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

A monoclonal antibody and procalcitonin technology, which is applied in the field of immunoanalysis medicine, can solve the problems of complex preparation process, low product yield and high cost, and achieve the effect of good specificity, high potency and high purity

Active Publication Date: 2015-04-22
重庆业为基生物科技集团有限公司
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  • Summary
  • 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 preparation process is complicated, and the amount of product obtained is low, which restricts the research and application development of PCT
There is no corresponding research

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

Examples

Experimental program
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Effect test

Embodiment 1

[0030] The preparation of embodiment 1 PCT recombinant protein

[0031] On the basis of obtaining the PCT coding gene from GENBANK, carry out codon bias modification, chemically synthesize the coding gene fragment, clone into the prokaryotic expression vector and induce protein expression after identification by DNA sequencing. The protein can be used as an immunogen and screening agent for antibody preparation, and can also be used as a calibrator for the establishment of PCT quantitative detection in subsequent experiments, specifically:

[0032] A PCT recombinant protein gene clone:

[0033] Obtain the gene sequence of human PCT protein from GENBANK (accession number is NM_004102), submit it to Graphical codon usage analyzer (http: / / guca.schoedl.del) to analyze its codon bias; specifically, human PCT The synonymous substitution of gene codons with a usage rate of <10% in Escherichia coli is replaced by codons preferred by Escherichia coli to make it easier to express in ...

Embodiment 2

[0061] Example 2 Preparation of procalcitonin B cell epitope peptide

[0062] The sequence of the chemically synthesized B cell epitope peptide is from the results of bioinformatics analysis, comprehensively analyzing the secondary structure, antigenicity, hydrophilicity, accessibility and flexibility of the PCT protein, and analyzing and scoring each segment, The region with high score was selected as the B cell epitope region. Specifically, Chou & Fasman was used to predict β-turn angle, 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 Bepipred linear epitope prediction technology. parameters to obtain potential B cell epitope peptides in human PCT protein;

[0063] The screening B cell epitope peptide, wherein the chemically synthesized epitope peptide is: N--apfrsalesspadpa—C (SEQ ID NO: 3), the B cell epito...

Embodiment 3

[0065] Example 3 Preparation and Identification of Monoclonal Antibody

[0066] 1. Immunization of Balb / c mice with peptide derivatives of procalcitonin B cell epitope

[0067] The procalcitonin B-cell epitope peptide antigen obtained in Example 2 was taken out from a -80°C refrigerator as an antigen, dissolved and then filtered.

[0068] Female Balb / c mice aged 6 weeks and weighing about 20 g were selected for immunization. Antigen emulsification adopts double syringe mutual pushing method. When immunizing for the first time, the procalcitonin B cell epitope peptide antigen shown in SEQ ID NO: 3 was emulsified and mixed with an equal volume of Freund's complete adjuvant to obtain an antigen mixture, and each mouse was dermally administered in an amount of 100 μg Add intraperitoneal injection at multiple points. The second and third immunizations were carried out on the 14th and 28th days respectively. The adjuvant was changed to incomplete Freund's adjuvant. The amount o...

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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 1:1024000, and has the advantages of good specificity, suitability for mass preparation, and the like; the monoclonal 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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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K19/00C07K16/26C12N5/20G01N33/74C12R1/91
Inventor 黄洪涛陈安易维京石延宾姚静魏勇张宪胡伟
Owner 重庆业为基生物科技集团有限公司
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