Anti-human nkx3.1 monoclonal antibody and its preparation method and application

A monoclonal antibody and sequence technology, applied in the field of immunology, can solve the problems of specificity and stability to be improved, high price, and large differences between antibody batches, so as to improve specificity and affinity, improve immune hit rate, The effect of stabilizing antibody properties

Active Publication Date: 2022-06-24
河南赛诺特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most of the NKX3.1 monoclonal antibodies prepared at present are immunized with prokaryotic expression of the full-length recombinant protein of NKX3.1, which have few types, are expensive, and need to be improved in specificity and stability
Moreover, the traditional preparation method of monoclonal antibody is not conducive to standardized production, and the difference between antibody batches is large and the stability is poor.

Method used

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  • Anti-human nkx3.1 monoclonal antibody and its preparation method and application
  • Anti-human nkx3.1 monoclonal antibody and its preparation method and application
  • Anti-human nkx3.1 monoclonal antibody and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Preparation of immunogen

[0039] 1. NKX3.1 head and tail selection

[0040] The head and tail sequences were selected based on the NKX3.1 sequence and structure published by Uniprot for fusion expression of NKX3.1. The accession number of the Uniprot of human NKX3.1 is Q99801, which contains 234 amino acids, of which 1-123 is its head, 124-183 is its stem, and 184-234 is its tail. The amino acid and nucleic acid sequences of the head are as shown in SEQ respectively. ID NOs: 11 and 12, and the tail amino acid and nucleic acid sequences are shown in SEQ ID NOs: 13 and 14, respectively.

[0041] 2. Fusion expression and purification

[0042] 1) The head and tail nucleic acid sequences of NKX3.1 were artificially synthesized, and enzyme cleavage sites NdeI and XhoI were introduced at both ends of the sequence. After synthesis, cloned into the pET-28a expression vector to construct pET-28a-NKX3.1 -A, pET-28a-NKX3.1-B expression vector.

[0043] 2) The recombina...

Embodiment 2

[0047] Example 2 NKX3.1 hybridoma cell line screening and monoclonal antibody preparation

[0048] 1. Animal immunity:

[0049] The NKX3.1-A and NKX3.1-B proteins purified above were mixed with an equal volume of Freund's complete adjuvant respectively, and the immunization dose of 6-8 weeks old BalB / c mice was 100 μg by subcutaneous injection. Each animal was immunized for the second time after an interval of two weeks. The antigen was emulsified with incomplete Freund's adjuvant, and the immunization dose was the same as that of the first time. After the second immunization, the tail blood was collected and the serum titer was determined by indirect ELISA method.

[0050] 2. Cell fusion:

[0051] Myeloma cells use sp2 / 0 derived from BalB / c, which is in the logarithmic growth phase when fused; the spleen of mice after shock immunization is aseptically harvested to prepare a single-cell suspension of spleen cells; mouse spleen cells and myeloma cells Mix at a ratio of 1:5, ...

Embodiment 3

[0059] Example 3 Detection of specificity of NKX3.1 monoclonal antibody by Western-Blot

[0060] 1. Sample preparation: prepare sp2 / 0 cell lysate and LNCaP cell lysate, lyse with RAPI containing 1mM PMSF, and add 5XSDS-PAGE Loading buffer for sample preparation. Sp2 / 0 is a mouse myeloma cell without fusion as a negative control, LNCaP cells are a human prostate cancer cell as a positive control, the two lysates will release intracellular proteins, and mouse myeloma cells will not express NKX3.1 , and LNCaP cells are cells that specifically express NKX3.1, which will bind to the screened positive monoclonal antibodies, and WB will show a positive reaction.

[0061] 2. Electrophoresis and membrane transfer: Load 30μg of each sample, run at 80V for 20min, and then run electrophoresis at 120V to bromophenol blue to the bottom of the gel. Then start to transfer the membrane, soak the NC membrane and filter paper with transfer buffer in advance, and place the sandwich form on the w...

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Abstract

The present invention relates to anti-human NKX3.1 monoclonal antibody and its preparation method and application. The invention provides an anti-human NKX3.1 monoclonal antibody, and further provides the amino acid sequences of the variable regions of its heavy chain and light chain. On this basis, the antibody can be prepared by conventional genetic engineering methods. Compared with the traditional monoclonal antibody preparation method, the anti-human NKX3.1 recombinant antibody prepared by the genetic engineering method of the present invention has the advantages of known sequence, stable antibody properties, good repeatability, etc., and can specifically recognize human NKX3.1 protein , can be used for IHC immunohistochemical detection. And the standardized antibody production process avoids the risk factors that appear in the traditional monoclonal antibody production and storage process. Furthermore, on the basis of the amino acid sequence of the antibody provided by the present invention, one or more amino acid additions, deletions, substitutions, etc., can also be modified to obtain active fragments or conservative variants, in order to further improve the specificity and Affinity lays the groundwork.

Description

technical field [0001] The invention belongs to the technical field of immunology, and in particular relates to an anti-human NKX3.1 monoclonal antibody and a preparation method and application thereof. Background technique [0002] The full length of human NKX3.1 cDNA is 3263 bp and has 2 exons. Exon I has 96 amino acids, and no obvious functional region has been found; exon II includes its homology domain, its N-terminal contains 28 amino acid residues, and its C-terminal contains 53 amino acid residues. Human and murine NKX3.1 proteins are identical at the homology domains, and their expression is regulated by androgen. The gene coding region of human NKX3.1 is located at 8P21, and the encoded product is a protein composed of 234 amino acids. NKX3.1 is linked to the gene coding regions of macrophage phagosome II MSR2, lipoprotein LPL and neurofilament light chain. The results show that NKX3.1 gene is expressed in various human prostate tissues and testis, but not expre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/85C12N5/10G01N33/577G01N33/574G01N33/68
CPCC07K16/18C12N15/85C12N5/0682G01N33/577G01N33/57434G01N33/57484G01N33/68C07K2317/565C07K2317/56C07K2317/33C12N2800/107C12N2510/02G01N2333/4703
Inventor 柴素真宋辉张楠常秋霜王迎利张倩曹晓菲刘文弟齐华
Owner 河南赛诺特生物技术有限公司
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