CN113234153B - Monoclonal antibodies for recognizing IFP35 family proteins and application of monoclonal antibodies in aspects of immune recognition and the like - Google Patents
Monoclonal antibodies for recognizing IFP35 family proteins and application of monoclonal antibodies in aspects of immune recognition and the like Download PDFInfo
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- CN113234153B CN113234153B CN202110373327.3A CN202110373327A CN113234153B CN 113234153 B CN113234153 B CN 113234153B CN 202110373327 A CN202110373327 A CN 202110373327A CN 113234153 B CN113234153 B CN 113234153B
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Abstract
The invention discloses a monoclonal antibody of 2 strains (the serial numbers of which are respectively mAb136 and mAb 218) capable of specifically recognizing Interferon inducible Protein IFP35 (Interferon-induced Protein 35) and NMI (N-Myc Interactor). The monoclonal antibodies have good stability and strong specificity, and by utilizing the monoclonal antibodies, IFP35 and NMI proteins in animal or human tissues can be accurately identified through immunoblot analysis, so that a basis is provided for relevant molecular biological research and clinical judgment of relevant diseases. The present patent provides the variable region sequences of these antibodies. The antibodies can be used as an immune recognition reagent, and can be used for preparing specific adsorption medical devices (capable of specifically adsorbing and removing or collecting target proteins and the like) and preparing antibody medicaments and the like.
Description
Technical Field
The invention belongs to the fields of life science, molecular biology and diagnostic reagents. Mainly relates to a batch of monoclonal antibodies for specifically recognizing IFP35 and NMI proteins and application thereof as an immunoblotting detection reagent.
Background
The IFP35 family protein is an interferon inducible protein, including IFP35 and NMI two family members. In the absence of interferon induction, the intracellular expression levels of IFP35 and NMI are very low, and the expression levels are obviously increased under the induction of IFN. IFP35 and NMI are reported to play a role in cancer development and progression, immune responses, and viral infections. When body tissues are infected or injured, IFP35 and NMI are rapidly released, natural immune response can be activated, and when the immune system is over-activated, cytokine storm can be triggered, so that systemic inflammatory diseases and even death can be caused. Sepsis, for example, is a serious inflammatory response complication in critically ill patients with infection, burn, trauma, poisoning, and the like. According to WHO statistics, about 3000 million people worldwide suffer sepsis, of which about 500 million people die with a high mortality rate. In these acute cases of infection, IFP35 and NMI are elevated in blood. Other chronic inflammatory and autoimmune diseases may also be associated with IFP35 and NMI in the blood or in body fluids. Therefore, clinically various detection reagents aiming at IFP35 and NMI can have important value for detecting the progress of diseases, and similar detection reagents are urgently needed in relevant medical basic research.
Immunoblotting and immunofluorescence are methods in which cells or biological tissue samples separated by gel electrophoresis or fixed cells or tissues are bound with specific antibodies and stained, and the location and depth of staining of the staining are analyzed to obtain information on the expression of a specific protein in the analyzed cells or tissues. The basis for the implementation of the immunoblotting analysis and the immunofluorescence method is a monoclonal antibody or a single domain antibody which needs to specifically recognize a target protein, a specific recognition polypeptide sequence or other specifically-combined small molecular compounds and the like.
The present invention is to solve the above problems and to provide monoclonal antibodies specifically recognizing IFP35 and NMI proteins and variable region sequences thereof. By using the monoclonal antibodies, specific bands or light spots can be exposed at the corresponding positions of IFP35 (35 kDa) and NMI (37 kDa) proteins by immunoblot analysis and immunofluorescence method.
Disclosure of Invention
The invention provides 2 monoclonal antibodies and variable region sequences thereof, wherein the monoclonal antibodies can specifically recognize IFP35 and NMI proteins through immunoblotting analysis. The specific information is as follows:
mAb136 heavy chain variable region amino acid sequence:
EVQLQQSGPELVKPGASVKISCKASGYAFSSSWLNWVKQRPGKGLEWIGRIYPGDGYTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARWGGFGYAMDYWGQGTSVTVSS(SEQ ID NO:1)
mAb136 heavy chain variable region CDR1: GYAFSSSW (SEQ ID NO: 3)
mAb136 heavy chain variable region CDR2: IYPGDGYT (SEQ ID NO: 4)
mAb136 heavy chain variable region CDR3: ARWGGFGYAMDY (SEQ ID NO: 5)
mAb136 light chain variable region amino acid sequence:
DIKMTQSHKFMSTSVGDRVSFTCKASQDVNTAVAWYQQKTGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSFPRTFGGGTKLEIK(SEQ ID NO:2)
mAb136 light chain variable region CDR1: QDVNTA (SEQ ID NO: 6)
mAb136 light chain variable region CDR2: SAS (SEQ ID NO: 7)
mAb136 light chain variable region CDR3: QQHYSFPRT (SEQ ID NO: 8)
mAb218 heavy chain variable region amino acid sequence:
EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWVKQRTEQGLEWIGKIDPEDGETKYAPKFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCTSLFDYWGQGTTLTVSS(SEQ ID NO:9)
mAb218 heavy chain variable region CDR1: GFNIKDYY (SEQ ID NO: 11)
mAb218 heavy chain variable region CDR2: IDPEDGET (SEQ ID NO: 12)
mAb218 heavy chain variable region CDR3: TSLFDY (SEQ ID NO: 13)
mAb218 light chain variable region amino acid sequence:
DIKMTQSPSSLSASLGERVSLTCRASQDIGNNLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPLTFGAGTKLEMK(SEQ ID NO:10)
mAb218 light chain variable region CDR1: QDIGNN (SEQ ID NO: 14)
mAb218 light chain variable region CDR2: ATS (SEQ ID NO: 15)
mAb218 light chain variable region CDR3: LQYASSPLT (SEQ ID NO: 16).
