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Targeted immunosuppressant TCABCD59 for preventing and treating infectious inflammation

A flexible and fusion protein technology, applied in the field of peptides, can solve the problems of 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

[0008]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 prior art is currently There is still a lack of effective technical means, and 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 an anti-C3d single-chain antibody and a complement inhibitor CD59, so that the anti-C3d single-chain antibody can Targeting the complement inhibitor CD59 to foci of influenza virus and bacterial co-infection yields superior therapeutic efficacy

Method used

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  • Targeted immunosuppressant TCABCD59 for preventing and treating infectious inflammation
  • Targeted immunosuppressant TCABCD59 for preventing and treating infectious inflammation
  • Targeted immunosuppressant TCABCD59 for preventing and treating infectious inflammation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Construction of human phage single-chain antibody C3d-ScFv

[0041] 1.1 For the construction of a large-capacity fully synthetic human phage single-chain antibody library, refer to Chinese patent 200910091261.8.

[0042] 1.2 Screening of human anti-human C3d single chain antibody

[0043] A total of three rounds of screening of human anti-human C3d single chain antibody

[0044] (1) Antigen coating: Human recombinant C3d protein-coated immunotubes were coated overnight at 4°C.

[0045] (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.

[0046] (3) Binding: add the blocked phage antibody library into the immunotube, and let stand at 4°C overnight for binding;

[0047] (4) Washing: washing with PBST and PBS.

[0048] (5) Elution: Elution with 1 ml of 0.2 mol / l glycine-hydrochloric a...

Embodiment 2

[0074] Example 2. Construction and Identification of C3d-ScFv-CD59 Targeting Complement Inhibitor

[0075] Use PCR amplification technology to amplify single-chain antibody gene fragments and CD59 gene fragments; use upstream primers: B6F and downstream primers: B6-CD59-R to amplify single-chain antibody fragments (see Table 2 for primer sequences); use upstream primers CD59 -F, downstream primer: CD59-his-R amplifies the CD59 gene fragment. The PCR reaction system is the same as in Example 1.

[0076] Table 2. Construction of primer sequences targeting complement inhibitors

[0077]

[0078]The single-chain antibody fragment and the CD59 fragment were respectively connected to the upstream primer B6F by PCR technology, and the downstream primers were respectively CD59-his-R. PCR system (same as above). Expression and purification of the targeted complement inhibitor C3d-ScFv-CD59: the method is the same as in Example 1.

[0079] The lengths of the coding gene sequences...

Embodiment 3

[0084] Example 3. Serum Total Complement Hemolytic Activity (CH50) Determination

[0085] 3.1 Preparation of buffer

[0086] (1) Storage solution:

[0087] Na 2 HPO 4 12H 2 O 2.85g

[0088] K H 2 PO 4 0.27g

[0089] NaCl 17.00g

[0090] (Add distilled water to 100ml, store at 4°C)

[0091] (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.

[0092] 3.2 Operation steps (improved Mayer method):

[0093] 1) Sensitized sheep erythrocytes: 2% sheep erythrocytes plus hemolysin (1:2000) after equal dilution, mix well, put in 37°C water bath for 30min.

[0094] 2) Diluted serum: 0.2ml of the serum to be tested was added with 3.8ml of buffer solution, and the dilution ratio was 1:20.

[0095] 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 he...

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Abstract

The invention discloses a targeted complement inhibitor fusion protein C3d-ScFv-CD59 of a single-chain antibody of a human anti-complement C3d molecule and a complement inhibitor CD59. The fusion protein has excellent antigen binding activity, and in-vitro inhibition experiments show that C3d-ScFv-CD59 has a more obvious inhibition effect corresponding to a single effector molecule CD59, and the effect is realized by identifying that the C3d component in a complement activation region plays a complement inhibition role. The C3d-ScFv-CD59 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 C3d-ScFv-CD59 has an obvious treatment effect compared with the single effector molecule CD59, 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 targeted 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 influenza, Staphyloc...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/17A61K39/395A61K47/55A61K47/68A61P11/00A61P31/04A61P31/16
CPCC07K16/18C07K14/70596C12N15/85A61K39/39533A61K38/177A61K47/6835A61K47/55A61P31/16A61P31/04A61P11/00C07K2317/565C07K2317/56C07K2317/622C07K2317/92C07K2317/24C07K2317/76C07K2319/00A61K2039/505
Inventor 贾雷立宋宏彬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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