Porcine pseudorabies virus resisting hybridoma cell line, preparation method thereof, monoclonal antibody and application thereof

A hybridoma cell line, porcine pseudorabies technology, applied in antiviral immunoglobulins, botanical equipment and methods, biochemical equipment and methods, etc., can solve the difficulty of renaturation of recombinant proteins, the difficulty of purifying the whole virus, and the lack of space. conformational epitopes, etc., to achieve the effect of strong specificity, high purity and good uniformity

Inactive Publication Date: 2012-10-24
ZHENGZHOU HOUYI PHARMA
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Problems solved by technology

[0010] The above conventional PRV monoclonal antibody preparation mostly uses prokaryotic expressed recombinant protein or cultured whole virus as the immunogen, but the recombinant protein has the disadvantages of difficult renaturation, lack of spatial conformational epitope, and the disadvantage of difficult purification of the whole virus.
Conventional industrial purification of whole viruses mostly uses the gel filtration purification method, which cannot completely remove serum proteins and cell host proteins in the source virus culture process, and density gradient centrifugation and other treatments are required to obtain further purified viruses. If the virus sample is mixed with other antigens with similar physical properties, it still cannot be removed

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  • Porcine pseudorabies virus resisting hybridoma cell line, preparation method thereof, monoclonal antibody and application thereof
  • Porcine pseudorabies virus resisting hybridoma cell line, preparation method thereof, monoclonal antibody and application thereof

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[0032] The present invention will be specifically described below in conjunction with specific embodiments.

[0033] The pathogenic bacteria, cells, experimental animals and biochemical reagents used in the present invention include: PRVYA strain, HCV, PPV, SE, PRRSV were isolated, identified and preserved by Zhengzhou Animal Husbandry Bureau; NP and myeloma cells SP2 / 0 were provided by the laboratory; BALB / C mice were purchased from the Animal Room, College of Animal Husbandry Engineering, Henan Agricultural University; HAT selective medium, Freund's complete adjuvant, Freund's incomplete adjuvant, FITC-labeled goat anti-mouse antibody, purchased from Sigma; calf Serum was purchased from GIBCO; BCA protein quantification kit was purchased from H iTrap Protein G HP; and other biological reagents were purchased from TaKaRa.

[0034] 1. Preparation of higher titer PR as immunogen

[0035] Pseudorabies virus suspension was inoculated on BHK-13 cells that had grown into a monolayer, an...

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Abstract

The invention relates to a porcine pseudorabies virus (PRV) resisting hybridoma cell line, a preparation method thereof, a monoclonal antibody and an application thereof. The preparation method of the porcine pseudorabies virus resisting hybridoma cell line specifically comprises the following steps of: (1) preparing an antigen immune BALB / c mouse by using PRV; (2) fusing immune mouse spleen cells with SP2 / 0 myeloma cells; and (3) screening out hybridoma cells secreting PRV resisting monoclonal antibodies. In the invention, high-purity PR antigens are obtained through purification by an Sepharose4FF chromatographic column, the screening of subsequent positive clones and the generation of antibodies are guaranteed, an indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method is determined to quickly determine the generation and the titer of antibodies, and a lot of RPV antibodies are obtained by inoculating BALB / c female mouse enterocoelia. The antibody is a single antibody aiming RP antigens, has the advantages of strong specificity, high purity, good homogeneity and the like and can be used for diagnosing and treating PR. Meanwhile, certain technical reference is provided for the preparation of other types of antibodies.

Description

Technical field [0001] The present invention belongs to the field of biotechnology-monoclonal antibody. The present invention relates to a hybridoma cell line against porcine pseudorabies virus and a preparation method thereof; in addition, the present invention also relates to the preparation and application of the monoclonal antibody. Background technique [0002] Porcine pseudorabies (Pseudorabies, PR) is an acute infectious disease caused by Pseudorabies Virus (PRV) of the herpesviridae and a-herpesvirus subfamily. The virus is highly resistant and the virus is between pH 4 and 9 keep it steady. The disease material stored in 50% glycerol saline, after 154 days at 0℃~6℃, the infectivity decreased only slightly, and it was still infectious after being stored for 3 years. . It is still infectious for more than 10 days in phosphate buffer and glucose saline. Under corrupt conditions, the virus in the disease material loses its infectivity after 11 days. It is still infectiou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/06C07K16/08G01N33/577
Inventor 吴红云哈斯通拉嘎王卫芳孙芳
Owner ZHENGZHOU HOUYI PHARMA
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