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Targeted immunosuppressant TCABCD55 for preventing and treating infectious inflammation

An activity modulator and flexible technology, applied in the field of peptides, can solve problems such as lack of technical means, and achieve the effect of improving survival rate, reducing lung index and increasing survival rate

Active Publication Date: 2021-12-17
中国人民解放军疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009]However, based on the differences in pathology and the distribution of lesions, the complement inhibitory treatment of severe pneumonia in the co-infection of influenza virus and bacteria in the existing technology is currently There is still a lack of effective technical means. The purpose of the present invention is to provide a new anti-C3d single-chain antibody with excellent targeting performance and a fusion protein of the anti-C3d single-chain antibody and the complement inhibitor CD55, so that the anti-C3d single-chain antibody can Targeting the complement inhibitor CD55 to foci of influenza virus and bacterial co-infection yields superior therapeutic efficacy

Method used

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  • Targeted immunosuppressant TCABCD55 for preventing and treating infectious inflammation
  • Targeted immunosuppressant TCABCD55 for preventing and treating infectious inflammation
  • Targeted immunosuppressant TCABCD55 for preventing and treating infectious inflammation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Construction of human phage single-chain antibody C3d-ScFv

[0042] 1.1 For the construction of a large-capacity fully synthetic human phage single-chain antibody library, refer to Chinese patent 200910091261.8.

[0043] 1.2 Screening of human anti-human C3d single chain antibody

[0044] A total of three rounds of screening of human anti-human C3d single chain antibody were carried out.

[0045] (1) Antigen coating: Human recombinant C3d protein-coated immunotubes were coated overnight at 4°C.

[0046] (2) Blocking: the immunotube was blocked with PBS containing 2% (w / v) BSA, and the phage antibody library was blocked with PBST (containing 0.1% Tween20) containing 2% (w / v) BSA at the same time, and blocked for 1 hour at 37°C.

[0047] (3) Binding: add the blocked phage antibody library into an immunotube, and let it stand at 4°C overnight for binding.

[0048] (4) Washing: washing with PBST and PBS.

[0049] (5) Elution: Elution with 1 ml of 0.2 mol / l gl...

Embodiment 2

[0075] Example 2. Construction and Identification of C3d-ScFv-CD55 Targeting Complement Inhibitor

[0076] Use PCR amplification technology to amplify single-chain antibody gene fragments and CD55 gene fragments; respectively use upstream primer: B6F, downstream primer: B6-CD55-R to amplify single-chain antibody fragments; use upstream primer CD55-F, downstream primer: CD55 -his-R amplifies the CD55 gene fragment. The PCR reaction system is the same as in Example 1 Table 1.

[0077] Table 2. Construction of primer sequences targeting complement inhibitors

[0078]

[0079] The single-chain antibody fragment and the CD55 fragment were respectively connected to the upstream primer B6F by PCR technology, and the downstream primer was CD55-his-R. PCR system (same as above). Expression and purification of the targeted complement inhibitor C3d-ScFv-CD55: the method is the same as in Example 1.

[0080] The lengths of the coding gene sequences of C3d single-chain antibody and CD...

Embodiment 3

[0086] Example 3. Serum Total Complement Hemolytic Activity (CH50) Determination

[0087] 3.1 Preparation of buffer

[0088] 3.1.1 Storage solution:

[0089] Na 2 HPO 4 12H 2 O 2.85g

[0090] K H 2 PO 4 0.27g

[0091] NaCl 17.00g

[0092] (Add distilled water to 100 ml, store at 4°C).

[0093] 3.1.2 Application solution (buffer): add 95 ml of distilled water to 5 ml of stock solution, and add 0.1 ml of 10% magnesium sulfate. Prepared today. Use within 12 hours.

[0094] 3.2 Operation steps (improved Mayer method):

[0095] 1) Sensitized sheep erythrocytes: 2% sheep erythrocytes plus hemolysin (1:2000) after equal dilution, mix well, and place in a water bath at 37°C for 30 minutes.

[0096] 2) Dilute serum: 0.2ml serum to be tested, add 3.8ml buffer solution, the dilution ratio is 1:20.

[0097] 3) Preparation of hemolysis standard tube: add 2ml of 2% sheep red blood cells to 8ml of distilled water, mix well, that is complete hemolysis. Take 2ml of total hemoly...

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Abstract

The invention discloses a single-chain antibody of a human anti-complement C3d molecule and a fusion protein of the single-chain antibody and a complement inhibitor CD55. The antibody and the fusion protein have excellent antigen binding activity, in-vitro inhibition experiments show that C3d-ScFv-CD55 has an obvious inhibition effect corresponding to a single effector molecule CD55, and the effect is realized by identifying that the C3d component in a complement activation region plays a complement inhibition role. The targeted complement inhibitor C3d-ScFv-CD55 is used for treating influenza / bacteria co-infected mice, the survival rate is obviously increased, lung lesions are obviously relieved, the targeted complement inhibition effect is obvious, and the targeted complement inhibitor C3d-ScFv-CD55 has an obvious treatment effect compared with the single effector molecule CD55, and it proves that the fusion protein provided by the invention has an excellent application prospect in preparation of drugs for treating influenza virus and bacteria co-infected pneumonia.

Description

technical field [0001] The invention provides a complement targeting immunosuppressant and belongs to the technical field of polypeptides. Background technique [0002] Influenza virus is an important class of pathogens that cause human diseases. Influenza virus and bacterial co-infection are very common during influenza pandemics. The most common bacterial pathogens are Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus influenzae, etc. . These bacteria reside in the nasopharynx of humans, from where they can travel to the lower respiratory tract. After virus infection, it can change from asymptomatic to invasive, and the number of people during respiratory infection is more than that of carriers, and a considerable proportion of death cases is accompanied by bacterial infection. Severe pneumonia following co-infection is an important cause of high mortality during influenza epidemics. Among the bacteria that co-infect with influenz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K19/00C12N15/62A61K38/17A61K47/68A61P11/00A61P29/00A61P31/04A61P31/16
CPCC07K16/18C07K14/70596A61K47/6811A61K38/177A61P31/16A61P31/04A61P11/00A61P29/00C07K2317/565C07K2317/76C07K2317/24C07K2317/56C07K2317/622C07K2317/92C07K2319/00Y02A50/30
Inventor 宋宏彬贾雷立李利忠白萱洋梁媛赵信平赵江云董灏郭卫光
Owner 中国人民解放军疾病预防控制中心
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