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Hog cholera virus E2 protein epitope and preparation and application of antibody

A technology of swine fever virus and antigenic epitope, which is applied in the direction of antiviral immunoglobulin, viral peptides, material inspection products, etc., can solve the problems of no detailed reports, etc., and achieve high coincidence rate, short production cycle and good specificity Effect

Active Publication Date: 2018-01-23
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no detailed report on which amino acid sequences of these three proteins serve as viral ligands to bind to viral receptors.

Method used

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  • Hog cholera virus E2 protein epitope and preparation and application of antibody
  • Hog cholera virus E2 protein epitope and preparation and application of antibody
  • Hog cholera virus E2 protein epitope and preparation and application of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Materials and methods

[0032] The strains, cells and experimental animals of CSFV Shimen strain, SP2 / 0 myeloma cells, PK-15 and Vero cells are all preserved by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Cells were kept at 37°C, 5% CO 2 and 10% FBS (FBS, Gibico, Shanghai, China) conditions. BALB / c female mice were purchased from Shanghai Slack Experimental Animal Company.

[0033] Vectors, reagents and strains pCold-TF vector, Escherichia coli BL21 (DE3) were purchased from Takara (Shanghai) Company; HRP-labeled goat anti-mouse IgG and FITC-labeled goat anti-mouse IgG were purchased from Sigma (Shanghai) Company; Enzyme EcoR I was purchased from NEB (Shanghai) Company.

Embodiment 2

[0034] Example 2: Amplification and protein preparation of the main epitope of E2

[0035] Amplification of E2 Main Antigen Epitope Region Gene and Construction of Recombinant Plasmid

[0036] According to the CSFV E2 gene sequence published by NCBI, the 1st-537th nucleotide sequence of the E2 gene was sent to the company (Jinweizhi) for optimization and synthesis, and the synthetic E2 gene was connected with the pCold-TF vector to construct a plasmid. After the plasmid was sequenced correctly , the positive plasmid was named pCold-E2.

[0037] Induced expression and purification of recombinant proteins

[0038] The recombinant plasmid pCold-E2 was transformed into BL21(DE3) Escherichia coli, cultured with shaking at 37°C until the OD600 value reached 0.6-0.8, then induced by adding IPTG with a final concentration of 1 mM, shaking at 16°C for 24 hours. The bacteria were collected and added to PBS for ultrasonic lysis, added 5×SDS buffer and boiled for 10 min, followed by SDS...

Embodiment 3

[0040] Embodiment 3: Preparation of monoclonal antibody

[0041] mouse immunization

[0042] 100 μg of purified protein was mixed and emulsified with Freund's complete adjuvant 1:1, and inoculated into 6-week-old healthy female BALB / c mice, and then every 2 weeks, the same amount of purified protein was emulsified with Freund's incomplete adjuvant for the second and For the third immunization, blood was collected on the 10th day after the third immunization, and the ELISA antibody titer was determined. For mice with a titer above 1:10000, 200 μg of pure antigen was injected intraperitoneally on the 3-4 day before cell fusion for booster immunity.

[0043] Preparation of McAbs

[0044] One day before fusion, mouse peritoneal macrophages were used as feeder cells, and then the mouse with the highest immune titer was selected for fusion of splenocytes and SP2 / 0 cells. The hybridoma cells were cultured in a 96-well plate lined with feeder cells at 37°C and 5% CO2 under the condit...

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Abstract

The invention discloses a hog cholera virus E2 protein epitope and preparation and application of an antibody. The sequence of the epitope is as shown in SEQ ID NO.1, and the epitope is positioned at105-109AA. The epitope can be reacted with CSFVs (Classical Swine Fever Viruses), but does not react with PK-15 cells. The epitope is applied to hog cholera virus antibody medicine detection agents. Due to the application, a hog cholera virus antibody diagnosis antigen can be prepared by using a common chemical synthesis method, and compared with a genetic engineering expression protein as a diagnosis antigen, the synthesized antigen is simple and convenient in process and relatively high in purity.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to the preparation and application of an antigenic epitope and antibody of classical swine fever virus E2 protein. Background technique [0002] CSF is a highly contagious infectious disease of pigs caused by CSFV, which has caused serious economic losses to the pig industry. CSFV is a member of the Flaviviridae family and the Pestivirus genus. CSFV is an enveloped single-stranded positive-sense RNA virus with a genome size of about 12.3 kb and only one large open reading frame (ORF). This ORF is translated into a polyprotein containing 3898 amino acid residues and a molecular weight of about 438kDa, and further processed into structural proteins and nonstructural proteins under the action of virus and host cell proteases, and its structural proteins and nonstructural proteins are on the viral RNA The coding sequence is Npro, C, Erns (E0), E1, E2, P7, NS2-3, NS4A, NS4...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C07K16/10G01N33/68G01N33/569
Inventor 童光志李国新武吉强徐晶晶姜一峰高飞童武郑浩
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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