Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof

A monoclonal antibody, human calcitonin technology, applied in the field of immunoassay medicine, can solve the problems of complex preparation process, low product yield and high cost, and achieve the effects of good specificity, high purity and high titer

Active Publication Date: 2012-10-03
重庆业为基生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In the prior art, most of the preparation methods of PCT are directly taken from tissue cells, such as parathyroid gland tissue, the cost is high, the preparati

Method used

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  • Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof
  • Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof
  • Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof

Examples

Experimental program
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Example Embodiment

[0031] Example 1 Preparation of PCT recombinant protein

[0032] On the basis of obtaining the PCT coding gene from GENBANK, we carried out codon preference modification, chemically synthesized the coding gene fragment, cloned into the prokaryotic expression vector, and induced protein expression after DNA sequencing identification. After identification of the nature, the PCT recombinant protein was purified in large quantities. The protein can be used as an immunogen and screening agent for antibody preparation, and can be used as a calibrator when establishing PCT quantitative detection in subsequent experiments. Specifically:

[0033] 1. Gene cloning of PCT recombinant protein:

[0034] Obtain the human PCT protein gene sequence (accession number NM_004102) from GENBANK and submit it to the Graphical codon usage analyzer (http: / / guca.schoedl.del) to analyze its codon bias; specifically, the human PCT Gene codon usage rate in E. coli The synonymous substitution of <10% is the pre...

Example Embodiment

[0063] Example 2 Preparation of procalcitonin B cell epitope peptide

[0064] The sequence of the chemically synthesized B cell epitope peptide segment is derived from the results of bioinformatics analysis, comprehensively analyzing the secondary structure, antigenicity, hydrophobicity, accessibility and flexibility of the PCT protein, and analyzing and scoring each segment. The region with high score is selected as the B cell epitope region. Specifically, use Chou & Fasman to predict β-turn, Emini method to predict antigen surface accessibility, Karplus& Schulz method to predict protein flexibility, Kolaskar & Tongaonkar protein antigenicity analysis, Parker method protein hydrophobicity analysis, and Beppred linear epitope prediction techniques Parameters to obtain potential B cell epitope peptides in human PCT protein;

[0065] Said screening of B cell epitope peptides, wherein the chemically synthesized epitope peptides are: N-- DMSSDLERDHRPHV—C (SEQ ID NO: 3). The chemical ...

Example Embodiment

[0068] Example 3 Preparation and identification of monoclonal antibodies

[0069] 1. Immunization of Balb / c mice with a derivative of procalcitonin B cell epitope peptide

[0070] The procalcitonin B cell epitope peptide antigen obtained in Example 2 was taken out from the refrigerator at -80°C as an antigen, dissolved and filtered.

[0071] 6-week-old female Balb / c mice weighing about 20g were selected for immunization. Antigen emulsification uses the double syringe mutual push method. For the first immunization, the procalcitonin B cell epitope peptide antigen shown in SEQ ID NO: 4 was emulsified and mixed with an equal volume of Freund's complete adjuvant to obtain an antigen mixture. Each mouse was skinned in an amount of 100 μg. Add intraperitoneal injection at more points. The second and third immunizations were performed on the 14th and 28th days. The adjuvant was changed to incomplete Freund's adjuvant. The antigen amount, injection volume and route were unchanged. After ...

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Abstract

The invention belongs to the field of immunology in medicine, and particularly relates to an application of a procalcitonin B cell epitope peptide fragment and a monoclonal antibody thereof; the epitope peptide fragment is shown in SEQ ID NO:3, and can be used for preparing hybridoma cells and secreting a corresponding monoclonal antibody; the monoclonal antibody can be used for preparing a diagnostic reagent for procalcitonin detection; the monoclonal antibody has purity of up to above 96%, has a titer of up to 1:256000, and has the advantages of good specificity, suitability for mass preparation, and the like; the monoclonal antibody, multiclonal antibody prepared according to the invention can be used for detection of patient blood procalcitonin content, and determination can be performed by using a double-antibody sandwich ELISA reaction mode, wherein a 'double-antibody sandwich' structure is formed by an enzyme-labeled human anti-PCT monoclonal antibody, an enzyme-label plate coated anti-PCT multiclonal antibody, and a PCT antigen of a sample to be detected.

Description

technical field [0001] The invention relates to the field of immunoanalysis medicine, in particular to the application of cell epitope peptide antigen and monoclonal antibody thereof. Background technique [0002] As long as bacterial infections can be detected, diagnosed, and treated early, most of them have a good prognosis. Otherwise, severe bacteremia and / or severe sepsis may develop. According to the US CDC report, sepsis has become the leading cause of non-cardiac ICU death ( N Engl J Med. 2003;348: 1546-1554 ), so early diagnosis of infectious inflammation is extremely important. In addition, with the increase of bacterial drug resistance and severe infection patients, how to guide clinical medication is becoming more and more important. [0003] Procalcitonin (PCT) is a specific marker of bacterial and fungal infections discovered in the 1990s. It is a non-hormone active calcitonin propeptide, consisting of 116 amino acids, 1~ 57 is the N-stump, 60-91 are calcito...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K19/00C07K16/26C12N5/20G01N33/74C12R1/91
Inventor 黄洪涛陈安易维京石延宾姚静张宪胡伟魏勇
Owner 重庆业为基生物科技有限公司
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