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Preparation method and applications of anti-pseudorabies virus pig origin monoclonal antibody

A pseudorabies virus and pig-derived technology, which is applied in the preparation and application of anti-pseudorabies virus pig-derived monoclonal antibody, can solve the problems of large losses, hinder the development of the pig industry, and cannot fundamentally solve the problem, so as to improve the phagocytosis effect of ability

Inactive Publication Date: 2018-06-26
SHENZHEN JASON INTELLIGENT BIOTECH CO LIMLTED PRC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] To sum up, since the active immunization method of vaccination not only has a long cycle and large losses, but also cannot fundamentally solve the problem; the passive immunization method is the best choice to solve this problem
However, the conventional passive immunization technology also has defects and cannot solve this problem; therefore, it is necessary to develop a more simple and easy-to-use technology with short-term effective technology to fundamentally solve the big problem that hinders the development of my country's pig industry.

Method used

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  • Preparation method and applications of anti-pseudorabies virus pig origin monoclonal antibody
  • Preparation method and applications of anti-pseudorabies virus pig origin monoclonal antibody
  • Preparation method and applications of anti-pseudorabies virus pig origin monoclonal antibody

Examples

Experimental program
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Effect test

preparation example Construction

[0049] figure 1 It is a flowchart of a preferred embodiment of the method for preparing anti-pseudorabies virus porcine monoclonal antibody of the present invention. Such as figure 1 As shown, the preparation method of anti-pseudorabies virus porcine monoclonal antibody of the present invention comprises the following steps:

[0050] S100, preparing hybridoma cells: immunizing the anti-pseudorabies virus through mouse immunization and cell fusion to prepare hybridoma cells that can secrete anti-pseudorabies virus monoclonal antibodies;

[0051] S200. Cloning of variable region sequences and constant region sequences: extracting the total RNA of the hybridoma cells, reverse transcription to prepare cDNA, and amplifying the sequences of the light chain and heavy chain of the variable region of the murine antibody respectively; taking lymphocytes Extract the total RNA, prepare cDNA by reverse transcription, and amplify the light chain and heavy chain sequences of the porcine an...

experiment example 1

[0144] Potency Detection of Porcine Monoclonal Antibody Against Variant Pseudorabies Virus

[0145] (1) anti-PRV glycoprotein gIII porcine monoclonal antibody, (2) anti-PRV glycoprotein GP50 porcine monoclonal antibody, (3) anti-PRV glycoprotein gE porcine monoclonal antibody produced by cell reactor fermentation Antibody, (4) anti-PRV glycoprotein gB porcine monoclonal antibody, (5) anti-PRV glycoprotein gH porcine monoclonal antibody, (6) anti-PRV glycoprotein gD porcine monoclonal antibody and other six pig sources The cultured monoclonal antibody fermentation broth was collected and filtered separately, and then the pig-derived antibody was captured by cation exchange chromatography, and then further purified and refined by anion exchange chromatography, and the buffer was replaced by ultrafiltration; A high-purity pig-derived monoclonal antibody protein against PRV glycoprotein. Using healthy mouse serum as a negative control, the antibody binding titers of the prepared ...

experiment example 2

[0150] Challenge Test of Porcine Monoclonal Antibody Against Variant Pseudorabies Virus

[0151] 1. Experimental animals

[0152] 90 gilts.

[0153] 2. Experimental grouping

[0154] The experimental sows were randomly divided into three groups:

[0155] A. Passive immunization group (challenge after immunization): 30 heads. Each sow was injected with 5.0 mL of anti-mutant pseudorabies virus pig-derived monoclonal antibody injection, and at the same time, 2.0 mL / head of anti-mutant pseudorabies virus pig-derived monoclonal antibody nasal spray was sprayed; PRV-JL strain (virus content is 10 6 TCID50 / mL) to attack poison, intramuscular injection of 3mL / head, and nasal drip of 3mL / head at the same time.

[0156] B. The control group of challenged poison (only challenged and not immunized): 30 heads. With PRV-JL strain (virus content is 10 6 TCID50 / mL) to attack poison, intramuscular injection of 3mL / head, and nasal drip of 3mL / head at the same time.

[0157] C. Normal co...

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Abstract

The invention belongs to the field of gene engineering, and discloses a preparation method and applications of an anti-pseudorabies virus pig origin monoclonal antibody. The preparation method comprises following steps: positive hybridoma cells capable of realizing stable secretion of anti-pseudorabies virus (PRV) glycoprotein monoclonal antibodies are screened; secretion of the monoclonal antibodies is carried out; enlarge culture is carried out; enrichment, purification, and sequencing are carried out; design of corresponding specific primers is carried out, pig origin modification is carried out, cloning is carried out using a virus gene carrier or a non-virus gene carrier (including plasmid DNA and minicircle DNA), large scale production of different pig origin monoclonal antibodies iscarried out using insect cells or CHO cell fermentation, or the above pig origin monoclonal antibody gene carrier is introduced into pigs; injection, or feeding through nasal feeding and spraying areadopted for neutralization of pseudorabies virus with the anti-pseudorabies virus pig origin monoclonal antibodies, so that passive immunity is adopted for purifying of pseudorabies virus positive pig farms, and preventing and treating of porcine pseudorabies.

Description

technical field [0001] The invention relates to the application of genetic engineering technology in the prevention and treatment of porcine pseudorabies and the purification of porcine pseudorabies-positive farms, more specifically, the preparation method and application of anti-pseudorabies virus porcine monoclonal antibody. Background technique [0002] Pseudorabies (PR) is a serious and severe infectious disease in pig farms in my country at present. It is extremely harmful to the pig industry. It often breaks out in pigs and causes serious economic losses to the pig industry. After pigs are infected with pseudorabies, the pseudorabies virus will replicate in the nasopharyngeal epithelium and tonsils and spread to the lymph nodes, impairing the body's ability to kill pathogens and cytotoxins, resulting in immunosuppression and hypoimmunity of the body, making many vaccines immune ineffective and soon Severe diseases such as swine fever and porcine contagious pleuropneumo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/13C07K16/46C07K16/08A61K39/42A61P31/22A23K50/30A23K20/147
CPCA61K2039/505A61K2039/552C07K16/085C07K16/462C07K2317/64C12N15/85
Inventor 包晟谢亦武曹天福包世啟
Owner SHENZHEN JASON INTELLIGENT BIOTECH CO LIMLTED PRC
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