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CREPT (Cell-cycle Related and Expression-elevated Protein in Tumor) antibody for identifying tumor cells or tumor tissues

A hybridoma cell and auxiliary identification technology, applied in the field of CREPT antibody, can solve problems such as difficult treatment, difficult cancer detection, and difficult prediction of patient prognosis, achieving high accuracy, low cost, and easy operation

Active Publication Date: 2012-07-11
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It is difficult to find cancer in the early stage, and the patient will not go to the hospital until various clinical symptoms appear, but it is usually already in the middle and late stage, and it is difficult to treat
After general treatment, it is difficult to predict the prognosis of patients

Method used

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  • CREPT (Cell-cycle Related and Expression-elevated Protein in Tumor) antibody for identifying tumor cells or tumor tissues
  • CREPT (Cell-cycle Related and Expression-elevated Protein in Tumor) antibody for identifying tumor cells or tumor tissues
  • CREPT (Cell-cycle Related and Expression-elevated Protein in Tumor) antibody for identifying tumor cells or tumor tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, the discovery of CREPT protein and its coding gene

[0029] P15 INK4b It is a member of the cell cycle-dependent kinase inhibitor (CKI) family and is located in the 9P21 region of chromosome 9. This site is prone to deletion, mutation and methylation, and plays an important role in cell proliferation and many tumors. p15 INK4b Gene is a cyclin-dependent kinase inhibitor that inhibits cell proliferation. It can make cells stay in G1 phase, thereby inhibiting cell proliferation and growth. The p15RS gene is p15 INK4b The related gene has obvious regulatory effect on the cell cycle, and it can act as a negative regulator in the G1 phase.

[0030] Using the p15RS gene as bait, a new gene (CREPT) and its encoded protein were obtained through bioinformatics methods. The amino acid sequence of the CREPT protein is shown in Sequence 1 of the Sequence Listing (consisting of 326 amino acid residues). The gene encoding CREPT protein is named CREPT gene, and its ...

Embodiment 2

[0031] Example 2, Preparation of Antibodies and Measurement of Their Potency and Sensitivity

[0032] 1. Construction of prokaryotic expression vector pGEX-5X-2 / CREPT

[0033] 1. Synthesize the double-stranded DNA shown in sequence 2 of the sequence listing.

[0034] 2. Using the double-stranded DNA in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0035] F1: 5'-TATA GATATC ATGTCCTCCTTCTCTGAGT-3';

[0036] R1: 5'-TATA CTCGAG TGAGTCAGTTGAAAACAGGT-3'.

[0037] PCR reaction conditions: denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 60 s; extension at 72°C for 10 min.

[0038] 3. Digest the PCR amplified product of step 2 with restriction endonucleases EcoRV and Xho I, and recover the digested product.

[0039] 4. The plasmid pGEX-5X-2 (purchased from clontech) was digested with restriction enzymes Sma I and X...

Embodiment 3

[0085] Embodiment 3, the determination of antibody specificity

[0086] 1. Construction of eukaryotic expression vector

[0087] 1. Construction of eukaryotic expression vector Flag-pcDNA3.1 / CREPT

[0088] (1) The double-stranded DNA shown in Sequence 2 of the Sequence Listing was synthesized.

[0089] (2) Using the double-stranded DNA in step (1) as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0090] F1: 5'-TATA GATATC CACCATGTCCTCCTTCTCTGAGT-3';

[0091] R1: 5'-TATA CTCGAG GTCAGTTGAAAACAGGTCCC-3'.

[0092] The PCR reaction conditions were the same as step 1 of Example 2.

[0093] (3) Digest the PCR amplified product of step (2) with restriction endonucleases EcoRV and Xho I, and recover the digested product.

[0094](4) The plasmid Flag-pcDNA3.1 (purchased from clontech company) was digested with restriction endonucleases EcoRV and Xho I, and the vector backbone (about 5.5 kb) was recovered. ...

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Abstract

The invention discloses a CREPT (Cell-cycle Related and Expression-elevated Protein in Tumor) antibody for identifying tumor cells or tumor tissues. The invention provides an application of a reagent for detecting CREPT protein in the following steps (a), (b) and (c): (a) assisting in identifying the tumor cells; (b) assisting in identifying the tumor tissues; and (c) assisting in identifying tumor patients. The CREPT antibody discovers that the expression level of a CREPT gene in a tumor cell line is much higher than that in normal cells, and the expression level of the CREPT gene in the tumor tissues is much higher than that in paracancerous tissues and normal tissues. Therefore, by applying the antibody provided by the invention, the tumor cells, the tumor tissues and the tumor patients can be subjected to assisted identification. The CREPT antibody has the advantages of simplicity and convenience in operation, low cost, high accuracy, prognosis prediction and the like.

Description

technical field [0001] The present invention relates to a CREPT antibody for identifying tumor cells or tumor tissues. Background technique [0002] Cancer, also known as malignant tumor in medical terms, is a disease caused by abnormalities in the mechanisms that control cell growth and proliferation. In addition to growing out of control, cancer cells can also locally invade the surrounding normal tissues and even transfer to other parts of the body through the internal circulatory system or lymphatic system. [0003] Cancer cells are characterized by unlimited and endless proliferation, which consumes a large amount of nutrients in the patient's body; cancer cells release a variety of toxins, causing a series of symptoms in the human body; cancer cells can also be transferred to grow and multiply throughout the body, It leads to emaciation, weakness, anemia, loss of appetite, fever, and severe damage to organ function. In contrast, there are benign tumors, which are eas...

Claims

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Application Information

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IPC IPC(8): C12N5/10C07K16/18G01N33/577G01N33/574C12R1/91
Inventor 常智杰胡建祥韩斌任芳丽王银银苏富琴张衍泉
Owner TSINGHUA UNIV
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