Anti-Calponin protein monoclonal antibody and cell strain, preparation method and application thereof

A monoclonal antibody and protein technology, which is applied in the field of biomedical engineering, can solve problems such as increased exercise and decreased stability of the actin cytoskeleton of tumor cells, and achieves highly specific effects

Active Publication Date: 2021-08-10
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may be related to the decreased stability of the actin cytoskeleton and the enhanced movement of tumor cells due to the decreased expression of Calponin

Method used

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  • Anti-Calponin protein monoclonal antibody and cell strain, preparation method and application thereof
  • Anti-Calponin protein monoclonal antibody and cell strain, preparation method and application thereof
  • Anti-Calponin protein monoclonal antibody and cell strain, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The preparation of embodiment 1 recombinant Calponin protein fragment

[0020] 1. Gene optimization and synthesis

[0021] According to the protein sequence with accession number P51911 in the Uniprot database, Calponin selects the protein fragment at positions 1-297, which is the amino acid sequence shown in SEQ ID NO.1; it is directly optimized into a gene fragment suitable for expression in Escherichia coli BL21. During the PCR process, BamH I and XhoI restriction sites were added to the 5' and 3' ends of the gene, respectively.

[0022] The PCR products were separated by agarose gel electrophoresis and recovered. The recovered fusion protein gene and the plasmid vector Pet30a used for expression were digested with BamHI and XhoI respectively, recovered by electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into Escherichia coli competent cell BL21, and the clones on the plate were picked and inoculated, and the PCR identifica...

Embodiment 2

[0027] The establishment of embodiment 2 hybridoma cell lines

[0028] 1. Immunity

[0029] The cross-linked polypeptide in Example 1 was emulsified with complete Freund's adjuvant (Sigma, F5881), and immunized with 4-6 week-old SPF female mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), abdominal Each mouse was injected subcutaneously at 6 points with a dose of 20 μg / mouse. Immunization was boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. 50μg / only.

[0030] 2. Cell Fusion

[0031] Aseptically prepare the mouse splenocyte suspension that reaches the immune standard, mix it with mouse myeloma cell sp2 / 0 (ATCCNumberCRL-8287) at a ratio of 5:1, and centrifuge at 1500rpm for 5min. After the super...

Embodiment 3

[0034] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0035] 1. Ascites preparation

[0036] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted about 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm for 10min at 4°C. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C.

[0037] 2. Purification of monoclonal antibodies

[0038] Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.

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Abstract

The invention relates to a monoclonal antibody capable of recognizing a human Calponin antigen, a secretory cell strain, a preparation method of the monoclonal antibody and application of the secretory cell strain in immunodetection. According to the technical scheme, amino acids from the first site to the 297th site at the C tail end of the Calponin protein are selected as antigen peptides, codon optimization is carried out, a gene segment suitable for being expressed in escherichia coli BL21 is formed, and finally the obtained recombinant protein comprises a Calponin protein segment and a histidine protein tag. A mouse is immunized by the recombinant protein, and the mouse hybridoma cell strain 24H5 capable of efficiently secreting the anti-Calponin protein monoclonal antibody and the anti-Calponin protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing Calponin protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

technical field [0001] The invention relates to the field of biomedical engineering, in particular to an anti-Calponin protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] Calponin (CaP), first discovered in chicken stomach smooth muscle by Takahashi et al. in 1986, is a protein with a molecular weight of about 34-kDa (292-330 amino acids), which binds to thin myofilaments as an actin-binding protein Involved in the contraction of smooth muscles. Its molecular structure is mainly composed of a single CH domain (calponin homology domain) at the amino terminal and single or multiple copies of tandem repeats of the calponin-like repeat motif (calponin-like repeat, CLR) at the carboxy terminal. CaP is a differentiation marker protein, which is abundantly expressed in differentiated smooth muscle cells. It participates in the remodeling of the actin cytoskeleton by combining with the cytoskeleton protein actin, and has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/574G01N33/577C12N5/20C12N15/70C12R1/19
CPCC07K16/18G01N33/57442G01N33/57484G01N33/577C12N15/70C07K2317/56C12N2800/22Y02A50/30
Inventor 付利利陈滢杨清海陈惠玲王小亚
Owner FUZHOU MAIXIN BIOTECH CO LTD
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