Preparation method of intestinal fatty acid binding protein IFABP antigen

Abstract

The invention provides a preparation method of an intestinal fatty acid binding protein IFABP antigen, which comprises the following steps: S1, designing a mature peptide into a signal peptide-6 * His-linker peptide-IFABP-linker peptide-IFABP-YINK-containing signal peptide dimer protein based on a gene sequence of an NCBI (National Center of Biotechnology Information) login number NP000125.2 encoding human IFABP mature peptide, and performing codon optimization on an amino acid sequence of the signal peptide-6 * His-linker peptide-IFABP-linker peptide-IFABP-YINK to obtain a nucleic acid sequence; s2, cloning the optimized gene into a eukaryotic expression vector, and constructing a recombinant expression plasmid; and S3, transfecting the recombinant plasmid into a mammalian cell 293F, carrying out cell culture after transfection, and purifying to obtain the IFABP antigen. According to the preparation method, the high-purity and high-activity IFABP recombinant antigen can be efficiently obtained on a large scale, and then the high-performance anti-IFABP monoclonal antibody is obtained.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medical immunology, and more specifically, to a method for preparing an intestinal fatty acid-binding protein (IFABP) antigen for the early diagnosis of diseases related to intestinal mucosal injury. Background Technology

[0002] Intestinal fatty acid-binding protein (IFABP) is a small molecule protein specifically found in the cytoplasm of intestinal epithelial cells. As a sensitive indicator of intestinal mucosal barrier integrity, it 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, IFABP can be rapidly released into the blood and intestinal lumen, becoming an early, sensitive, and specific biomarker of intestinal mucosal injury.

[0003] IFABP testing has wide applications in various clinical scenarios, including but not limited to: acute gastroenteritis (viral or bacterial infection), intestinal ischemia, inflammatory bowel disease (IBD), necrotizing enterocolitis (NEC) in newborns, and intestinal function assessment after abdominal trauma or surgery. Early and accurate detection of IFABP levels is of great significance for the timely diagnosis, disease monitoring, treatment evaluation, and prognosis of the above-mentioned diseases.

[0004] Currently, NEC diagnostics based on IFABP mainly rely on commercially available immunoassay kits. However, the preparation technology of its core raw material—high-performance anti-IFABP monoclonal antibodies—is largely monopolized by foreign companies. Antibodies used domestically suffer from high costs, unstable delivery times, or poor performance (such as affinity and specificity). Furthermore, the specific antigenic epitopes recognized by existing antibodies are unclear, which limits further optimization of the test kit performance (such as the selection of paired antibodies and improvement of detection sensitivity) and the development of novel detection methods (such as epitope-specific sensors).

[0005] In terms of antigen preparation, natural IFABP is difficult to extract from tissues, has low purity, and limited yield. Conventional recombinant expression methods often face problems such as inclusion body formation, poor solubility, cumbersome purification steps, and incomplete removal of endotoxins, which affect the quality of subsequent immunogens and the efficiency of antibody screening.

[0006] Therefore, designing and developing an efficient and scalable method for obtaining high-purity, high-activity IFABP recombinant antigens is of significant practical value and strategic importance for breaking the technological monopoly and developing domestically produced high-performance NEC diagnostic reagents. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing intestinal fatty acid-binding protein IFABP antigen.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing intestinal fatty acid-binding protein IFABP antigen, comprising the following steps: S1. Based on the gene sequence encoding the mature human IFABP peptide from NCBI accession number NP_000125.2; the mature peptide was designed as a signal peptide-6×His-linker peptide-IFABP-linker peptide-IFABP-YINK dimer protein containing the signal peptide, named dIFABP, whose amino acid sequence is shown in SEQ ID NO. 1. The amino acid sequence was optimized by codons to obtain the nucleic acid sequence shown in SEQ ID NO: 2. S2. The optimized gene was cloned into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant expression plasmid pcDNA3.1(+)-dIFABP; S3. The recombinant plasmid was transfected into mammalian 293F cells, and after transfection, the cells were cultured and purified to obtain the IFABP antigen.

[0009] After transfection, the cells are placed in a carbon dioxide incubator for sterile culture for 2-5 days before purification.

[0010] The purification process was carried out at 4°C.

[0011] The purification steps included centrifugation, collection of supernatant, and filtration. The filtrate was purified using high-affinity nickel ion affinity chromatography medium FF. The His-tagged fusion protein was initially purified by elution using an imidazole gradient. The elution peak was collected to obtain the IFABP protein solution. The IFABP protein solution was then finely purified by molecular sieve chromatography, while being replaced with buffer. An endotoxin removal column was used to reduce the endotoxin level to below <0.1 EU / μg protein.

[0012] The cell viability was greater than 95% both after transfection and before purification.

[0013] The incubation conditions for the carbon dioxide incubator are 37℃, 5% CO2, and a shaker at 100-150 rpm.

