Human intragastric factor protein polyclonal antibody and preparation method thereof

Abstract

The invention discloses a human intragastric factor protein polyclonal antibody and a preparation method thereof. The preparation method comprises the steps of artificial antigen design, animal immune optimization and antibody purification and verification. Accurate epitope design is achieved, structural interference of natural GIF is avoided through artificial polypeptide, and the cross reaction rate is lt; 5%; an efficient immune strategy is adopted, so that the immune period is shortened to 8-10 weeks, and the titer is increased by 2-3 times; low-cost production is realized, natural protein is replaced by the synthesized polypeptide, and the cost is reduced by 60%; the specificity is high, the cross reaction is reduced due to the design of the artificial antigen, and verification data supports specific recognition of natural GIF; large-scale production, polypeptide synthesis and a double-stage purification process are adopted, so that the consistency between batches is high (CV is less than or equal to 8%); the application range is wide, and diagnosis, pathological research and treatment monitoring scenes are covered.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a polyclonal antibody against human gastric intrinsic factor protein and a preparation method thereof. Background Art

[0002] GIF is a glycoprotein secreted by gastric parietal cells and is responsible for the absorption of vitamin B12. Its abnormality is related to diseases such as pernicious anemia and autoimmune gastritis. GIF autoantibodies (such as type I blocking antibodies) are key markers for diagnosing such diseases. However, the existing technology has the following defects:

[0003] Cross-reaction problem: Traditional polyclonal antibodies are prone to cross-react with similar proteins such as pepsinogen, affecting the detection accuracy.

[0004] Insufficient antigen design: The extraction cost of natural GIF is high and it contains interfering epitopes (such as the glycosylation site Asn-120).

[0005] Complex process: Existing methods rely on multiple immunizations and complex purification steps (such as multiple affinity chromatographies), with a long cycle and high cost. Summary of the Invention

[0006] The purpose of the present invention is to provide a polyclonal antibody against human gastric intrinsic factor protein and a preparation method thereof, to solve the problems of cross-reaction, insufficient antigen design, and complex process existing in the existing technology.

[0007] To achieve the above purpose, the present invention proposes a method for preparing a polyclonal antibody against human gastric intrinsic factor protein, including:

[0008] S1. Artificial antigen design: Predict the linear epitopes of GIF (such as amino acid sequences 25-40, 150-165) through bioinformatics, screen epitopes by avoiding glycosylation sites (such as Asn-120), chemically synthesize polypeptide fragments containing the target epitopes, and couple them to KLH or BSA carrier proteins through the Sulfo-SMCC or mixed anhydride method to enhance immunogenicity for polypeptide synthesis and coupling;

[0009] S2. Animal immunization optimization: The experimental animals are New Zealand white rabbits or alpacas. Considering both antibody yield and affinity, the immunization program includes primary immunization with 200 μg of antigen + Freund's complete adjuvant, subcutaneous multi-point injection, booster immunization every 2 weeks with the dose decreasing to 100 μg, and then changing to Freund's incomplete adjuvant. For titer monitoring, blood is collected when the ELISA titer ≥ 1:10^6 after the 4th immunization;

[0010] S3. Antibody Purification and Verification: Two-stage purification, Protein A / G affinity chromatography for crude IgG extraction, antigen-specific affinity chromatography, and further purification using immobilized synthetic polypeptide packing material. Verification indicators include ELISA for detecting antibody affinity (EC50 ≤ 10 nM), Western Blot for verifying specific binding to native GIF, and immunohistochemistry for localizing GIF expression in gastric tissue sections.

[0011] SEQ ID NO:1: Amino acid sequence of the GIF antigen;

[0012] SEQ ID NO:2: DNA sequence encoding the GIF antigen (if recombinant expression is involved).

[0013] The present invention has the following beneficial effects: Precise epitope design, artificial polypeptides avoid structural interference of native GIF, and the cross-reactivity rate < 5%; Efficient immunization strategy, the immunization period is shortened to 8 - 10 weeks, and the titer is increased by 2 - 3 times; Low-cost production, synthetic polypeptides replace natural proteins, and the cost is reduced by 60%; High specificity, artificial antigen design reduces cross-reactivity, and verification data supports specific recognition of native GIF; Large-scale production, synthetic polypeptides and two-stage purification process, with high batch-to-batch consistency (CV ≤ 8%); Wide application, covering diagnostic, pathological research, and treatment monitoring scenarios. Detailed Embodiment

[0014] The following further elaborates on the present invention in conjunction with embodiments, enabling those skilled in the art to implement it with reference to the text of the specification.

