Anti-fish viral haemorrhagic septicemia virus single-chain antibody

A technology of viral hemorrhage and single-chain antibody, which is applied in antiviral agents, antiviral immunoglobulins, antibodies, etc.

Inactive Publication Date: 2015-06-17
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The development of single-chain antibody ScFv against fish VHSV virus through phage antibody library technology has not been reported at home and abroad. The research of the present invention undoubtedly has important theoretical significance in the development of VHSV virus diagnostic reagents, new drug research and antigen epitope identification. and application value

Method used

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  • Anti-fish viral haemorrhagic septicemia virus single-chain antibody
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  • Anti-fish viral haemorrhagic septicemia virus single-chain antibody

Examples

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Effect test

Embodiment 1

[0017] Example 1 Construction of anti-fish viral hemorrhagic sepsis virus phage single-chain antibody library

[0018] 1. Emulsify the fish viral hemorrhagic septicemia virus obtained by ultracentrifugation with Freund's complete adjuvant 1:1 and immunize BALB / c mice, and boost the immunization twice until the antibody titer reaches 1:100,000 or more. Take the mouse spleen, grind it with liquid nitrogen, add 1ml Trizol reagent to 50-100mg spleen, let stand at room temperature for 5min, add 0.2ml chloroform, shake vigorously repeatedly for 15s, and let stand at room temperature for 2-3min. Centrifuge at 12,000g / min at 4°C for 15min, draw the water phase into another clean 1.5ml centrifuge tube, add 0.5ml of isopropanol and mix by inversion, settle at -20°C for 30-60min, and centrifuge at 12,000g / min for 10min. The precipitate was retained, washed once with 70% ethanol, air-dried, and the precipitated RNA was dissolved in DEPC-treated water.

[0019] 2. Removal of genomic DNA a...

Embodiment 2

[0038] Example 2 Analysis of single-chain antibody diversity

[0039] Ten clones were randomly selected, and the plasmid was extracted after shaking the bacteria, and digested with Sfi I and Not I, and the positive clones were preliminarily identified. Using S1 (S1: 5-CAACGTGAAAAAATTATTATT-3) and S6 (S6: 5-GTAAATGAATTTTCTGTATGAGG-3) as primers for sequencing analysis, the sequencing results of 10 clones showed that the sequences were consistent with the mouse immunoglobulin variable region sequence, Conforms to the mouse light and heavy chain variable region gene structure, the arrangement is VH-Linker-VL. Among them, the VH part is about 357-367bp, the VL is about 320-330bp, and the linker base sequences between the heavy chain and the light chain are all correct. Sequence alignment shows that the homology is more than 80%.

Embodiment 3

[0040] Example 3 Enrichment of Anti-Fish Viral Hemorrhagic Septicemia Virus Single Chain Antibody Library

[0041] 1. The fish viral hemorrhagic septicemia virus cell culture supernatant (titer is 1 × 10 6 PFU / mL) was diluted 1:5 (volume ratio) and coated with carbonate coating buffer to immunotubes, 2ml / tube, overnight at 4°C.

[0042] 2. After coating, wash the tube 3 times with PBS and pat dry; seal the immunotube with blocking (2% skim milk PBS, MPBS), and block at 37°C for 2 hours.

[0043] 3. Pour off the blocking solution, wash the tube 3 times with PBS, and pat dry;

[0044] 4. Mix the supernatant of the primary phage single-chain antibody library obtained above with MPBS and phage supernatant according to the volume ratio of MPBS: supernatant = 2:3, and perform de-interference treatment at room temperature for 20 minutes.

[0045] 5. Add the processed liquid in 4 into the sealed immunotube, 2ml / tube, incubate with gentle shaking for 30min, and then incubate for 1.5 ...

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Abstract

The invention discloses an anti-fish viral haemorrhagic septicemia virus single-chain antibody, a preparation method and application thereof, a gene for coding the single-chain antibody, and a vector, host cell and the like containing the gene. The anti-fish viral haemorrhagic septicemia virus single-chain antibody is formed by connecting an antibody heavy chain variable region and a light chain variable region through a joint peptide, and can be efficiently expressed through a prokaryotic expression system. The molecular weight of the single-chain antibody is 28kD or so; and the single-chain antibody can specifically identify the fish viral haemorrhagic septicemia virus and intercept the combination between the virus and natural serum, and can be further used for diagnosis of the virus, development of the therapeutic preparation and research of the antigen epitope.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a single-chain antibody against fish viral hemorrhagic sepsis virus and its preparation method and application. Background technique [0002] Viral hemorrhagic septicemia (VHS) is an infectious disease mainly caused by viral hemorrhagic septicemia virus (VHSV), which often causes salmon and trout A variety of seawater fish is morbid, and the mortality rate is as high as 90%, which causes huge economic losses to the development of the world's aquaculture industry. The World Organization for Animal Health (Office International Des Epizooties, OIE) included it in the list of aquatic animal diseases, and my country listed it as a second-class animal disease ("List of First and Second Class Infectious and Parasitic Diseases of Imported Animals of the People's Republic of China" , 2008). [0003] The genome of VHSV is an unsegmented single-stranded negative-strand RNA with a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70G01N33/569A61K39/42A61P31/14
Inventor 王宏华张全启于海洋齐洁王旭波王志刚贺艳
Owner OCEAN UNIV OF CHINA
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