[0014] This invention has outstanding substantive features and significant progress compared with the prior art. Specifically, this invention provides a method for preparing intestinal fatty acid binding protein IFABP antigen. By adopting a soluble expression strategy and a two-step purification method of fusion tag-enzyme digestion, high-purity and high-activity IFABP recombinant antigen can be obtained efficiently and on a large scale, with extremely low endotoxin levels, which can meet the standards of high-quality immunogen and diagnostic antigen. Attached Figure Description

[0015] Figure 1 The diagram shows the design and structural model of the recombinant human IFABP protein in this embodiment.

[0016] Figure 2 This is a diagram showing the purification results of the target protein in the example.

[0017] Figure 3 The images show the final SDS-PAGE and Western blot analysis results of the proteins in the examples.

[0018] Figure 4 This is a graph showing the reactivity assay of the target protein with IFABP antibody in the examples. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below through specific embodiments. Example 1

[0020] A method for preparing intestinal fatty acid-binding protein IFABP antigen, comprising the following steps: S1. The gene used is the mature peptide sequence encoding human IFABP (NCBI Protein ID: NP_000125.2). It was designed as a signal peptide-6×His-linker peptide-IFABP-linker peptide-IFABP-YINK dimer protein, named dIFABP. The amino acid sequence is shown in SEQ ID NO: 1. Its structure was modeled using de novo modeling, and the structural model is shown in [link to structural model]. Figure 1 Its amino acid sequence was codon-optimized to adapt to high-efficiency expression in mammalian human embryonic kidney 293 (HEK293) cells. The optimized nucleic acid sequence is shown in SEQ ID NO: 2.

[0021] The amino acid sequence of the mature IFABP peptide is as follows: AFDSTWKVDRSENYDKFMEKMGVNIVKRKLAAHDNLKLTITQEGNKFTVKESSAFRNIEVVFELGVTFNYNLADGTELRGTWSLEGNKLIGKFKRTDNGNELNTVREIIGDELVQTYVYEGVEAKRIFKKD; the signal peptide sequence is: GWSCIILFLVATATGVHSA; the LINKER sequence is GGGGSGGGGSGGGGS; S2. The optimized gene was cloned into the eukaryotic expression vector pcDNA3.1(+). In this vector, the target gene was positioned downstream of the CMV enhancer. A 6×His tag was fused to the C-terminus of the gene.

[0022] S3. Transfect transiently using PEI transfection reagent. For every 20 mL culture system, dilute 20 μg of recombinant plasmid with 60 μL of PEI (1 mg / mL) in 1 mL of serum-free medium (SMM 293-TII Expression Medium (Serum-free, complete medium), Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number RZ7OC1301-A). Vortex to mix, incubate at room temperature for 10 minutes, and then add to 20 mL of mammalian 293F cells while shaking. The viable cell density at transfection is approximately 3 × 10⁻⁶ cells / mL. 6 cells / mL, viability >95%; the transfected 293F cells were then placed in FreeStyle... TM In 293 Expression Medium, after 72 hours of culture at 37°C, 5% CO2, and 120 rpm on a shaker, the viable cell density was approximately 6 × 10⁻⁶. 6 When the cell count / mL is >95%, purification is initiated.

[0023] Mammalian Cell 293F using FreeStyle TM 293-F suspension cells (Zhejiang Meisen Cell Technology Co., Ltd., catalog number: CTCC-001-0430).

[0024] The purification process is carried out in a 4°C cold room or chromatography cabinet, and specifically includes the following steps: Step 1: Clarification and Filtration Centrifuge at 3000×g for 30 minutes and collect the cell supernatant; filter the cell culture supernatant sequentially through 0.45 μm and 0.22 μm PES membrane filters to remove cell debris and particulate matter.

[0025] Step 2: Affinity chromatography (Ni-NTA) Chromatographic column: HisTrap TM HP, 5 mL (Catalog No.: 17-5248-02, Cytiva).

[0026] Equilibration buffer: Buffer A: 20 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 7.4.

[0027] Loading: Load the filtered supernatant into a column that has been equilibrated with Buffer A at a flow rate of 5 mL / min.

[0028] Wash: Wash with 10-15 column volumes of Buffer A until the UV absorption baseline is stable.

[0029] Elution: Linear gradient elution was performed using Buffer B (20 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 7.4) containing 250 mM imidazole, and the eluted fractions corresponding to the UV absorption peaks were collected.

[0030] Buffer replacement: Replace the elution fraction with a PD-10 desalting column or ultrafiltration tube into the Strep-Tactin affinity chromatography equilibration buffer.

[0031] Step 3: Molecular sieve chromatography (fine purification) Column: HiLoad TM 16 / 600 Superdex TM 75 pg (Catalog No.: 28-9893-33, Cytiva).

[0032] Run buffer: 1×PBS, pH 7.4.

