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Edwardsiella tarda antidiotypic antibody genetic engineering vaccine and preparation method thereof

A genetically engineered vaccine, idiotype antibody technology, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems that have not been reported in the literature

Inactive Publication Date: 2009-06-17
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] At the same time, the Shaanxi Science and Technology Information Institute is entrusted to search the subject of the present invention in Chinese and foreign literature databases, and there is no document report exactly the same as the present invention

Method used

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  • Edwardsiella tarda antidiotypic antibody genetic engineering vaccine and preparation method thereof
  • Edwardsiella tarda antidiotypic antibody genetic engineering vaccine and preparation method thereof
  • Edwardsiella tarda antidiotypic antibody genetic engineering vaccine and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0027] In the previous research of the present invention, the Edwardsiella tarda anti-idiotypic monoclonal antibody hybridoma cell line 1E11 has been successfully prepared, which can secrete an anti-idiotypic antibody with antigen "internal image" and high protection rate. In order to construct the Edwardsiella tarda anti-idiotypic antibody genetic engineering vaccine, its implementation method is:

[0028] 1. Delayed recovery of Edwardsiella anti-idiotypic monoclonal hybridoma cells:

[0029] Take out the monoclonal hybridoma cell line 1E11 that secretes Edwardsiella tarda anti-idiotype from the frozen liquid nitrogen tank, put it in a 37°C water bath to thaw quickly, add 10 mL of RPMI-1640 serum-free culture medium preheated at 37°C, Centrifuge at 1000rpm for 10min, discard the supernatant; add 5mL of 20% RPMI-1640 culture solution containing calf serum, centrifuge at 1000rpm for 10min, discard the supernatant; add 10mL of RPMI-1640 culture solution Gently pipette the pelle...

Embodiment 2

[0108] Materials and methods

[0109] 1 Strains, plasmids and main reagents Plasmid PET28a and strain E.coli BL21(DE3) are products of Novagen, USA. RPMI1640 is a product of GibcoBRL Company, RNA extraction reagents are purchased from Takara Company, and RT-PCR kits are products of Invitrogen Company of the United States. Plasmid extraction kits and gel recovery kits are purchased from Beijing Biotech Company. PCR reagents and ligation reagents were purchased from Beijing Tianwei Times Company.

[0110]Lysis solution: 50mmol Tris-HCl, 0.5mmol EDTA, 5mmol DTT, 5% glycerol, pH8.0.

[0111] Refolding solution: 25mmol Tris-HCl, 10mmol NaCl, 0.1mol urea, 1% glycerol, pH 8.0.

[0112] Gradient dialysate: 1.25mmol Tris-HCl, 10mmol NaCl, 0.05mol urea, 1% glycerin, pH8.0.

[0113] 2.25mmol Tris-HCl, 10mmol NaCl, 0.01mol urea, 1% glycerin, pH8.0.

[0114] 2. Preparation method of Edwardsiella tarda anti-idiotypic antibody genetic engineering vaccine

[0115] Other situa...

Embodiment 3

[0121] Immunoprotective effect of Edwardsiella tarda anti-idiotypic antibody genetically engineered vaccine

[0122] 1. Fish used in the test: American redfish, also known as red meager, belongs to Perciformes and Totoaba family. Sciaenops ocellatus (Linnaeus)), a total of 200 tails were used in the experiment, divided into two groups, the injection group and the control group, 100 tails in each group.

[0123] 2. Method

[0124] The Edwardsiella tarda anti-idiotypic antibody and Freund's incomplete adjuvant were mixed 1:1, and the American redfish was immunized by intraperitoneal injection. Each fish was injected with 0.1mL (containing 10-20μg of antibody). One month after the immunization, 5 times the half-lethal dose The American redfish was challenged by intraperitoneal injection of Edwardsiella tarda, and the protective effect of anti-idiotypic antibody genetic engineering vaccine on American redfish was observed.

[0125] Ten fish were randomly selected from each group...

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Abstract

The invention provides a genetic engineering vaccine for an anti-idiotypic antibody of edwardsiella tarda and a method for preparing the same. The method comprises the following steps: adopting an RT-PCR method to clone VH and VL variable region genes from a hybridoma cell strain which excretes idiotype monoclonal antibodies of the edwardsiella tarda; introducing a connecting peptide (Gly4Ser)3 into the VH and VL variable region genes; constructing a single chain antibody scFv gene in the form of VL-Linker-VH in vitro, and cloning the single chain antibody scFv gene to an expression vector PET28a and expressing the same in Escherichia coli; and after purifying expressed product, identifying the product through SDS-PAGE, Western blot and ELASA: expressing an anti-idiotypic single chain antibody gene scFv of the edwardsiella trada in a BL21 bacterium, preserving the expressed product in the form of an insoluble inclusion body, obtaining a high purity single chain antibody fragment by dissolving the inclusion body, purifying, and renaturing in vitro. The relative molecular weight of the recombinant protein is 27 kD. The immune protection rate for sea fishes is averagely more than 80 percent.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and mainly relates to biological preparations for preventing and treating fish bacterial diseases, in particular to an Edwardsiella tarda anti-idiotypic antibody genetic engineering vaccine and a preparation method thereof. Background technique [0002] Bacterial epidemic diseases have broken out in seawater cultured fish and shrimp, and the pathogens are Vibrio (Vibrio anguillarum, Vibrio alginolyticus, Vibrio vulnificus, Vibrio riverina, Vibrio parahaemolyticus) and Edwardsiella tarda. Among them, Edwardsiella tarda infection has the characteristics of fulminant attack, and its primary infection or secondary infection caused by it has serious pathogenicity to humans and fish. Among the common fish diseases, Edwardsiella tarda is one of the most important pathogenic bacteria in freshwater and marine cultured fish. The bacterium is a Gram-negative, perinatal flagellar motile brevibacteriu...

Claims

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Application Information

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IPC IPC(8): A61K39/395C12N15/31A61P31/04
Inventor 秦红黄威权金晓航刘玉林张欣刘子冬
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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