B-cell epitope peptide of heart fatty acid binding protein (H-FABP), antibody and applications thereof

A fatty acid combination and B cell technology, applied in the field of medical testing, can solve problems such as no related reports, and achieve good antigenicity and specificity

Active Publication Date: 2012-10-24
重庆业为基生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no relevant report

Method used

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  • B-cell epitope peptide of heart fatty acid binding protein (H-FABP), antibody and applications thereof
  • B-cell epitope peptide of heart fatty acid binding protein (H-FABP), antibody and applications thereof
  • B-cell epitope peptide of heart fatty acid binding protein (H-FABP), antibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, the preparation of H-FABP recombinant protein

[0026] Amplify the nucleotide sequence of the H-FABP gene coding region by reverse transcription PCR, clone it into the prokaryotic expression vector pET28a, induce protein expression after sequencing and identification, and purify the prepared H-FABP recombinant protein after characterization. The recombinant protein can be used It is used to prepare the immunogen and screening source of polyclonal antibodies, and can also be used as a calibrator in the subsequent establishment of H-FABP detection, specifically:

[0027] According to the reported gene sequence of heart type fatty acid-binding protein (heart type fatty acid-binding protein, H-FABP) (Genbank: NM_004102), the nucleotide sequence of its coding region is shown in SEQ ID NO.1. The corresponding polypeptide sequence after translation into amino acids is shown in SEQ ID NO.2, which encodes 133 amino acids, specifically:

[0028] MVDAFLGTWK LVDSKN...

Embodiment 2

[0049] Embodiment 2, the preparation of the B cell epitope peptide of H-FABP

[0050] According to the amino acid sequence of H-FABP, using the Protean software in the DNAStar Lasergene software package, the hydrophilicity, antigenic index and flexible region in the secondary structure of H-FABP were analyzed, and combined with the antigenic index prediction method to predict its B cell epitope. And use Chou & Fasman 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 and other technical parameters, Screening the B cell epitope peptide of H-FABP, specifically amino acid residues at positions 32-47 in SEQ ID NO.2 (QVASMTKPTTIIEKNG, SEQ ID NO.5), in order to improve the interaction between the B cell epitope peptide and the carrier protein Linking ability, a...

Embodiment 3

[0053] Example 3, Preparation and Identification of Monoclonal Antibody

[0054] one, Immunization of Balb / c Mice with B Cell Epitope Peptide Derivatives

[0055] The H-FABP' stored in a -80°C refrigerator in Example 1 was taken out as an antigen, dissolved and filtered. Then select 6-week-old female Balb / c mice weighing about 20 g for immunization. For the first immunization, H-FABP' was emulsified and mixed with an equal volume of Freund's complete adjuvant, and the antigen was emulsified by double syringe push method to obtain the antigen mixture. Each mouse was injected with 100 μg of antigen mixture; the second and third immunizations were carried out on the 14th and 28th days after injection, respectively, the same antigen was used for immunization, the adjuvant was changed to incomplete Freund's adjuvant, and the amount of antigen, injection volume and route were different. Change. After the third immunization, the titer was measured by indirect ELISA method, and...

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Abstract

The invention belongs to the immunology field in medicine and particularly relates to a B-cell epitope peptide of H-FABP and applications of an antibody thereof. The epitope peptide is shown in SEQ ID NO.5 and can be used for preparing hybridoma cell lines and secreting a corresponding monoclonal antibody. The monoclonal antibody is capable of being used for preparing a diagnostic reagent which is used for detecting the H-FABP and prepared on a large scale, provided with purity higher than 96%, good in specificity and the like; and a detection titer of a purified antibody through an indirect enzyme-linked immunosorbent assay (ELISA) reaches 1:256,000. Monoclonal antibodies and polyclonal antibodies prepared through the epitope peptide can be used for H-FABP detection in patients' blood, and a foundation is laid for early diagnosis of acute myocardial infarction (AMI).

Description

[0001] technical field [0002] The invention relates to the field of medical detection, in particular to a heart-type fatty acid binding protein B cell epitope peptide, and also relates to a monoclonal antibody prepared from the epitope peptide and its application. Background technique [0003] Cardiovascular disease is an important disease that seriously threatens human life and health, and has become the number one killer of human health in the 21st century. Around the world, 17.5 million people die from heart disease and other cardiovascular diseases every year, accounting for about 1 / 3 of the global death toll. It is expected that this number will exceed 25 million by 2020, when cardiovascular diseases will become the primary cause of human death and disability. reason. Acute myocardial infarction (AMI) is the main type of cardiovascular disease, with a sudden onset and a mortality rate of about 30% in the acute phase. In my country, the incidence of acute myocardial ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K19/00C12N5/20C07K16/18G01N33/68G01N33/577
Inventor 黄洪涛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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