Anti-setd3 monoclonal antibody and use thereof

A monoclonal antibody and protein technology, applied in applications, anti-enzyme immunoglobulins, instruments, etc., to achieve good recognition specificity and strong recognition specificity

Active Publication Date: 2022-06-14
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The authors found that the H73 methylation modification of β-Actin protein regulates the polymerization and depolymerization of myofibrils, and also affects the migration ability of cells
The authors also found that Setd3-knockout female mice had a dystocia rate of more than 80% and fewer pups

Method used

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  • Anti-setd3 monoclonal antibody and use thereof
  • Anti-setd3 monoclonal antibody and use thereof
  • Anti-setd3 monoclonal antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Monoclonal antibody and preparation method thereof

[0047] 1. Cloning construction and protein prokaryotic expression

[0048] (1) Cloning the SETD3 CDS sequence into the pGEX-6p-1 vector to obtain the recombinant vector pGEX-6p-1-SETD3.

[0049] The primer pair sequences used were: forward primer (5'-ATGGGTAAGAAGAGTCGAG-3'; SEQ ID NO: 9); reverse primer (5'-TTACTCCTTAACTCCAGCAGTG-3'; SEQ ID NO: 10), cloned into pGEX-6p- 1 The EcoRI-NotI restriction site of the vector.

[0050] (2) Protein expression and purification:

[0051] ①Plasmid transformation:

[0052] The constructed SETD3 prokaryotic expression plasmid was transformed into BL21 competent cells, spread on the LB plate containing ampicillin resistance, and after the monoclonal growth, the monoclonal shake was picked.

[0053] ② Shaking bacteria & expression:

[0054] Shake the bacteria overnight, transfer a large amount the next morning, and dilute 1:100 to 1:50. Shake at 37°C to OD 600 =0.4...

Embodiment 2

[0122] Embodiment 2, antibody titer detection

[0123] Use ELISA to determine the titer of the antibody, coat each well of the plate with 50ng of antigen (prokaryotic purified human SETD3 protein), dilute the purified monoclonal antibody according to the ratio in Table 2, and measure the A450 absorbance of each well, the results are shown in Table 3 and image 3 shown;

[0124] table 3

[0125] potency 1K 2K 4K 8K 16K 32K 64K 128K 256K Absorbance 0.580 0.579 0.480 0.441 0.340 0.230 0.163 0.109 0.077

[0126] From Table 3 and image 3 It can be seen that the antibody titer can reach 1:128000.

Embodiment 3

[0127] Embodiment 3, antibody specific detection

[0128] 1. Use in vitro purified SETD3 protein to detect antibody specificity.

[0129] Purify GST-tagged SETD3 protein or pure GST-tagged protein in vitro, and use Western Blot to test the recognition specificity of the purified antibody, such as Figure 4 As shown, the left picture shows the loading of the two proteins by Coomassie Brilliant Blue staining, and the right picture shows the specificity of the antibody detected by Western Blot experiment. The results show that the SETD31-H9 monoclonal antibody has a good recognition specificity for GST-SETD3 , did not recognize the equivalent amount of GST protein.

[0130] 2. Detection of antibody specificity at the cellular level

[0131] (1) Construct SETD3 knockdown or overexpression cell line in mammalian cell PC9 cell line, detect with 1-H9 antibody, such as Figure 5 shown. The method for constructing the SETD3 knockdown or overexpression cell line in the embodiment of...

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PUM

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Abstract

The present invention provides an anti-SETD3 monoclonal antibody and its use. The monoclonal antibody can recognize SETD3 protein. The monoclonal antibody includes a heavy chain variable region and a light chain variable region: the heavy chain variable region The region has three complementary determining regions with the amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:3; the light chain variable region has three complementary determining regions with the amino acid sequence shown in SEQ ID NO:4-SEQ ID NO:6. The anti-SETD3 monoclonal antibody provided by the present invention has good recognition specificity to in vitro purified protein, in vitro cultured mammalian cell exogenous SETD3 protein, and mouse tissue SETD3 protein; compared with commercially available antibodies, the monoclonal antibody recognizes more specific.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an anti-SETD3 monoclonal antibody and its application. Background technique [0002] SETD3 is a member of the methyltransferase family containing the SET domain, which is close to SETD4 and SETD6 proteins in terms of kinship and structure. The secondary structure of SETD3 mainly includes catalytic domain (SET domain) and substrate binding domain (Rubis-subs-bind domain). SETD3 is relatively conserved in different species, and its homologous protein has not been found in yeast. In humans, SETD3 is expressed in a variety of tissues, with the highest expression in testis and skeletal muscle. In the process of myoblast differentiation, SETD3 can promote the differentiation of skeletal muscle; SETD3 and FoxM1 are combined in the promoter region of vascular endothelial growth factor (vascular endothelial-derived growth factor, VEGF) signaling pathway and negatively regulate the expression ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/577G01N33/573G01N33/558G01N33/58
CPCC07K16/40G01N33/577G01N33/573G01N33/558G01N33/587C07K2317/565C07K2317/56C07K2317/35G01N2333/91017
Inventor 杜海宁赵梦洁段宏国殷兆红
Owner WUHAN UNIV
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