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CN-122011154-A - Preparation method of intestinal fatty acid binding protein IFABP antigen

CN122011154ACN 122011154 ACN122011154 ACN 122011154ACN-122011154-A

Abstract

The invention provides a preparation method of an intestinal fatty acid binding protein IFABP antigen, which comprises the following steps of S1, encoding a gene sequence of human IFABP mature peptide based on NCBI accession number NP_000125.2, designing the mature peptide into a signal peptide-6 XHis-connecting peptide-IFABP-YINK containing signal peptide dimer protein, carrying out codon optimization on the amino acid sequence to obtain a nucleic acid sequence, S2, cloning the optimized gene into a eukaryotic expression vector to construct a recombinant expression plasmid, and carrying out cell culture and purification after transfection on the recombinant plasmid to obtain the IFABP antigen, wherein S3, the recombinant plasmid is transfected into a mammalian cell 293F cell. The preparation method can obtain the high-purity and high-activity IFABP recombinant antigen in a large scale with high efficiency, and further obtain the high-performance anti-IFABP monoclonal antibody.

Inventors

  • JIA RUI
  • Bai yilin
  • JIA YANGYANG

Assignees

  • 河南省儿童医院郑州儿童医院

Dates

Publication Date
20260512
Application Date
20260209

Claims (6)

  1. 1. A method of preparing an intestinal fatty acid binding protein IFABP antigen comprising the steps of: S1, encoding a gene sequence of human IFABP mature peptide based on NCBI accession number NP_000125.2, wherein the mature peptide is designed as a signal peptide-6 XHis-connecting peptide-IFABP-YINK dimer protein containing the signal peptide, is named as dIFABP, the amino acid sequence is shown as SEQ ID NO. l, and the amino acid sequence is subjected to codon optimization to obtain a nucleic acid sequence shown as SEQ ID NO. 2; s2, cloning the optimized gene into a eukaryotic expression vector pcDNA3.1 (+) vector to construct a recombinant expression plasmid pcDNA3.1 (+) -dIFABP; S3, the recombinant plasmid is transfected into a mammalian cell 293F cell, and the cell culture is carried out after transfection, and the IFABP antigen is obtained after purification.
  2. 2. The method for producing an intestinal fatty acid-binding protein IFABP antigen according to claim 1, wherein the cells are placed in a carbon dioxide incubator for 2-5 days after transfection and subjected to purification treatment.
  3. 3. A method of producing an intestinal fatty acid binding protein IFABP antigen according to claim 1, wherein the purification step is carried out at 4 ℃.
  4. 4. The method for preparing an intestinal fatty acid binding protein IFABP antigen according to claim 1, wherein the purification step comprises centrifugation, supernatant taking, filtration, purification of filtrate by using a high affinity nickel ion affinity chromatography medium FF, preliminary purification of His-tagged fusion protein by using imidazole gradient elution, collection of elution peaks to obtain an IFABP protein solution, fine purification of the IFABP protein solution by molecular sieve chromatography while displacement into a buffer solution, and endotoxin removal column treatment to reduce endotoxin level below <0.1 EU/. Mu.g protein.
  5. 5. The method of claim 1, wherein the cell viability is greater than 95% after transfection and before purification.
  6. 6. The method for producing an intestinal fatty acid-binding protein IFABP antigen according to claim 1, wherein the culture conditions in the carbon dioxide incubator are 37℃and 5% CO 2 and 100-150-rpm shaker.

Description

Preparation method of intestinal fatty acid binding protein IFABP antigen Technical Field The invention relates to the field of biotechnology and medical immunology, in particular to a preparation method of an Intestinal Fatty Acid Binding Protein (IFABP) antigen for early diagnosis of related diseases of intestinal mucosa injury. Background The Intestinal Fatty Acid Binding Protein (IFABP) is a small molecular protein which specifically exists in intestinal epithelial cell cytoplasm, is used as a sensitive index of intestinal mucosa barrier integrity, and has important diagnostic value in various intestinal diseases and injury states. When the intestinal mucosa is damaged due to ischemia, infection, inflammation, trauma or surgery, the IFABP can be rapidly released into blood and intestinal lumen, and becomes an early, sensitive and specific biomarker of the intestinal mucosa damage. Detection of IFABP has wide application in a variety of clinical settings including, but not limited to, acute gastroenteritis (viral or bacterial infection), intestinal ischemia, inflammatory Bowel Disease (IBD), neonatal Necrotizing Enterocolitis (NEC), abdominal trauma or post-operative bowel function assessment, and the like. The IFABP level is detected early and accurately, and the method has important significance for timely diagnosis, disease monitoring, curative effect evaluation and prognosis judgment of the diseases. Currently, NEC diagnosis based on IFABP mainly depends on commercial immunodetection kits, but the preparation technology of a core raw material, namely a high-performance anti-IFABP monoclonal antibody, is mostly monopolized by foreign companies. Antibodies used in China have the problems of high cost, unstable shelf life, poor performance (such as affinity and specificity) and the like. In addition, the information of specific epitopes recognized by the existing antibodies is ambiguous, which limits further optimization of the performance of the detection kit (e.g., selection of the paired antibodies, improvement of detection sensitivity) and development of novel detection methods (e.g., epitope-specific sensors). In the aspect of antigen preparation, natural IFABP has the advantages of high extraction difficulty from tissues, low purity and limited yield. The conventional recombinant expression method often faces the problems of inclusion body formation, poor solubility, complicated purification steps, incomplete endotoxin removal and the like, and influences the quality of the subsequent immunogens and the antibody screening efficiency. Therefore, the method for designing and developing the high-efficiency and large-scale IFABP recombinant antigen with high purity and high activity has important practical application value and strategic significance for breaking technical monopoly and developing domestic high-performance NEC diagnostic reagent. Disclosure of Invention The invention aims at overcoming the defects of the prior art, and provides a preparation method of an intestinal fatty acid binding protein IFABP antigen. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: A method of preparing an intestinal fatty acid binding protein IFABP antigen comprising the steps of: s1, encoding a gene sequence of human IFABP mature peptide based on NCBI accession number NP_000125.2, wherein the mature peptide is designed as a signal peptide-6 XHis-connecting peptide-IFABP-YINK dimer protein containing the signal peptide, and is named as dIFABP, the amino acid sequence is shown as SEQ ID NO. l, and the amino acid sequence is subjected to codon optimization to obtain a nucleic acid sequence shown as SEQ ID NO. 2; s2, cloning the optimized gene into a eukaryotic expression vector pcDNA3.1 (+) vector to construct a recombinant expression plasmid pcDNA3.1 (+) -dIFABP; S3, the recombinant plasmid is transfected into a mammalian cell 293F cell, and the cell culture is carried out after transfection, and the IFABP antigen is obtained after purification. After transfection, the cells are placed into a carbon dioxide incubator for aseptic culture for 2-5 days, and then are subjected to purification treatment. The purification step was performed at 4 ℃. The purification steps comprise centrifugation, supernatant taking, filtration, purification of filtrate by adopting a high-affinity nickel ion affinity chromatography medium FF, preliminary purification of His-tagged fusion protein by utilizing imidazole gradient elution, collection of elution peaks to obtain an IFABP protein solution, fine purification of the IFABP protein solution by molecular sieve chromatography, simultaneous replacement of the IFABP protein solution into a buffer solution, and endotoxin removal column treatment to reduce the endotoxin level to below 0.1 EU/mug protein. After transfection and before purification, the cell viability was greater than 95%. The culture conditions of the carbon dioxide incub