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CK20 antigen, hybridoma cell strain, monoclonal antibody, and application thereof

A monoclonal antibody, CK20 technology, applied in the field of immunology, can solve the problems of increasing monoclonal cell line screening, affecting judgment and diagnosis, and low specificity antibodies, so as to avoid non-specific antibodies, good accuracy and strong specificity Effect

Active Publication Date: 2021-02-23
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

However, due to the high homology of the amino acid sequence of the intermediate fiber rod, when using the full-length recombinant protein of CK20 as an immunogen to prepare antibodies, it is easy to cause low specificity due to homology, which leads to increased screening of monoclonal cell lines
In the development of diagnostic reagents, the full-length CK20 protein is used to prepare antibodies, and it is easy to obtain low-specificity antibodies that can recognize various cytokeratins, resulting in the failure of development
In addition, the use of synthetic peptides as immunogens can determine antibody recognition epitopes, but the differences in the three-dimensional structure of the protein and the interaction between different molecules of the polypeptide fragments at different positions may bring differences to the results and affect the judgment and diagnosis

Method used

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  • CK20 antigen, hybridoma cell strain, monoclonal antibody, and application thereof
  • CK20 antigen, hybridoma cell strain, monoclonal antibody, and application thereof
  • CK20 antigen, hybridoma cell strain, monoclonal antibody, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The CK20 antigen in this example is a fusion protein obtained by adding linking elements between the head and tail fragments of human CK20 protein.

[0035] The Uniprot accession number of human CK20 is P35900, which contains 424 amino acids, of which 1-69 is the head, 70-377 is the stem, and 378-424 is the tail. The amino acid and nucleotide sequence of the head is shown in SEQ ID For NO: 1 and SEQ ID NO: 2, see SEQ ID NO: 3 and SEQ ID NO: 4 for the amino acid and nucleotide sequences of the tail. According to the CK20 sequence and structure published by Uniprot, the head and tail sequences were selected as the first sequence and the second sequence, respectively.

[0036] The commonly used elastic linker (GGGGS)n is (GGGGS)n, and the elastic linker will not affect the conformation and function of the proteins at both ends. In order to ensure the distance between the head and tail of cytokeratin, reduce the steric hindrance of the head and tail, and exert the function...

Embodiment 2

[0042] The CK20 antigen in this example is artificially synthesized by adding the nucleic acid sequence SEQ ID NO: 6 of the connecting element between the head nucleotide sequence SEQ ID NO: 2 and the tail nucleotide sequence SEQ ID NO: 4 of CK20 After cloning into the pET-28a expression vector, the pET-28a-CK20-Linker expression vector was constructed and transformed into Escherichia coli to express and purify.

[0043] 1) Add the linker nucleic acid sequence between the head and tail nucleic acid sequences of CK20 for artificial synthesis, and introduce restriction sites NdeI and XhoI at both ends of the sequence, clone into the pET-28a expression vector after synthesis, and construct pET-28a - CK20-Linker expression vector.

[0044] 2) Transform the recombinant plasmid constructed above into BL21(DE3) Escherichia coli competent, pick a single colony and culture it in 4 mL of LB liquid medium containing kanamycin (50 μg / mL), add IPTG at a final concentration of 1 mM for For...

Embodiment 3

[0049] The hybridoma cell line C7C11 in the present embodiment is made by the method comprising the following steps:

[0050] 1) Animal immunization: the above-mentioned purified CK20-Linker protein was mixed with an equal volume of Freund's complete adjuvant, and the 6-8 week-old BalB / c mice were immunized by subcutaneous injection. The immunization dose was 100 μg / mouse, and two weeks later For the second immunization, the antigen was emulsified with Freund's incomplete adjuvant, and the immunization dose was the same as the first time, and intraperitoneal injection was adopted. After the second immunization, the tail blood was taken to determine the serum titer by gradient dilution by indirect ELISA method, and the mice with the highest antibody titer were selected for tail vein pulse immunization, and the pulse dose was 50 μg / mouse. .

[0051] 2) Cell fusion: the myeloma cells were sp2 / 0 derived from BalB / c, and they were in the logarithmic growth phase during fusion; the...

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Abstract

The invention relates to a CK20 antigen, a hybridoma cell strain, a monoclonal antibody, and application thereof. High-variation head and tail sequences of CK20 are selected according to the characteristics of a cytokeratin sequence and connected by adopting connecting components, gene synthesis is carried out according to the sequence of head-connecting-components-tail, and a prokaryotic expression vector is inserted, expressed and purified to obtain the CK20 antigen; and the hybridoma cell strain C7C11 capable of stably secreting the mouse anti-human CK20 monoclonal antibody is finally obtained by using the CK20 antigen as an immunogen to immunize a BALB / c mouse, fusing spleen cells of the mouse with SP2 / 0 cells, obtaining monoclonal cells by using a limited dilution method and screeningpositive hybridoma cells by using an indirect ELISA method, and the preservation number is CCTCC NO: C2020201. The monoclonal antibody is prepared through induction in an animal body, and the specificity and the sensitivity of the monoclonal antibody are verified through Western Blot and immunohistochemistry (IHC). The CK20 monoclonal antibody provided has better specificity and sensitivity, andcan be used for preparing a CK20 in-vitro immunoassay reagent or a kit.

Description

technical field [0001] The invention relates to CK20 antigen, hybridoma cell line, monoclonal antibody and application thereof, and belongs to the technical field of immunology. Background technique [0002] Cytokeratin is a component of intermediate fibers, which consist of a rod-shaped region of about 310 amino acids in the middle, with hypervariable N-terminal heads and C-terminal tails at both ends. Due to the unique composition of the intermediate fiber, its unique structure is formed: non-helical head-helical rod-non-helical tail, the amino acid sequence of the rod has high homology, and the same type of intermediate fiber rod has 70 %-90% homology, but cytokeratin is an important component of intermediate fibers, and 54 members have been found so far, so it is easy to cause low specificity due to homology when performing immunological experiments. It is particularly important to identify a certain cytokeratin. [0003] As an important component of cytokeratin, cytok...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/18C12N15/70C12N5/20G01N33/577G01N33/68G01N33/574
CPCC07K14/4741C07K16/18C12N15/70G01N33/577G01N33/68G01N33/57496G01N33/574C07K2319/00G01N2333/4742Y02A50/30
Inventor 王亚杰徐星星柴素真张东辉常秋霜翟晋豫刘文弟刘畅李三华牛银银齐华
Owner 河南赛诺特生物技术有限公司
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