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Antibody phosphorylated at the 74th tyrosine residue of TOPK and its preparation method and application

A tyrosine residue, phosphorylation technology, applied in anti-enzyme immunoglobulins, instruments, measuring devices, etc., can solve the problem of inability to accurately reflect TOPK activity, slow research in the field of inhibitors, and hinder the development of clinical disease diagnosis and treatment prospects, etc.

Active Publication Date: 2022-04-19
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One is the difficulty in analyzing the crystal structure of TOPK, which leads to slow research in the field of its inhibitors, hindering its development prospects for clinical disease diagnosis and treatment
Secondly, the upstream regulation mechanism of TOPK is still not completely clear. So far, hDlg[2], ERK2[9], c-Myc-E2F1[20], P53[21], E2F-CREB / ATF[22] and Cdk1 have been reported / CyclinB[1,23] can interact with TOPK and regulate its function
However, the expression of non-phosphorylated TOPK does not accurately reflect the activity of TOPK in cells. Currently, only studies have shown that protein kinases Cdk1 / CyclinB and ERK2 can activate TOPK by phosphorylation at the Thr9 site, and studies have confirmed that phosphorylation at the Thr9 site is related to TOPK promotes tumorigenesis independent of relationship

Method used

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  • Antibody phosphorylated at the 74th tyrosine residue of TOPK and its preparation method and application
  • Antibody phosphorylated at the 74th tyrosine residue of TOPK and its preparation method and application
  • Antibody phosphorylated at the 74th tyrosine residue of TOPK and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example one determines that Src is toPK upstream protein kinase, phosphorylated TOPK at the Y74 and Y272 sites.

[0092] 1. Active Src phosphorylated TOPK was found by in vitro kinase experiments.

[0093] The specific steps of in vitro kinase experiments are as follows:

[0094] 1.1 Expression and purification of TOPK protein in E.coli BL21 bacteria: pET46-His-TOPK-WT plasmid was converted into E.coli BL21 competent cells, and single colonies were cultured overnight at 37 °C. The bacteria were inoculated again overnight, incubated at 37 °C to OD600 to 0.6~0.8, then added 1mM IPTG 30 °C oscillation induction for 4h, followed by repeated freeze-thawing of bacterial bodies, added Ni-NTA-His-binding beads (purchased from QIAGEN), incubated at 4 °C overnight, and then washed with PNI 20 and PNI 40 respectively, and finally PNI 400 elution, quantification, electrophoresis, And Coomassie bright blue staining identification.

[0095] 1.2 We incubate the prepared TOPK protein 2ug, ...

Embodiment 2

[0122] Example 2 explores the effect of Src phosphorylation on TOPK function.

[0123] 1. Single mutation and double mutation of THE Y74 and Y272 sites of TOPK are carried out by subclonal technology.

[0124] First of all, according to the requirements of the two sites of tyrosine mutation to phenylalanine, the corresponding primers were designed and synthesized (synthesized by Shanghai Yingjun Biotechnology Co., Ltd.), and then PCR was performed with pcDNA3-HA-TOPK as a template, and the mutant products were obtained: single-mutant pcDNA3-HA-TOPK (Y74F), pcDNA3-HA-TOPK (Y272F) and double-mutant pcDNA3-HA-TOPK (FF), and then converted to TOP10, and the plasmid was extracted after amplification.

[0125] 2. Establish overexpression stable cell lines in JB6 and SW480 cell lines, and make growth curves to compare the proliferation changes of each group of cells.

[0126] 2.1 PcDNA3.1, pcDNA3-HA-TOPK (WT), pcDNA3-HA-TOPK (Y74F), pcDNA3-HA-TOPK (Y272F), pcDNA3-HA-TOPK (Y272F) and pcDNA...

Embodiment 3

[0148] Example Preparation of three TOPK phosphorylated antibodies.

[0149] 1. According to the TOPK target sequence and the known phosphorylation sites Y74 and Y272, design the phosphorylated and non-phosphorylated polypeptide sequences that need to be synthesized.

[0150] p-TOPK(Y74):NH 2 -Cys Asn Asp His p[Tyr]Ser Val Tyr Gln Lys Arg Leu MetAsp Glu-CONH 2

[0151] NP-TOPK(Y74):NH 2 -Cys Asn Asp His Tyr Ser Val Tyr Gln Lys Arg Leu MetAsp Glu-CONH 2

[0152] p-TOPK(Y272):NH 2 -Cys Asp Glu Ser Asp Phe Asp Asp Glu Ala Tyr p[Tyr]Ala-CONH 2

[0153] NP-TOPK(Y272):NH 2 -Cys Asp Glu Ser Asp Phe Asp Asp Glu Ala Tyr TyrAla-CONH 2

[0154] 2. Prepare phosphorylated antibodies.

[0155] The specific steps are as follows:

[0156] 2.1 According to the design sequence, the polypeptide is synthesized by solid phase synthesis method, purified with RP-HPLC conditions, and identified by LC / MS, with a purity of >85%.

[0157] 2.2 Use KLH carrier protein coupling to obtain polypeptides for a...

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Abstract

The invention discloses an antibody against the phosphorylation of the 74th tyrosine residue of TOPK, which belongs to the field of biology and disease diagnosis. The antibody is obtained by coupling a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 with a carrier protein as an antigen, and immunizing animals with the antigen to obtain polyantiserum, which is purified. The antibody can be applied to the immunoblotting method detection at the tumor cell level. At present, there is no antibody for the phosphorylation detection of TOPK Y74 on the market. The invention lays a solid foundation for the study of tumor occurrence, early diagnosis and prognosis prediction treatment of tumors .

Description

Technical field [0001] The present invention belongs to the field of biological and disease diagnosis, specifically relates to an antibody anti-TOPK 74th position tyrosine residue phosphorylation, the present invention further relates to the preparation method and application of the antibody. Background [0002] TOPK, also known as PBK, is a killer T lymphocyte-derived protein kinase activated by PDZ ligation kinase and lymph factors, which is a new silk-threonine protein kinase [1,2], which is highly expressed in breast cancer, colorectal cancer, melanoma, lung cancer, leukemia, etc.[3], and the expression is significantly related to the malignancy of breast and colorectal cancer [4]. TOPK is involved in the regulation of tumor cell cycle progression [5-8], tumor cell transformation [9,10], proliferation [11], and apoptosis [12,13]. [0003] In recent years, more and more studies have found that TOPK plays an important role in the clinical diagnosis and prognosis of tumors. In ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40G01N33/68G01N33/574
CPCC07K16/40
Inventor 朱峰段秋红肖娟娟王哲薛佩佩孙惠敏邵晨曾凡帆全纯涛卢涛刘琳袁萍柯昌庶熊华路慧尹燕华潘华雄
Owner HUAZHONG UNIV OF SCI & TECH
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