Specific antibody of pepsinogen II as well as preparation method and application thereof

A pepsinogen and specific technology, applied in the biological field, can solve the problems of unfavorable long-distance transportation and popularized detection, large-scale mass production of finished products, unfavorable large-scale mass production, etc. The effect of chemical production and simple structure

Active Publication Date: 2022-06-07
BIOISLAND LAB +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the above-mentioned technology based on the immune system of animals such as mice to produce PGII antibodies and separate and extract them has the following disadvantages: (1) The stability of the antibodies is poor, and it needs to be stored, transported, tested and verified at low temperature (4 degrees Celsius) It is not conducive to long-distance transportation and popularization of detection; (2) large-scale mass product

Method used

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  • Specific antibody of pepsinogen II as well as preparation method and application thereof
  • Specific antibody of pepsinogen II as well as preparation method and application thereof
  • Specific antibody of pepsinogen II as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1: Preparation of antigens

[0095] The DNA sequence encoding pepsinogen II was constructed into a pFASTBac-HTA expression vector, and PGII with a histidine tag (6*His-tag) was recombinantly expressed in SF9 insect cells, and then extracted and purified as an antigen.

[0096] The antigen has amino acid sequences as shown in SEQ ID NO: 1, specifically:

[0097] MKWMVVVLVCLQLLEAAVVKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRFGDLSVTYEPMAYMDAAYFGEISIGTPPQNFLVLFDTGSSNLWVPSVYCQSQACTSHSRFNPSESSTYSTNGQTFSLQYGSGSLTGFFGYDTLTVQSIQVPNQEFGLSENEPGTNFVYAQFDGIMGLAYPALSVDEATTAMQGMVQEGALTSP VFSVYLSNQQGSSGGAVVFGGVDSSLYTGQIYWAPVTQELYWQIGIEEFLIGGQASGWCSEGCQAIVDTGTSLLTVPQQYMSALLQATGAQEDEYGQFLVNCNSIQNLPSLTFIINGVEFPLPPSSYILSNNGYCTVGVEPTYLSSQNGQPLWILGDVFLRSYYSVYDLGNNRVGFATAA

Embodiment 2

[0098] Example 2: Alpaca immunization

[0099] The present embodiment will be Example 1 prepared antigen immune alpaca. The specific steps are as follows:

[0100] (1) The average dividing of the prepared antigens in Example 1 into 4 parts, each serving is about 0.25mg;

[0101](2) Cumulative immunization of alpacas is carried out 4 times, and the antigen is injected subcutaneously into the animal, and the first immunization is recorded as the first day, and the subsequent immunization is on the 10th, 19th and 28th days, respectively; Among them, on the 28th day, about 200 mL of venous peripheral blood of alpacas was collected before the fourth immunization, and on the 42nd day, that is, 14 days after the fourth immunization, about 300 mL of alpaca venous peripheral blood was collected.

[0102] Compared with the immunization technology scheme of traditional animal antibodies such as rats and rabbits, the method provided in the present embodiment is to collect a large amount of al...

Embodiment 3

[0103] Example 3: Construction of antibody libraries

[0104] Taking two batches of alpaca venous peripheral blood collected in Example 2 as raw materials, a highly diverse nano-antibody library was constructed. Both batches of intravenous peripheral blood are treated in the same way, specifically:

[0105] (1) Lymphocytes were isolated from the peripheral blood of the alpaca vein using density gradient centrifugation;

[0106] (2) The total mRNA of lymphocytes is extracted and reverse transcription to cDNA;

[0107] (3) Using appropriate DNA primers, using the above cDNA as a template, the VHH fragments of alpaca immunoglobulins IgG2 and IgG3 were amplified by polymerase chain reaction (PCR), that is, DNA fragments of nano-antibodies;

[0108] (4) Connecting VHH's DNA to the surface of the phage to display the screening vector constitutes the VHH-pIII fusion protein expression vector plasmid library; Among them, pIII is a protein present on the flagella on the surface of the phag...

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Abstract

The invention relates to an antigen binding protein, an antibody or an antibody active fragment obtained by immunizing a camelidae animal with pepsinogen II. The antibody for specific recognition and combination with pepsinogen II is screened, identified and prepared by relying on an immune system of a camelidae animal, and the obtained antibody is high in specificity, can be used for stomach lesion detection and has potential clinical diagnosis and treatment value; the antibody provided by the invention is simple in structure, easy to carry out genetic engineering modification, easy to realize humanization, high in stability, low in mass production cost and beneficial to realizing large-scale production.

Description

Technical field [0001] The present invention relates to the field of biotechnology, specifically to a specific antibody of pepsinogen II and a method and application thereof. Background [0002] An antibody is a protein secreted primarily by plasma cells that is used by the immune system to identify and neutralize foreign substances such as bacteria, viruses, and other foreign substances, called antigens. The binding of antibodies and antigens relies entirely on the interaction of non-covalent bonds, and this specific binding mechanism allows the antibody to capture foreign microorganisms as well as infected cells, further inducing other immune mechanisms to attack them, or directly neutralizing their targets. Antibodies and antibody-related products have been widely used in life sciences and medicine and other research fields, based on antigen-antibody specific combination and derived from many experimental techniques laid an important foundation for scientific research and clin...

Claims

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Application Information

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IPC IPC(8): C07K16/40C07K16/00C12N15/13G01N33/573C40B50/06C12R1/19
CPCC07K16/40C07K16/005G01N33/573C40B50/06C07K2317/22C07K2317/565C07K2317/569C07K2317/92G01N2333/96477G01N2800/06Y02A50/30
Inventor 刘剑峰张胜蓝
Owner BIOISLAND LAB
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