Detailed Description
Example 1: the method provides a method for transferring the protein to an NC membrane or other membrane carriers (such as PVDF and the like) after carrying out gel electrophoresis, and further detecting the target protein by a Western Blotting method. The method comprises the following specific steps: the recombinant expression IFP35 protein and the NMI protein are transferred to an NC membrane after being subjected to polyacrylamide gel electrophoresis, the NC membrane is incubated with 5% skim milk at 37 ℃ for 1 hour, and the NC membrane is washed 5 times with 1 XPBST solution for 5 minutes each time. The monoclonal antibody of the present invention was diluted to 1. Mu.g/mL using 1-cent BSA solution as a primary antibody, incubated with the NC membrane at 4 ℃ for 12 hours, and the NC membrane was washed 5 times with 1 XPBST solution for 5 minutes each. The NC membrane was incubated with HRP enzyme-labeled secondary antibody that specifically recognizes the murine antibody at 37 ℃ for 1 hour, and the NC membrane was washed with 1 XPBST solution 3 times for 5 minutes each. Specific bands can be shown on the position of IFP35 and NMI protein on the membrane by adding a chromogenic substrate of HRP and developing through a gel imaging system.
Drawings
FIG. 1 monoclonal antibodies recognize hIFP35 and hNMI in immunoblot experiments. The mAb136 and mAb218 can recognize hIFP35 and hNMI simultaneously, and show specific bands at 35kDa and 37kDa molecular weights respectively.
SEQUENCE LISTING
<110> Zhongshan university
<120> a batch of monoclonal antibodies for identifying IFP35 family proteins and application thereof in aspects of immune recognition and the like
<130> 20220614
<160> 16
<170> PatentIn version 3.3
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Claims (5)
1. A monoclonal antibody that specifically recognizes IFP35 and NMI of human origin, characterized by:
the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;
the heavy chain variable region comprises CDR1, CDR2, and CDR3;
the amino acid sequence of CDR1 of the heavy chain variable region is as follows: GYAFSSSW;
the amino acid sequence of the CDR2 of the heavy chain variable region is as follows: IYPGDGYT;
the amino acid sequence of CDR3 of the heavy chain variable region is as follows: ARWGGFGYAMDY;
the light chain variable region comprises CDR1, CDR2, and CDR3;
the amino acid sequence of the CDR1 of the light chain variable region is as follows: QDVNTA;
the amino acid sequence of the CDR2 of the light chain variable region is as follows: an SAS;
the amino acid sequence of the CDR3 of the light chain variable region is as follows: QQHYSFPRT.
2. The monoclonal antibody of claim 1, characterized in that:
the amino acid sequence of the heavy chain variable region is as follows: <xnotran> EVQLQQSGPELVKPGASVKISCKASGYAFSSSWLNWVKQRPGKGLEWIGRIYPGDGYTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARWGGFGYAMDYWGQGTSVTVSS; </xnotran>
The amino acid sequence of the light chain variable region is as follows: <xnotran> DIKMTQSHKFMSTSVGDRVSFTCKASQDVNTAVAWYQQKTGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSFPRTFGGGTKLEIK. </xnotran>
3. A monoclonal antibody that specifically recognizes IFP35 and NMI of human origin, characterized by:
the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;
the heavy chain variable region comprises CDR1, CDR2, and CDR3;
the amino acid sequence of CDR1 of the heavy chain variable region is as follows: GFNIKDYY;
the amino acid sequence of CDR2 of the heavy chain variable region is as follows: IDPEDGET;
the amino acid sequence of CDR3 of the heavy chain variable region is as follows: TSLFDY;
the light chain variable region comprises CDR1, CDR2, and CDR3;
the amino acid sequence of the CDR1 of the light chain variable region is as follows: QDIGNN;
the amino acid sequence of the CDR2 of the light chain variable region is as follows: ATS;
the amino acid sequence of the CDR3 of the light chain variable region is as follows: LQYASSPLT.
4. The monoclonal antibody of claim 3, characterized in that:
the amino acid sequence of the heavy chain variable region is as follows: <xnotran> EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWVKQRTEQGLEWIGKIDPEDGETKYAPKFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCTSLFDYWGQGTTLTVSS; </xnotran>
The amino acid sequence of the light chain variable region is as follows: <xnotran> DIKMTQSPSSLSASLGERVSLTCRASQDIGNNLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPLTFGAGTKLEMK. </xnotran>
5. Use of the monoclonal antibody according to any one of claims 1 to 4 for the preparation of a reagent for the specific detection of IFP35 and NMI proteins of human origin.
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| WO2020200186A1 (en) * | 2019-04-01 | 2020-10-08 | 中山大学 | Diagnosis and treatment for chronic inflammation and virus infection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2017528129A (en) * | 2014-08-22 | 2017-09-28 | インスティチュート・オブ・バイオフィジックス，チャイニーズ・アカデミー・オブ・サイエンシズ | Methods and compositions for treating and / or preventing diseases or disorders associated with abnormal levels and / or activity of IFP35 family proteins |
| WO2020200186A1 (en) * | 2019-04-01 | 2020-10-08 | 中山大学 | Diagnosis and treatment for chronic inflammation and virus infection |
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