[0033] Sample loading and collection: Concentrate the sample collected in step 2 to 3 mL and load it at a flow rate of 1 mL / min. Collect the main peak corresponding to the monomeric IFABP protein based on the UV absorption chromatogram. Note: The concentration range of the sample collected in step 2 can be between 2 and 5 mL.

[0034] Endotoxin removal: The purified protein solution was passed through ToxiErase. TM Endotoxin Removal Resin, treated with G-Biosciences, reduced endotoxin levels to below <0.1 EU / μg protein.

[0035] Concentration and buffer replacement: Using an Amicon® Ultra-15 Centrifugal Filter Unit, 10kDa MWCO, Merck ultrafiltration centrifuge tube, the protein solution was concentrated to the target concentration and replaced with final storage buffer (1×PBS, pH 7.4).

[0036] Protein concentration assay: using NanoDrop TM The One micro spectrophotometer measured the absorbance at a wavelength of 280 nm, and the final concentration of the IFABP protein solution was 0.43 mg / mL.

[0037] Purity analysis: 5 μg of purified protein was analyzed by 12% SDS-PAGE. The test results are shown below. Figure 2 After Coomassie brilliant blue staining, it showed a single main band with a purity >95%. Figure 2 In the diagram, lane M: protein marker; lane Me: cell culture medium; lane FT: flow-through medium; lanes W1 / 2: washing medium; lanes 1-8: elution medium. Molecular weight uniformity analysis: Size exclusion chromatography-high performance liquid chromatography was used for analysis. A single symmetrical peak was observed on the TSKgel G2000SWXL column, indicating that the protein is in monomeric form and there are no significant aggregates.

[0038] Endotoxin detection: The endotoxin content was <0.1 EU / μg protein, detected using the Limulus amebocyte lysate (LAL) reagent colorimetric method.

[0039] The final protein was analyzed by SDS-PAGE and Western blot (HRP-labeled 6×His, His-Tag monoclonal antibody). Results are as follows: Figure 3 As shown, the final results of SDS-PAGE and Western blot of the target protein are as follows: Lane M: Protein Marker; Lane 1: Reduced target protein; Channel 2: Unreduced target protein.

[0040] Reactivity assay: 5 μg of purified protein was subjected to 12% SDS-PAGE electrophoresis followed by Western blotting. A commercially available IFABP antibody was used as the primary antibody. The results showed that the recombinant dIFABP protein expressed in this study exhibited good reactivity with the IFABP antibody. See the attached table for test results. Figure 4 . Figure 4 In the diagram, lane M represents the protein marker, and lane 1 represents the recombinant target protein dIFABP. Example 2

[0041] This embodiment is basically the same as Example 1, except that: in the purification steps, step 4 uses a Ni-NTASuperflow gravity column (catalog number: 30230, Qiagen); step 5 uses a solution containing 0.1% Triton X-114 for phase partitioning to reduce the endotoxin level to below <0.1 EU / μg protein.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A method for preparing intestinal fatty acid-binding protein IFABP antigen, comprising the following steps: S1. Based on the gene sequence encoding the mature human IFABP peptide of NCBI accession number NP_000125.2; the mature peptide was designed as a signal peptide-6×His-linker peptide-IFABP-linker peptide-IFABP-YINK containing a signal peptide dimer protein, named dIFABP, and the amino acid sequence is shown in SEQ ID NO.

1. The amino acid sequence was optimized by codons to obtain the nucleic acid sequence shown in SEQ ID NO:2; S2. The optimized gene was cloned into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant expression plasmid pcDNA3.1(+)-dIFABP; S3. The recombinant plasmid was transfected into mammalian 293F cells, and after transfection, the cells were cultured and purified to obtain the IFABP antigen.

2. The method for preparing intestinal fatty acid-binding protein IFABP antigen according to claim 1, characterized in that: After transfection, the cells are placed in a carbon dioxide incubator for sterile culture for 2-5 days before purification.

3. The method for preparing intestinal fatty acid-binding protein IFABP antigen according to claim 1, characterized in that: The purification process was carried out at 4°C.

4. The method for preparing intestinal fatty acid-binding protein IFABP antigen according to claim 1, characterized in that: The purification steps include centrifugation, supernatant collection, and filtration. The filtrate is purified using high-affinity nickel ion affinity chromatography medium FF. The His-tagged fusion protein is initially purified by elution using an imidazole gradient. Collect the elution peaks to obtain the IFABP protein solution; IFABP protein solutions were finely purified by molecular sieve chromatography and simultaneously replaced with buffer solution; endotoxin removal column treatment was used to reduce endotoxin levels to below <0.1 EU / μg protein.

5. The method for preparing intestinal fatty acid-binding protein IFABP antigen according to claim 1, characterized in that: The cell viability was greater than 95% both after transfection and before purification.

6. The method for preparing intestinal fatty acid-binding protein IFABP antigen according to claim 1, characterized in that: The incubation conditions for the carbon dioxide incubator are 37℃, 5% CO2, and a shaker at 100-150 rpm.

Images