Anti-PD-L1 antibody as well as application, preparation method, kit and drug

A PD-L1 and antibody technology, applied in biochemical equipment and methods, antibodies, anti-tumor drugs, etc., can solve the problems of difficult gene detection, inability to detect silence or expression, increase workload, etc., and achieve high specificity and affinity. Effect

Active Publication Date: 2017-10-27
联合益康(北京)生物科技有限公司
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AI Technical Summary

Problems solved by technology

[0008] Gene-based detection methods can only reflect the gene content in the sample at the nucleic acid level, and cannot detect whether it is silenced or expressed. Therefore, the detection results are not directly related to the actual content of the protein
In addition, genes will be difficult to detect due to degradation during processing, and nucleic acid detection methods must go through specialized nucleic acid extraction and purification processes, which increases additional workload

Method used

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  • Anti-PD-L1 antibody as well as application, preparation method, kit and drug
  • Anti-PD-L1 antibody as well as application, preparation method, kit and drug
  • Anti-PD-L1 antibody as well as application, preparation method, kit and drug

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preparation example Construction

[0043] In another aspect, the present invention provides a method for preparing the above-mentioned anti-PD-L1 antibody, which comprises: immunizing animals with an antigenic peptide having an amino acid sequence as shown in positions 29-290 of SEQ ID No.2.

[0044] The present invention shows that the 29-290 position of PD-L1 has good antigenicity and contains B cell epitope through bioinformatics analysis, and the 29-290 region of PD-L1 is used as the recombinant protein (antigenic peptide) to immunize mice to prepare mouse monoclonal Anti-PD-L1 antibody, the obtained anti-PD-L1 antibody can recognize PD-L1 and has better specificity.

[0045] Of course, it should be noted that other animals such as rabbits, rats, pigs and other mammals can also be selected for immunization to prepare antibodies.

[0046] Further, in some embodiments of the present invention, before immunization, the preparation method further includes: transforming E. coli with an expression vector containi...

Embodiment 1

[0055] Construction of PD-L1 recombinant protein expression plasmid

[0056] According to the nucleic acid sequence in Genebank (GI: 20070268) and the encoded protein sequence of Q9NZQ7, the PD-L1 monoclonal is synthesized by gene synthesis corresponding to the amino acid sequence from the 29th to the 290th position of the PD-L1 protein (SEQ ID NO.2). The DNA sequence ((SEQ ID NO.1) was introduced with restriction sites EcoR I and Xho I, cloned into the expression vector pET41a (Novagen Company), and sequenced and analyzed, and the sequenced correct clones were selected for protein expression and purification.

Embodiment 2

[0058] PD-L1 recombinant protein expression and purification

[0059] Escherichia coli containing the plasmid with the correct sequence of PD-L1 was cultivated to OD 600 0.5, add 10 μmol / L IPTG, culture overnight at 16°C, and after harvesting, sonicate and carry out Ni2 + Affinity purification. And carry out 12% SDS-PAGE detection, Coomassie brilliant blue staining, the result is as follows figure 1 As shown, the recombinant PD-L1 protein molecular weight is about 57kDa,

[0060] figure 1 The result (in the figure: marker is the molecular weight marker) shows that there is a band at the position of 55KD, indicating that the expression of PD-L1 recombinant protein obtained in this example is correct.

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Abstract

The invention discloses an anti-PD-L1 antibody as well as application, a preparation method, a kit and a drug, and relates to the technical fields of biological detection and treatment. The anti-PD-L1 antibody provided by the invention contains a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 6. The anti-PD-L1 antibody can be combined with PD-L1 recombinant proteins and inflammation tissue cells or tumor tissue cells expressing PD-L1 molecules via specific recognition, so that the anti-PD-L1 antibody has a relatively high specificity and avidity, can be used for immunoblotting, enzyme-linked immuno sorbent assay, immunohistochemistry and flow cytometry and can also be used for treating diseases of tumors, immunological diseases, chronic infectious diseases and the like.

Description

technical field [0001] The present invention relates to the technical field of biological detection and treatment, in particular, to an anti-PD-L1 antibody and its application, preparation method, kit and medicine. Background technique [0002] Programmed cell death ligand 1 (PD-L1) is a transmembrane glycoprotein consisting of 290 amino acids. PD-L1 belongs to the B7 family and has IgV and IgC-like regions, transmembrane regions, and cytoplasmic region tails. PD-L1 interacts with programmed cell death 1 (PD-1) on T cells, It plays an important role in the negative regulation of the immune response (Riley J L.PD-1 signaling infantry T cells[J]. Immunol Rev.2009, 229(1):114-1125.), extensively low expression in normal On the surface of immune and non-immune cells, overexpressed in some tumor cells. [0003] In the normal immune system, PD-L1 plays an extremely important role in immune tolerance. Studies have shown that PD-L1 is expressed on the surface of periodontal ligam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N5/20G01N33/68G01N33/574A61K39/395A61P35/00A61P37/00A61P31/00
CPCA61K2039/505C07K16/2827C07K2317/56
Inventor 王新华
Owner 联合益康(北京)生物科技有限公司
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