Humanized single-chain antibody 8B of clostridium perfringens alpha-toxin

A Clostridium perfringens and single-chain antibody technology, which is applied in the direction of antibodies, anti-toxins, anti-enzyme immunoglobulins, etc., can solve the problems of no treatment methods, etc., to overcome various side effects, strong penetrating ability in the body, The effect of small molecular weight

Active Publication Date: 2015-04-22
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alpha toxin is the most important pathogenic factor of Clostridium perfringens, so far there is no effective treatment

Method used

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  • Humanized single-chain antibody 8B of clostridium perfringens alpha-toxin
  • Humanized single-chain antibody 8B of clostridium perfringens alpha-toxin
  • Humanized single-chain antibody 8B of clostridium perfringens alpha-toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Construction of recombinant expression plasmid pET-28a-CPA

[0019] According to the complete sequence of Clostridium perfringens α-toxin (CPA) published in Genbank (Genbank accession number L43548.1), the complete gene sequence of Clostridium perfringens α-toxin (CPA) was designed and synthesized. Insert NdeI and EcoRI restriction sites at the end, and clone into the pMD19-T vector to construct the plasmid pMD19-T-CPA, and then transfer it into E.coli JM109 to make a puncture strain for preservation.

[0020] The large fragment of vector plasmid pET-28a purified and recovered after enzyme digestion and the CPA digestion product purified and recovered after enzyme digestion were ligated with T4 DNA ligase to obtain recombinant plasmid pET-28a-CPA, which was transformed into Escherichia coli JM109 competent cells, containing kan-resistant agar plates for initial screening. Select a single colony and culture it in LB liquid medium; extract the plasmid with a p...

Embodiment 2

[0021] Example 2: Expression, purification and protein properties of CPA

[0022] The recombinant plasmid pET-28a-CPA 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 CPA had an obvious expression band at about 43KD, and the size was consistent with the theoretical value. The expression level accounted for 15.6% of the total bacterial protein (see figure 2 ).

[0023] SDS-PAGE electrophoresis was performed on the lysate supernatant and the precipitate of the recombinant protein expressing bacteria at the same time. The results showed that most of the target protein was in the supernatant, and a small amount of target protein also existed in the corresponding position in the precipitate. environment can be expressed in soluble form (see image 3 ).

[0024] Purification of recombinant protein CPA, recombinant protein expressing cells were lysed by ultraso...

Embodiment 3

[0025] Example 3: Screening of fully human anti-Clostridium perfringens alpha toxin neutralizing scFv antibody

[0026] The purified CPA recombinant protein was coated on a 96-well microtiter plate as an antigen, and kept overnight at 4°C. Discard the supernatant the next day, block with 2% Milk-PBS at 37°C for 2 hours, add the prepared phage antibody library Source bioscience (UK), purchased from Beijing Ximei Technology Co., Ltd. (Chinese distributor), the titer is 1.0×10 13 , incubated at room temperature for 60 minutes with vigorous shaking, and left to stand for 60 minutes. Finally discard the liquid, wash with PBS containing 0.1% Tween-20 10 times, after washing, gently pat dry the remaining liquid in each well, add 50 μL eluent (5 mg / mL trypsin-PBS) to each well, Vigorously shake at room temperature for 10 min to elute the phage, collect and store at 4°C.

[0027] Infect E.coli TG1 with the eluted phage, spread on TYE plates (containing 100 μg / mL Amp and 1% glucose) a...

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Abstract

The invention discloses a humanized single-chain antibody 8B of clostridium perfringens alpha-toxin. The humanized single-chain antibody 8B is screened from a phage antibody library Source bioscience, is a full-humanized antibody of the clostridium perfringens alpha-toxin and can be used for achieving a toxin neutralization treating effect, overcoming various side effects of a heterologous antibody and eliminating complex steps and high cost of humanization modification on the heterologous antibody; and the single-chain antibody is small in molecular weight, strong in in-vivo penetrability and capable of rapidly reaching to damaged tissues and cells to exert an antitoxin effect, so that the double aims of economy and high efficiency are achieved. The alpha-toxin has a certain homology with other types of toxins of clostridium perfringens, so that the antibody drug possibly generates inhibiting and treating effects for other types of toxins and can also be used as a detecting reagent to detect the clostridium perfringens alpha-toxin.

Description

technical field [0001] The invention belongs to the fields of bioengineering and disease prevention, and in particular relates to the humanized single-chain antibody 8B of Clostridium perfringens alpha toxin and its application in the diagnosis and treatment of Clostridium perfringens alpha toxin poisoning. Background technique [0002] Clostridium perfringens (Clostridium perfringens), also known as Clostridium welchii (C.welchii), is a Gram-positive bacterium with a thick rod-shaped shape with blunt ends, obligate anaerobic, capsulated and non-flagellated , is the main pathogen causing human food poisoning, gas gangrene, antibiotic-associated diarrhea, and animal enterotoxemia and dysentery. Clostridium perfringens is difficult to form spores under normal conditions, and it is beneficial to form spores in a sugar-free medium. Most strains of Clostridium perfringens can form capsules. The G+C mol% of the bacterial DNA is 24- 27. It can grow normally on the basic medium, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C07K16/40A61K39/40A61P39/02G01N33/573
Inventor 张国利于佳朱进田园刘雨玲陈萍岳玉环吴广谋史飞徐艳玲赵鑫
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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