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Clostridium perfringens alpha toxin humanized single chain antibody 8b

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: 2018-07-17
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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  • Clostridium perfringens alpha toxin humanized single chain antibody 8b
  • Clostridium perfringens alpha toxin humanized single chain antibody 8b
  • Clostridium perfringens alpha toxin humanized single chain antibody 8b

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 alpha toxin (CPA) published in Genbank (Genbank accession number L43548.1), the complete gene sequence of Clostridium perfringens alpha toxin (CPA) was designed and synthesized. The NdeI and EcoRI restriction sites were inserted into the end, and cloned into the pMD19-T vector to construct the plasmid pMD19-T-CPA, which was then transferred into E.coliJM109 to make a puncture strain for preservation.

[0020] The large fragment of the carrier plasmid pET-28a purified and recovered after digestion and the CPA digestion product purified and recovered after digestion were ligated with T4 DNA ligase to obtain the recombinant plasmid pET-28a-CPA, which was transformed into E. coli JM109 competent cells, containing kan-resistant agar plates were used for preliminary screening. A single colony was selected and cultured in LB liquid mediu...

Embodiment 2

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

[0022] The recombinant plasmid pET-28a-CPA was transformed into the expression bacteria-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 around 43KD, and the size was consistent with the theoretical value. The expression amount accounted for 15.6% of the total bacterial protein (see figure 2 ).

[0023] The supernatant and the precipitate of the recombinant protein expressing bacteria lysate were subjected to SDS-PAGE electrophoresis 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. can be expressed in a soluble form under ambient conditions (see image 3 ).

[0024] Purification of the recombinant protein CPA, the recombinant protein expression b...

Embodiment 3

[0025] Example 3: Screening of fully human anti-C. perfringens alpha toxin neutralizing scFv antibodies

[0026] The purified CPA recombinant protein was coated on a 96-well microtiter plate as an antigen, overnight at 4°C. The supernatant was discarded the next day, blocked with 2% Milk-PBS at 37°C for 2 h, and the prepared phage antibody library Source bioscience (UK), purchased from Beijing Ximei Technology Co., Ltd. (China distributor), was added to the phage antibody library at a titer of 1.0 × 10 13 , incubate with vigorous shaking for 60 min at room temperature, and let stand for 60 min. Then, the liquid was discarded and washed 10 times with PBS containing 0.1% Tween-20. After washing, the remaining liquid in each well was gently patted dry, and 50 µL of eluate (5 mg / mL trypsin-PBS) was added to each well. Shake vigorously for 10 min at room temperature to elute the phage and collect and store at 4°C.

[0027] E. coli TG1 was infected with the eluted phage and spread...

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Abstract

The invention discloses a humanized single-chain antibody 8B of Clostridium perfringens α toxin, which is screened from the phage antibody library Source bioscience, and is a fully human anti-Clostridium perfringens α toxin antibody, which can be realized Neutralizing the therapeutic effect of toxins, while overcoming various side effects of heterologous antibodies, eliminating the cumbersome steps and high cost of humanized transformation of heterologous antibodies, due to the small molecular weight of single-chain antibodies and strong penetrating ability in vivo, It quickly reaches damaged tissues and cells to play an anti-toxin effect, thus achieving the dual purpose of economy and high efficiency. The α-toxin has certain homology with other types of toxins of Clostridium perfringens, and the antibody drug may have inhibitory and therapeutic effects on other types of toxins, and can also be used as a detection reagent to detect α-toxins of Clostridium perfringens.

Description

technical field [0001] The invention belongs to the fields of bioengineering and disease prevention, and in particular relates to a humanized single-chain antibody 8B of Clostridium perfringens alpha toxin and its application in a method for diagnosing and treating 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-like shape with blunt rounded ends, obligate anaerobic, capsular aflagellate , 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 general conditions, and it is beneficial to form spores in sugar-free medium. Most of the 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 med...

Claims

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Application Information

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Patent Type & Authority Patents(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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