antibody secreted by the hybridoma cell line of claim 1.
3. Use of the hybridoma cell strain of claim 1 or the monoclonal antibody of claim 2 in preparing a kit for detecting tilapia immunoglobulin igλ.
4. The use according to claim 3 for the preparation of tilapia Ig lambda + B cell sorting kit.
5. The use according to claim 3 for the preparation of tilapia Ig lambda + B cell proportion analysis kit.
6. The use according to claim 3 for the preparation of a kit for detecting antibody levels after investigation of tilapia immunization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211340017.2A CN115786274B (en) | 2022-10-29 | 2022-10-29 | Tilapia Ig lambda monoclonal antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211340017.2A CN115786274B (en) | 2022-10-29 | 2022-10-29 | Tilapia Ig lambda monoclonal antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115786274A CN115786274A (en) | 2023-03-14 |
| CN115786274B true CN115786274B (en) | 2024-01-12 |
Family
ID=85434366
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211340017.2A Active CN115786274B (en) | 2022-10-29 | 2022-10-29 | Tilapia Ig lambda monoclonal antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115786274B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198741A (en) * | 2014-07-23 | 2014-12-10 | 福建省淡水水产研究所 | Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit |
| CN106834235A (en) * | 2016-12-26 | 2017-06-13 | 广西大学 | Anti- Tilapia mossambica IgM monoclonal antibody cell line and its screening technique and application |
| CN109852588A (en) * | 2018-12-24 | 2019-06-07 | 中国水产科学研究院珠江水产研究所 | The monoclonal antibody and its cell strain of a kind of anti-Tilapia mossambica Immunoglobulin IgM and application |
| CN112795543A (en) * | 2021-02-04 | 2021-05-14 | 华中农业大学 | Hybridoma cell strain, monoclonal antibody secreted by hybridoma cell strain and resisting grass carp IL-15R alpha and application of monoclonal antibody |
| CN116041526A (en) * | 2022-11-14 | 2023-05-02 | 华中农业大学 | Mouse anti-grass carp IgT monoclonal antibody, preparation method and application thereof |
| CN116218790A (en) * | 2022-09-30 | 2023-06-06 | 华东师范大学 | Hybridoma cell strain secreting monoclonal antibody against T lymphocyte surface membrane protein of tilapia mossambica as well as preparation method and application thereof |
-
2022
- 2022-10-29 CN CN202211340017.2A patent/CN115786274B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198741A (en) * | 2014-07-23 | 2014-12-10 | 福建省淡水水产研究所 | Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit |
| CN106834235A (en) * | 2016-12-26 | 2017-06-13 | 广西大学 | Anti- Tilapia mossambica IgM monoclonal antibody cell line and its screening technique and application |
| CN109852588A (en) * | 2018-12-24 | 2019-06-07 | 中国水产科学研究院珠江水产研究所 | The monoclonal antibody and its cell strain of a kind of anti-Tilapia mossambica Immunoglobulin IgM and application |
| CN112795543A (en) * | 2021-02-04 | 2021-05-14 | 华中农业大学 | Hybridoma cell strain, monoclonal antibody secreted by hybridoma cell strain and resisting grass carp IL-15R alpha and application of monoclonal antibody |
| CN116218790A (en) * | 2022-09-30 | 2023-06-06 | 华东师范大学 | Hybridoma cell strain secreting monoclonal antibody against T lymphocyte surface membrane protein of tilapia mossambica as well as preparation method and application thereof |
| CN116041526A (en) * | 2022-11-14 | 2023-05-02 | 华中农业大学 | Mouse anti-grass carp IgT monoclonal antibody, preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Monoclonal antibody against Nile tilapia (Oreochromis niloticus) IgM heavy chain: A valuable tool for detection and quantification of IgM and IgM+ cells;Janet Vel´azquez等;Fish and Shellfish Immunology;44-54页 * |
| Preparation of the monoclonal antibody against Nile tilapia Igλ and study on the Igλ+ B cell subset in Nile tilapia;Yan-Ling Song等;Fish and Shellfish Immunology;1-9页 * |
| 尼罗罗非鱼IgT重链基因的克隆与表达分析;李焕等;免疫学杂志;1067-1072页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115786274A (en) | 2023-03-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113150136B (en) | Preparation of novel coronavirus N protein monoclonal antibody | |
| CN105112375B (en) | Hybridoma cell strain ZJED0-02, anti-Ebola virus GP protein monoclonal antibody and its preparation and application | |
| CN108004215B (en) | Hybridoma cell strain, antibody produced by hybridoma cell strain and preparation method of antibody | |
| CN110551212A (en) | Preparation method and application of anti-GII.4 norovirus capsid protein VP1 and VLP (virus-like particle) monoclonal antibody | |
| CN115786274B (en) | Tilapia Ig lambda monoclonal antibody and application thereof | |
| CN116041526B (en) | Mouse anti-grass carp IgT monoclonal antibody, preparation method and application thereof | |
| CN112048483B (en) | Epitope of 1-type PAStV capsid protein, monoclonal antibody and preparation thereof | |
| CN109932504B (en) | Kit for detecting mandarin fish rhabdovirus | |
| CN114540377A (en) | Flavobacterium columnare VI type secretion system effector molecule, immune protein and application thereof | |
| CN113980908A (en) | Actinobacillus pleuropneumoniae ApxIV protein monoclonal antibody and blocking ELISA kit thereof | |
| CN113462654B (en) | Helicobacter hepaticum CdtB protein monoclonal antibody and hybridoma cell strain secretion and application thereof | |
| CN113980126B (en) | Pasteurella multocida toxin monoclonal antibody and blocking ELISA kit thereof | |
| CN116621976B (en) | Monoclonal antibody 2H7 against PDCoV nucleocapsid protein and application method thereof | |
| CN115856296B (en) | Monoclonal antibody for resisting shigella and application thereof in detection | |
| CN114702574B (en) | Recombinant antibody of single-chain variable region of white spot virus, preparation method and application thereof | |
| CN112458060B (en) | Monoclonal antibody of type 1 PAStV, preparation and ELISA application thereof | |
| CN114853894B (en) | Monoclonal antibody of toxoplasma gondii FtsH1 and application thereof | |
| CN117487006B (en) | Monoclonal antibody for resisting A-type sai virus, epitope and application | |
| CN112940085B (en) | BTV1 protective epitope polypeptide, specific recognition monoclonal antibody thereof, antibody secreting cell and application thereof | |
| CN116769019B (en) | ASFVp30 protein monoclonal antibody and application thereof | |
| CN116121197B (en) | Monoclonal antibody of anti-iridovirus SDDV isolate of yellow-fin sea bream and application thereof | |
| CN117887668A (en) | Hybridoma cell HB3 for detecting HIV-1p24 antigen and application thereof | |
| CN116444654A (en) | anti-African swine fever virus p72 protein monoclonal antibody, preparation method and application | |
| CN117551622A (en) | Hybridoma cell for detecting HIV-1p24 antigen, monoclonal antibody and application | |
| CN115850429A (en) | Antigen protein for inducing mouse Rhox10 protein polyclonal antibody, polyclonal antibody and preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |