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Specific antibody of pepsinogen I 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 popularization of detection, large-scale mass production of high finished products, unfavorable large-scale mass production, etc. The effect of modernized 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 mice and other animals to produce PGI 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 production of finished products is high
The above-mentioned monoclonal antibodies are full-length immunoglobulins (IgG), which need to be repeatedly immunized and injected into animals and isolated and purified from animal body fluids, or isolated after recombinant expression in mammalian cells and other expensive expression systems , Purification and extraction, complex operation and high cost, which is not conducive to large-scale batch production

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1: Preparation of antigens

[0096] The DNA sequence encoding pepsinogen I was constructed into pFASTBac-HTA expression vector, and PGI with histidine tag (6*His-tag) was recombinantly expressed in SF9 insect cells, and then extracted and purified as antigen.

[0097]所述抗原如有如SEQ ID NO:1所示的氨基酸序列,具体为:MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNPARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGSSNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNWVPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANIQSDIGASENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEGSCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA

Embodiment 2

[0098] Example 2: Alpaca immunization injection

[0099] In this example, alpacas were immunized with the antigen prepared in Example 1. Specific steps are as follows:

[0100] (1) The antigen prepared in Example 1 was equally divided into 4 parts, each of about 0.25 mg;

[0101] (2) The alpacas were immunized 4 times in total, and the antigens were subcutaneously injected into the animals. The first immunization was recorded as the first day, and the subsequent immunizations were on the 10th, 19th, and 28th days respectively; On the 28th day, before the fourth immunization injection, about 200 mL of alpaca venous peripheral blood was collected, 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 traditional immunization technical solutions for animal antibodies such as mice and rabbits, the method provided in this example collects a large amount of alpaca venous periphe...

Embodiment 3

[0103] Example 3: Construction of Antibody Libraries

[0104] Using the two batches of alpaca venous peripheral blood collected in Example 2 as raw materials, a high diversity Nanobody library was constructed. The two batches of alpaca venous peripheral blood were treated in the same way, specifically:

[0105] (1) Using density gradient centrifugation to separate lymphocytes from the peripheral blood of alpaca veins;

[0106] (2) Extracting the total mRNA of lymphocytes and reverse transcribing into cDNA;

[0107] (3) Using appropriate DNA primers, the above-mentioned cDNA is used as a template, and the VHH fragments of alpaca immunoglobulin IgG2 and IgG3 are obtained by polymerase chain reaction (PCR) amplification, that is, the DNA fragments of Nanobodies;

[0108] (4) connecting the DNA of VHH to the phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library; wherein, pIII is a protein present on the phage surface flagella;...

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PUM

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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 I. The antibody for specific recognition and combination with pepsinogen I 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 invention relates to the field of biotechnology, in particular to a specific antibody of pepsinogen I and a preparation method and application thereof. Background technique [0002] Antibodies are proteins mainly secreted by plasma cells and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses, which are called antigens. The binding of antibodies and antigens is entirely dependent on non-covalent interactions. This specific binding mechanism allows antibodies to capture foreign microorganisms as well as infected cells, further inducing other immune mechanisms to attack them, or directly neutralize their targets . Antibodies and antibody-related products have been widely used in research fields such as life science and medicine. Many experimental techniques derived from antigen-antibody specific binding have laid an important foundation for scientific research and clinical treatment, such as immunodia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/573C40B50/06C40B40/10C12N15/70C12N7/01C12N1/21C12R1/92C12R1/19
CPCC07K16/40C07K16/005G01N33/573C40B50/06C40B40/10C12N15/70C12N7/00C07K2317/22C07K2317/92C07K2317/565C07K2317/56C07K2317/94G01N2333/96477C07K2317/569G01N2800/06C12N2795/00021C12N2795/00043Y02A50/30
Inventor 刘剑峰张胜蓝
Owner BIOISLAND LAB
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