Anti-human carcino-embryonic antigen antibody as well as coding gene and application thereof

A carcinoembryonic antigen antibody, coding gene technology, applied in the field of genetic engineering, can solve the problems of unreported and applied imaging diagnosis, and achieve the effect of high affinity

Inactive Publication Date: 2015-05-20
宁波盛华达科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Currently, imaging diagnosis based on CEA has not been reported and applied

Method used

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  • Anti-human carcino-embryonic antigen antibody as well as coding gene and application thereof
  • Anti-human carcino-embryonic antigen antibody as well as coding gene and application thereof
  • Anti-human carcino-embryonic antigen antibody as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Screening of anti-human CEA protein single-chain antibody

[0032] In this example, a single-chain antibody library was constructed, and the anti-human CEA protein single-chain antibody was screened against the single-chain antibody library.

[0033] The method disclosed in the Chinese patent literature with the publication number CN 1444651 A is used to construct a human single-chain antibody library, and to screen the anti-human CEA protein single-chain antibody in the human single-chain antibody library. The specific implementation process is as follows:

[0034] 1. Amplify human antibody heavy chain and light chain variable region DNA

[0035] Using poly A+RNA (purchased from Clontech) from human bone marrow, human fetal liver, human spleen and human peripheral blood leukocytes as templates, using oligo(dT) and random primers (random primers), using reverse transcriptase kit (purchased from From Clontech), poly A+ RNA was reverse transcribed into cDNA acc...

Embodiment 2

[0134] Embodiment 2 specific detection

[0135] In this example, the single-chain antibody screened in the above-mentioned examples was expressed and purified in a bacterial system, and its specificity was determined.

[0136] 1. Expression and purification of single chain antibody

[0137] Cloning the gene encoding the single-chain antibody hCEA-A into the expression vector pET27b(+) to construct pET27b-hCEAa;

[0138] Transform pET27b-hCEAa into expression bacteria E.coli BL21(DE3), and induce expression with IPTG (0.5mM) according to the method provided by Novagen; in the expressed target protein, the N-terminal of the scFv is the pelB sequence, and the pelB sequence can be The expressed scFv is secreted into the periplasmic space of BL21 (DE3); the C-terminus of the scFv contains an HSV tag and a 6×His tag to facilitate the purification of the target protein; Methods The single-chain antibody against human CEA protein was obtained by separating and purifying with Ni-NTA ...

Embodiment 3

[0149] Example 3 Anti-human CEA protein single-chain antibody detects serum CEA

[0150] In this example, the above anti-human CEA protein single chain antibody was used to detect the CEA tumor marker in the serum of tumor patients.

[0151] The rabbit polyclonal antibody against human CEA (catalogue number sc-20131, H300) was purchased from Santa Cruz Biotechnology, USA. Human CEA protein was purchased from Shanghai Kexing Biotechnology Co., Ltd. Serum samples of tumor patients and normal serum samples were from Hangzhou Armed Police Hospital.

[0152] The ELISA test method is as follows:

[0153] (1) Coat a 96-well plate with a rabbit polyclonal antibody against human CEA, overnight at 2-8°C;

[0154] (2) The coated 96-well plate is sealed with SuperBlock;

[0155] (3) Serum samples (100-fold dilution) or human CEA protein were diluted in 0.02% BSA-physiological saline and then added to the 96-wells that had been coated with rabbit anti-human CEA polyclonal antibody. CE...

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Abstract

The invention discloses an anti-human carcino-embryonic antigen antibody as well as a coding gene and application thereof. The anti-human carcino-embryonic antigen antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of three hypervariable regions CDRLH1, CDRH2 and CDRH3 of the heavy chain variable region are GFAVSSN, HRSGN and VPGFSRGQYEESWYFDL respectively; the amino acid sequences of three hypervariable regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are RASQSIDSYLN, GASNLRN and HQAYSPFT respectively. The anti-human carcino-embryonic antigen antibody disclosed by the invention is a fully humanized anti-human carcino-embryonic antigen antibody, has high affinity with a human carcino-embryonic antigen and can be used for detecting and treating the cancer disease of a human body.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an anti-human carcinoembryonic antigen antibody and its coding gene and application. Background technique [0002] Tumor markers are chemical substances that reflect the presence of tumors. They either do not exist in normal adult tissues but are only found in embryonic tissues, or their content in tumor tissues greatly exceeds that in normal tissues. Their existence or quantitative changes can indicate the nature of tumors, so as to understand the histogenesis and cell differentiation of tumors , cell function, to help tumor diagnosis, classification, prognosis judgment and treatment guidance. [0003] In the fight against malignant tumors, doctors, patients and family members are often most concerned about how to detect the occurrence, development and metastasis of tumors as early as possible. The development of diagnostic instruments and technologies such as X-ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/63G01N33/574A61K49/00
Inventor 王炜侯伟王丽江余乃绚
Owner 宁波盛华达科技有限公司
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