[0015] The present invention provides a method for preparing a polyclonal antibody against human gastric intrinsic factor protein, including:

[0016] S1. Artificial Antigen Design: Predict the linear epitopes of GIF (such as amino acid sequences 25 - 40, 150 - 165) through bioinformatics, screen epitopes by avoiding glycosylation sites (such as Asn-120), chemically synthesize polypeptide fragments containing the target epitopes, and couple them to KLH or BSA carrier proteins through Sulfo-SMCC or mixed anhydride methods to enhance immunogenicity for polypeptide synthesis and coupling;

[0017] S2. Animal Immunization Optimization: Experimental animals are New Zealand white rabbits or alpacas, considering both antibody yield and affinity. Immunization procedure: Primary immunization, 200 μg antigen + Freund's complete adjuvant, subcutaneous multi-point injection; Booster immunization, once every 2 weeks, with the dose decreasing to 100 μg, and changing to Freund's incomplete adjuvant; Titer monitoring, blood collection is performed when the ELISA titer ≥ 1:10^6 after the 4th immunization;

[0018] S3. Antibody Purification and Verification: Two-stage purification, Protein A / G affinity chromatography for crude IgG extraction, antigen-specific affinity chromatography, and further purification using immobilized synthetic polypeptide packing material. Verification indicators include ELISA for detecting antibody affinity (EC50 ≤ 10 nM), Western Blot for verifying specific binding to native GIF, and immunohistochemistry for localizing GIF expression in gastric tissue sections.

[0019] SEQ ID NO:1: Amino acid sequence of the GIF antigen;

[0020] SEQ ID NO:2: DNA sequence encoding the GIF antigen (if recombinant expression is involved).

[0021] The present invention has the following beneficial effects: Precise epitope design, artificial polypeptide avoiding the structural interference of native GIF, with a cross-reactivity rate < 5%; Efficient immunization strategy, immunization cycle shortened to 8 - 10 weeks, and titer increased by 2 - 3 times; Low-cost production, synthetic polypeptide replacing natural protein, cost reduced by 60%; High specificity, artificial antigen design reducing cross-reactivity, and verification data supporting specific recognition of native GIF; Large-scale production, synthetic polypeptide and two-stage purification process, with high batch-to-batch consistency (CV ≤ 8%); Wide application, covering diagnostic, pathological research, and therapeutic monitoring scenarios.

[0022] Example 1:

[0023] Antigen preparation, synthetic polypeptide sequence: NH2-Cys-Gly-Pro-Ser-Leu-Tyr-...-Lys-COOH (containing SEQ ID NO:1 and NO:2), coupled to KLH carrier through Sulfo-SMCC.

[0024] Example 2:

[0025] Animal immunization, immunization protocol for New Zealand rabbits: Boost every 2 weeks after the primary immunization, and the ELISA titer reaches 1:1.28×10^6 at the 6th week.

[0026] Example 3:

[0027] Antibody purification, after initial purification with a Protein A column, purified by an antigen affinity column, and SDS-PAGE shows a purity ≥ 95%.

[0028] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. A human intrinsic factor protein polyclonal antibody and a preparation method thereof, comprising: S1. Artificial antigen design: predict the linear epitopes of GIF by bioinformatics, avoid glycosylation sites for epitope screening, chemically synthesize peptide fragments containing target epitopes, couple to KLH or BSA carrier protein by Sulfo-SMCC or mixed anhydride method, and enhance immunogenicity to enable peptide synthesis and coupling; S2. Animal immunization optimization: New Zealand white rabbits or alpacas were used as experimental animals, taking into account both antibody production and affinity. The immunization procedure was as follows: primary immunization, 200 μg antigen + Freund's complete adjuvant, subcutaneous multi-point injection, booster immunization, once every 2 weeks, the dose was gradually reduced to 100 μg, and Freund's incomplete adjuvant was used instead. For titer monitoring, blood was collected when the ELISA titer was ≥1:10^6 after the fourth immunization; S3. Antibody purification and validation: two-stage purification, Protein A / G affinity chromatography, crude IgG extraction, antigen-specific affinity chromatography, further purification by immobilized synthetic peptide filler, validation indicators, ELISA, antibody affinity detection, Western Blot, validation of specific binding with native GIF, immunohistochemistry, localization of GIF expression in gastric tissue sections.