Application of B cell epitope peptide of human PCT (Procalcitonin) and monoclonal antibody of B cell epitope peptide

A monoclonal antibody and B cell technology, applied in the field of immunoanalysis medicine, can solve the problems of low product yield, complicated preparation process, and high cost, and achieve high titer, high purity, and good specificity

Active Publication Date: 2012-08-22
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

AI Technical Summary

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

Method used

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  • Application of B cell epitope peptide of human PCT (Procalcitonin) and monoclonal antibody of B cell epitope peptide
  • Application of B cell epitope peptide of human PCT (Procalcitonin) and monoclonal antibody of B cell epitope peptide
  • Application of B cell epitope peptide of human PCT (Procalcitonin) and monoclonal antibody of B cell epitope peptide

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Experimental program
Comparison scheme
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Embodiment 1

[0031] The preparation of embodiment 1 PCT recombinant protein

[0032] 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:

[0033] A PCT recombinant protein gene clone:

[0034] 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

[0063] Example 2 Preparation of B cell epitope peptides of human PCT

[0064] 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 the B cell epitope peptide of human PCT;

[0065]The screening B cell epitope peptide, wherein the chemically synthesized epitope peptide is: N-- cmlgtytqdfnkfh—C (SEQ ID NO: 3), the B cell epitope peptide chemi...

Embodiment 3

[0068] Example 3 Preparation and Identification of Monoclonal Antibody

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

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

[0071] Female Balb / c mice aged 6 weeks and weighing about 20 g were selected for immunization. Antigen emulsification adopts double syringe mutual pushing method. For the first immunization, the 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, which was added intradermally in an amount of 100 μg to each mouse intraperitoneal injection. 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 of antigen, injection volume and route remained unchanged. After the thir...

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Abstract

The invention belongs to the field of immunology in medicines and particularly relates to application of a B cell epitope peptide of a human PCT (Procalcitonin) and a monoclonal antibody of the B cell epitope peptide. The epitope peptide is shown as SEQ ID NO:3 and can be used for preparing a hybridoma cell and secreting a corresponding monoclonal antibody. The monoclonal antibody can be used for preparing a diagnostic reagent for detecting the procalcitonin and has the advantages of purity as high as 98 percent, valence as high as 1:512000, favorable specificity, capability of being prepared in a large batch and the like. The monoclonal antibody and a polyclonal antibody which are prepared by the invention can be used for detecting the content of the PCT in the blood of a patient, for example, a double antibody sandwich ELISA (Enzyme-Linked Immuno Sorbent Assay) reaction mode, namely enzyme-labelled anti-human PCT antibody and an elisa plate to used to coat an anti-PCT polyclonal antibody and a measured sample PCT antigen to form a double antibody sandwich structure for measuring.

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 Applications(China)
IPC IPC(8): C07K7/08C07K19/00C12N5/20C07K16/26G01N33/577
Inventor 黄洪涛陈安石延宾姚静易维京魏勇张宪胡伟
Owner 重庆业为基生物科技集团有限公司
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