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Anti-clostridium perfringens alpha toxin fully humanized monomolecular antibody 7D-mFc and application thereof

A technology of Clostridium perfringens and α-toxin, which is applied in the fields of applications, antibodies, anti-toxins, etc., and can solve problems such as high cost

Active Publication Date: 2020-09-15
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the various side effects of heterologous antibodies, avoid the cumbersome steps and high cost of heterologous antibody humanization, and provide a small molecular weight, strong penetrating ability in the body, and quickly reach the human body. Clostridium perfringens α-toxin fully human ScFv-mFc monomolecular antibody for damaged tissues and cells to exert anti-toxin effect

Method used

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  • Anti-clostridium perfringens alpha toxin fully humanized monomolecular antibody 7D-mFc and application thereof
  • Anti-clostridium perfringens alpha toxin fully humanized monomolecular antibody 7D-mFc and application thereof
  • Anti-clostridium perfringens alpha toxin fully humanized monomolecular antibody 7D-mFc and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Construction of recombinant expression plasmid P300-trxA-SUMO-7D-mFc

[0016] According to the full sequence of human IgG published in Genbank, an mFc fragment containing a mutated hinge region was designed and synthesized. The synthetic gene sequence was EcoRI-CH2-SacⅠ-CH3-XhoⅠ, and cloned into the pMD19-T vector to construct the plasmid pMD19- T-mFc was then transferred into E.coli JM109 and preserved as a puncture strain.

[0017] The P300-trxA-SUMO (PTS) expression vector was digested with EcoRI and XhoI and then purified and recovered. The mFc fragment purified and recovered after double digestion with pMD19-T-mFc was ligated with T4 DNA ligase to obtain the recombinant plasmid PTS- mFc, transformed into Escherichia coli JM109 competent cells, and the agar plate containing Amp resistance was used for preliminary screening. Select a single colony and culture it in LB liquid medium; extract the plasmid with a plasmid recovery kit, and identify it by PCR. T...

Embodiment 2

[0022] Example 2 Expression, purification and protein properties of 7D-mFc

[0023] The recombinant plasmid PTS-D7-mFc was transformed into the expression strain Escherichia coli BL21 (DE3), and the expression was induced by IPTG. The results of SDS-PAGE showed that the recombinant protein 7D-mFc had an obvious expression band at about 80KD, and the size was consistent with the theoretical value. The expression level of 7D-mFc accounted for 25.6% of the total bacterial protein (see figure 2 ).

[0024] Purification of the fusion protein: After the induced expression cells were ultrasonically lysed, the supernatant was taken, precipitated step by step with 20-45% saturated ammonium sulfate, and the precipitate was resuspended with 20mM Tris-cl +0.5M Nacl (pH 8.0) 2+ Metal chelation chromatography, eluted with 50mM and 200mM imidazole respectively, the target protein is in the elution peak of 200mM imidazole, equilibrated with 20mM Tris-cl + 10mM Nacl (pH8.0) to desalt the G-2...

Embodiment 3

[0025] Example 3 Identification of Biological Activity of Fully Human Anti-Clostridium perfringens α-toxin Monomolecular Antibody 7D-mFc

[0026] Take 50% Egg Yolk Emulsion (Haiqing Biology, product number HB8295) and centrifuge at 10000×g for 20min at 4°C, take the supernatant, dilute it 1:10 times with normal saline, add 100µL diluted egg yolk liquid to each well of the cell culture plate, Add 5µg of purified CPA toxin and 5µg of 7D-mFc to each well. At the same time, 7D ScFv was used as another group. Normal saline was used as a negative control, and only CPA toxin was added as a positive control. Each sample was measured in duplicate wells, and placed at 37°C for 2 h, read the average value of OD620, the results show that 7D-mFc has a significantly higher inhibitory activity on lecithin decomposition than simple 7D.

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Abstract

The invention discloses an anti-clostridium perfringens alpha toxin fully humanized 7D-mFc monomolecular antibody and an application thereof. The base sequence of the antibody is shown as SEQ ID NO.3,and the amino acid sequence is shown as SEQ ID NO.4. The invention also discloses an application of the anti-clostridium perfringens alpha toxin fully humanized 7D-mFc monomolecular antibody in preparation of drugs for treating and preventing clostridium perfringens alpha toxin poisoning. The invention discloses a detection reagent of the clostridium perfringens alpha toxin, and the detection reagent comprises the clostridium perfringens alpha toxin completely humanized 7D-mFc monomolecular antibody with the amino acid sequence shown as the SEQ ID NO.4.

Description

technical field [0001] The invention belongs to the fields of bioengineering and disease prevention and control, and in particular relates to a preparation method and application of fully human monomolecular antibody 7D-mFc against Clostridium perfringens alpha toxin. Background technique [0002] Clostridium perfringens (C .perfringens), also known as Clostridium welchii (C .welchii), widely exists in the natural environment and is found in the digestive tract of almost all warm-blooded animals. It belongs to the normal intestinal flora of humans and animals a member of. Clostridium perfringens can cause lamb dysentery, necrotic enteritis and enterotoxemia in lambs, calves, piglets, rabbits and chicks. In recent years, the main pathogen of livestock "sudden death syndrome" in my country has brought huge economic losses to the development of animal husbandry in various countries. All types of Clostridium perfringens produce α-toxin (CPA). This bacterium can cause gas gangre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13A61K39/40A61P39/02
CPCC07K16/1282A61P39/02C07K2317/76C07K2317/94C07K2317/622A61K2039/505
Inventor 张国利田园岳玉环陈萍刘楚含吴广谋李泽鸿刘雨玲王冬冬雍伟邓欣卢士伟于佳那漫
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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