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Novel tumour serum marker and application thereof

A technology for serum markers and tumors, applied in biological testing, biomaterial analysis, peptide preparation methods, etc., can solve problems such as limiting clinical application value, unclear early detection of tumors, unclear CT antigen autoantibodies, etc.

Inactive Publication Date: 2014-03-26
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a common problem is that it is not clear which CT antigens can induce the body to produce a certain titer of detectable autoantibodies; by what method can the existence of these autoantibodies be confirmed, and how to detect these autoantibodies
In addition, the relationship between these new autoantibodies and the early detection, diagnosis and prognosis of tumors is unclear, which greatly limits their clinical application value

Method used

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  • Novel tumour serum marker and application thereof
  • Novel tumour serum marker and application thereof
  • Novel tumour serum marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1, Prokaryotic expression vector construction, expression and purification of POTE fragment antigen

[0065] (1) Cloning of POTE gene cDNA

[0066] HELA cell mRNA was extracted with TRIzol reagent and reverse-transcribed (RT) into cDNA. Using this cDNA as a template, the cDNA fragments of two subtypes 2A and 2C of POTE were amplified by PCR. After determination, their sequences were shown as SEQ ID No.13 (POTE-2A ORF sequence) and SEQ ID No.14 (POTE-2C ORF sequence) shown.

[0067] (2) Construction of prokaryotic expression vectors for POTE polypeptide fragments

[0068] By PCR method, the gene sequence encoding POTE-E protein molecule 1-144aa (or 2-144aa) (see SEQ ID No.10), the gene sequence encoding POTE-E protein molecule 390-524aa region (see SEQ ID No.11) and the gene sequence encoding the 528-641aa region of the POTE-E protein molecule (see SEQ ID No.12) were cloned into the pGEX-6P-3 vector to construct pGEX-GST-POTE_144, pGEX-GST-POTE_390 and pGEX- ...

Embodiment 2

[0071] Example 2, Purification and identification of POTE autoantibodies

[0072] In this example, in order to prove the existence of POTE autoantibodies, a solid-phase affinity chromatography column for POTE polypeptide fragments was first prepared, and this column was used to enrich and purify tumor patient serum to obtain POTE autoantibodies. The specific operation is as follows:

[0073] Preparation of POTE polypeptide fragment solid-phase affinity chromatography column: (1) POTE polypeptide preparation: after prokaryotically expressed GST-POTE-144 fragment (GST-144) is affinity-bound with Glutathione-Sepharose beads, it is incubated with precision enzyme, The GST tag linked to the 144 polypeptide was excised, and then the digestion mixture was centrifuged at 14,000 rpm with a 10kDa pore size ultrafiltration tube, and the precision enzyme was removed by ultrafiltration to obtain a purified POTE-144 (POTE-E-144) polypeptide fragment. In the same way, POTE-390 (390-524aa) a...

Embodiment 3

[0077] Embodiment 3, the identification of POTE autoantibody epitope

[0078] The POTE autoantibodies purified from the serum of tumor patients by GST-144 affinity chromatography may be polyclonal antibodies against different sequence epitopes in the 1-144aa fragment polypeptide of POTE. In order to identify the epitope of the POTE autoantibody, in this example, the possible regional epitope within the 144 amino acid residues of the N-terminal of the POTE-E protein, that is, the 20th-53aa (SEQ ID No.2), was respectively encoded by PCR method The gene sequences corresponding to the 57-90aa (SEQ ID No.3) and 94-127aa (SEQ ID No.4) regions were cloned into the pGEX-6P-3 vector to construct pGEX-GST-POTE_20-53, _57- 90 and _94-127aa fusion protein expression vector. The above plasmids were transformed into Rosetta Escherichia coli, and IPTG induced the expression of the prokaryotic protein, namely the corresponding fragment protein of POTE-E. Escherichia coli was disrupted and l...

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Abstract

The invention provides a novel tumour serum marker and application thereof, particularly relates to application of autoantibodies of POTE protein being taken as tumour serum markers, further relates to application of a reagent for detecting the autoantibodies of the POTE protein in preparation of a composition for detection, auxiliary diagnosis and / or prognosis judgement of cancers, and meanwhile relates to the reagent for detecting the autoantibodies of the POTE protein, a method for detecting the autoantibodies of the POTE protein, and a method for enriching and purifying the autoantibodies of the POTE protein. Besides, the invention further relates to the POTE autoantibodies obtained after the enrichment and the purification according to the method, and application of the POTE autoantibodies.

Description

technical field [0001] The present invention relates to a new tumor serum marker and its application, and belongs to the fields of biotechnology and medicine. Specifically, the present invention relates to a new autoantibody of the tumor serum marker POTE, the sequence and the corresponding recognition antigen of the autoantibody. The recombinant technology of the antigen protein, the preparation method of the affinity chromatography column for enriching and purifying the POTE autoantibody, the method of enriching and purifying the autoantibody from human serum and the technical method of detecting the POTE autoantibody in human serum. The invention also discloses the use of the POTE autoantibody and the corresponding detection method, such as for diagnosing and treating tumors. Background technique [0002] Cancer is currently the second leading cause of death in the world. In 2008, there were approximately 12.7 million new cases and 7.6 million deaths due to cancer. Desp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/574G01N1/34C07K16/18C07K1/22
CPCC07K16/18G01N33/531G01N33/57484G01N33/6893G01N2800/7028
Inventor 邵根泽朱柏力李莉王亚清王晓珍蔡小青林明张沙周柔丽严考文匡静宇易娟卢广
Owner PEKING UNIV
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