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Anti-TOPK antibody with 74th tyrosine residue phosphorylated as well as preparation method and application of anti-TOPK antibody

A tyrosine residue, phosphorylation technology, applied in anti-enzyme immunoglobulins, instruments, measuring devices, etc., can solve the problems that hinder the development prospects of clinical disease diagnosis and treatment, the regulatory mechanism is not completely clear, and the research in the field of inhibitors is carried out. slowness etc.

Active Publication Date: 2016-06-08
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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  • Anti-TOPK antibody with 74th tyrosine residue phosphorylated as well as preparation method and application of anti-TOPK antibody
  • Anti-TOPK antibody with 74th tyrosine residue phosphorylated as well as preparation method and application of anti-TOPK antibody
  • Anti-TOPK antibody with 74th tyrosine residue phosphorylated as well as preparation method and application of anti-TOPK antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Confirmation that Src is an upstream protein kinase of TOPK, and TOPK is phosphorylated at Y74 and Y272.

[0092] 1. Through in vitro kinase experiments, it was found that active Src can phosphorylate TOPK.

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

[0094] 1.1 Express and purify TOPK protein in E.coliBL21 bacteria: transform pET46-His-TOPK-WT plasmid into E.coliBL21 competent cells, pick a single colony and culture overnight at 37°C. Overnight bacteria were re-inoculated, cultured with shaking at 37°C until OD600 was 0.6-0.8, then added 1mMIPTG and induced by shaking at 30°C for 4h, then repeatedly freeze-thawed to lyse the bacteria, added Ni-NTA-His-bindingbeads (purchased from QIAGEN Company), 4°C Incubate overnight, then wash with PNI20 and PNI40 respectively, and finally PNI400 is eluted, quantified, electrophoresis, and identified by Coomassie brilliant blue staining.

[0095] 1.2 We prepared 2ug of TOPK protein, 0.2ug of...

Embodiment 2

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

[0123] 1. Perform single mutation and double mutation on the Y74 and Y272 sites of TOPK by subcloning technology.

[0124] Firstly, design and synthesize corresponding primers (synthesized by Shanghai Yingjun Biotechnology Co., Ltd.) according to the requirements of the mutation of tyrosine to phenylalanine at two sites, and then use pcDNA3-HA-TOPK as a template to perform PCR to obtain the mutated products respectively : Single mutant pcDNA3-HA-TOPK(Y74F), pcDNA3-HA-TOPK(Y272F) and double mutant pcDNA3-HA-TOPK(FF), then transformed into TOP10, extracted plasmid 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 cells in each group.

[0126] 2.1 pcDNA3.1, pcDNA3-HA-TOPK (WT), pcDNA3-HA-TOPK (Y74F), pcDNA3-HA-TOPK (Y272F) and pcDNA3-HA- TOPK(FF) was transferred int...

Embodiment 3

[0148] Example 3 Preparation of TOPK phosphorylated antibody.

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

[0150] p-TOPK(Y74):NH 2 -CysAsnAspHisp[Tyr]SerValTyrGlnLysArgLeuMetAspGlu-CONH 2

[0151] NP-TOPK(Y74):NH 2 -CysAsnAspHisTyrSerValTyrGlnLysArgLeuMetAspGlu-CONH 2

[0152] p-TOPK(Y272):NH 2 -CysAspGluSerAspPheAspAspGluAlaTyrp[Tyr]Ala-CONH 2

[0153] NP-TOPK(Y272):NH 2 -CysAspGluSerAspPheAspAspGluAlaTyrTyrAla-CONH 2

[0154] 2. Preparation of phosphorylated antibody.

[0155] Specific steps are as follows:

[0156] 2.1 The peptide was synthesized by solid-phase synthesis according to the designed sequence, purified by RP-HPLC, and identified by LC / MS with a purity of >85%.

[0157] 2.2 Use KLH carrier protein to couple to obtain polypeptide, which is used as antigen protein.

[0158] 2.3 Rabbits were immunized, rabb...

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Abstract

The invention discloses an anti-TOPK antibody with 74th tyrosine residue phosphorylated as well as a preparation method and an application of anti-TOPK antibody and belongs to the field of biology and disease diagnosis. The antibody is obtained as follows: polypeptides with the amino acid sequence shown in SEQ ID NO: 1 are coupled with carrier protein, a product serves as an antigen, the antigen is used for immunize an animal, polyclonal antibody serums are obtained and purified, and the antibody is obtained. The antibody can be applied to immunoblotting detection of the tumor cell level. Currently, an antibody for detecting Y74 phosphorylation of TOPK is not available in the market. The antibody lays a solid foundation for research of tumor occurrence as well as early diagnosis and prognosis predicted treatment of the tumor.

Description

technical field [0001] The invention belongs to the field of biology and disease diagnosis, and specifically relates to an antibody against the phosphorylation of the 74th tyrosine residue of TOPK, and also relates to a preparation method and application of the antibody. Background technique [0002] TOPK, also known as PBK, is PDZ-linked kinase and lymphokine-activated killer T lymphocyte-derived protein kinase. Carcinoma, melanoma, lung cancer, leukemia and other tumor cells are highly expressed, and the expression level is significantly correlated with the malignancy of breast cancer 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 prediction of tumors. In 2010, ZlobecI et al. discussed the prognostic value of TOPK in...

Claims

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Application Information

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Patent Type & Authority Applications(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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