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African swine fever prevention and/or treatment neutralizing antibody and preparation method and application thereof

A monoclonal antibody and carrier technology, applied in the field of neutralizing antibodies, African swine fever prevention and/or therapeutic neutralizing antibodies, can solve the problems of short half-life of antibodies and inability to provide continuous protection, and achieve small virus particles and long-lasting Protective, cost-effective effects

Active Publication Date: 2020-02-28
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent provides a bispecific antibody preparation method without providing the antibody sequence. This method uses recombinant CHO cells to express the antibody, which is then purified and injected into pigs. However, because the half-life of the antibody is very short, it cannot continue to provide protection, so the interval Requires repeated injections over a shorter period of time

Method used

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  • African swine fever prevention and/or treatment neutralizing antibody and preparation method and application thereof
  • African swine fever prevention and/or treatment neutralizing antibody and preparation method and application thereof
  • African swine fever prevention and/or treatment neutralizing antibody and preparation method and application thereof

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preparation example Construction

[0053] Another aspect of the embodiments of the present invention provides a method for preparing virus particles, which includes: culturing host cells, waiting for the cells to grow to 70% confluence, adding a transfection reagent, adding the expression vector, pAAV2-RC vector and pHelper The host cells were co-transfected with the vector, cultured at 37°C for 5 hours, and then replaced with fresh medium, then continued to culture and collected cells, and then the collected cells were repeatedly freeze-thawed and lysed, and then post-processed to extract virus particles.

[0054] In some embodiments, the host cells are conventional host cells in the art, preferably eukaryotic cells, more preferably HEK293 cells.

[0055] In some embodiments, the transfection reagent is a conventional transfection reagent in the art, as long as it can transfect the foreign plasmid into the host cell, preferably polyethyleneimine reagent (PEI reagent). The preparation method of the polyethylene...

Embodiment 1

[0072] Example 1 Expression of P54 Protein Recombinant Plasmid Construction

[0073] The amino acid sequence of the P54 protein of an ASFV isolated from China (Gene Bank accession number: MK128995) was codon-optimized (SEQ ID No: 11), and synthesized and cloned into the pCI expression vector at Nanjing GenScript Company. Add a His tag to the C-terminal, insert between the restriction endonucleases MluI and XhoI of the pCI vector to obtain the pCI-P54His plasmid, refer to figure 1 shown. Wherein, the pCI expression vector can also be replaced by various vectors conventional in the art that can be replicated and expressed in the host. The host can be a prokaryotic cell, such as a bacterial cell; a eukaryotic cell, such as an insect, yeast, mammalian cell, etc.; more specifically, it can be a genetically engineered bacterium obtained by transforming the aforementioned recombinant plasmid into Escherichia coli.

Embodiment 2

[0074] Preparation of embodiment 2 immune antigen (ASFV P54 protein)

[0075] Escherichia coli was used to amplify the pCI-P54His plasmid, and the plasmid was extracted. HEK293 cells were transfected with PEI and pCI-P54His plasmid, and after 3 days, centrifuged at 3000 r / min for 15 min to obtain the supernatant to obtain P54His protein (SEQ ID No: 12). Harvested cell cultures were subjected to SDS-PAGE, while empty HEK293 cells were used as negative controls. The specific operation is as follows: take 40 μl of harvested cell culture, add 10 μl of 5×loading buffer, bathe in boiling water for 5 minutes, centrifuge at 12000 r / min for 1 minute, take the supernatant and carry out SDS-PAGE gel (12% concentration gel) electrophoresis, After electrophoresis, the gel was stained and decolorized to observe the target band. Such as figure 2 As shown, the target band appears around the molecular weight of about 20kDa, and the negative control has no band at the corresponding position...

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Abstract

The invention provides an African swine fever prevention and / or treatment neutralizing antibody which is an embedded monoclonal antibody and comprises a heavy chain and a light chain. A light chain variable region has a sequence shown as SEQ ID No:6, a heavy chain variable region has a sequence shown as SEQ ID No:2, and both a light chain constant region and a heavy chain constant region come froma swine derived antibody. The invention further provides a gene coding the embedded monoclonal antibody and an expression vector or a transformant comprising the coding gene. The expression carrier can be obtained by inserting the coding gene into pAAV-CAG. The invention further provides a method of utilizing the expression vector to prepare AAV. The antibody sequences or the transformant can beused for preparing detection reagents for African swine fever virus, agents for inducing immunoreaction aiming at African swine fever virus antigen in tested animals or agents for preventing animals from being infected by the African swine fever virus, and rapid and lasting antibody protection such as African swine fever virus vaccines can be provided.

Description

technical field [0001] The invention relates to a neutralizing antibody, in particular to a preventive and / or therapeutic neutralizing antibody for African swine fever, its preparation method and application, and belongs to the technical field of animal immune drugs. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by African swine fever virus (ASFV), clinically manifested as high fever, skin congestion, edema, and dirty Extensive organ bleeding and changes in the function of the respiratory system and nervous system. The onset of the disease is short, with an incubation period of 5-15 days, high morbidity and mortality, and the mortality rate can even be as high as 100%. In countries or regions infected by the pathogen, it can cause huge economic losses, seriously threaten food safety and affect the pig industry. The World Organization for Animal Health (OIE) lists it as a Class A epidemic disease, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13A61K39/395A61P31/20
CPCA61K2039/505A61P31/20C07K16/081C07K2317/52C07K2317/56
Inventor 曹文龙孔迪孙祥明滕小锘张大鹤易小萍
Owner 苏州沃美生物有限公司
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