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FliC protein and GAPDH protein microcapsule vaccine

A technology of microcapsules and vaccines, which is applied in the direction of microcapsules, vaccines, capsule delivery, etc., can solve the problems of death, economic loss in aquaculture industry, etc., achieve strong immunogenicity, facilitate large-scale promotion and application, and reduce stress

Active Publication Date: 2016-11-09
佳纬生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The bacteria can infect a variety of fish including freshwater and seawater, and can cause a large number of deaths in a short period of time, which has brought huge economic losses to my country's aquaculture industry

Method used

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  • FliC protein and GAPDH protein microcapsule vaccine
  • FliC protein and GAPDH protein microcapsule vaccine
  • FliC protein and GAPDH protein microcapsule vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Obtaining and sequence analysis of fliC protein and GAPDH protein

[0013] 1) Isolation of Edwardsiella tarda

[0014] In 2015, flounder raised in a flounder farm in Shandong Province had obvious symptoms of Edwardsiella tarda infection, but the diseased fish had been immunized with Edwardsiella tarda vaccine before. Take the dying diseased fish, obtain the liver tissue under aseptic conditions, streak it on the ordinary nutrient agar plate, culture at 30°C, pick the dominant colony for purification and culture until the pure culture bacteria are obtained.

[0015] The isolated pure cultures of each strain were expanded on a common nutrient agar plate, and then the colonies were eluted with sterile physiological saline to obtain a bacterial suspension for injection infection. The finally screened strains showed high virulence to healthy flounder, which could cause 90% death of flounder, and dead flounder had the same symptoms as diseased flounder. Therefore...

Embodiment 2

[0026] Example 2: Expression of recombinant protein and coupling with lipopolysaccharide

[0027] 1) Construction of GAPDH and fliC gene expression vectors: The amplified Edwardsiella tarda GAPDH and fliC gene products were gel-cut, recovered and purified, and the purified products were digested with BamHI and XholⅠ, then connected to the prokaryotic expression vector pET32a, and constructed respectively The expression vectors pET32a-GAPDH, pET32a-fliC, and the expression vectors were transformed into Escherichia coli BL21 (DE3), cultivated on the LB plate containing ampicillin, after the colonies grew out, the PCR method was used to screen the positive clone expressing bacteria, and the The strains detected as positive clones by PCR were further confirmed by sequencing.

[0028] 2) Expression and purification of recombinant protein: After confirming the correct insertion sequence in the vector by sequencing, the screened Edwardsiella tarda GAPDH and fliC positive clone expres...

Embodiment 3

[0031] Example 3: Preparation of fliC and GAPDH double subunit oral microcapsule vaccine

[0032]After mixing the coupling products of GAPDH, fliC and LPS prepared above by equal weight, the total amount of the mixture was mixed with astragalus polysaccharide in a ratio of 3:1, and the antigen mixture was mixed with sodium alginate at a ratio of 1:4 (W / W ) were mixed in proportion, and distilled water was used to prepare a sodium alginate-antigen emulsion, wherein the final concentration of sodium alginate was 2%, and it was embedded for 30 min under the agitation of a mixer. The emulsion is directly sprayed into 3% CaCl stirred by a mixer using the spray-type sharp-hole coagulation bath method. 2 solution, forming calcium alginate microcapsules, emulsions and CaCl 2 The volume ratio of the solution is 1:1, and the stirring is continued for 20 minutes after the spraying is completed. Collect calcium alginate microcapsules by centrifugation, then disperse the microcapsules in...

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Abstract

The invention provides a fliC protein and GAPDH protein microcapsule vaccine, wherein the antigens are fliC protein and GAPDH protein, the amino acid sequence of the fliC protein is shown as SEQ ID NO:1, and the amino acid sequence of the GAPDH protein is shown as SEQ ID NO:2. The prepared microcapsule vaccine is convenient to use and suitable in grain size, has good slow release feature and antigen protectiveness, and can effectively stimulate a gut immunity system of the fish body to generate immune response, so that the fish body has the specific immunity protection force for Edwardsiella tarda, and therefore, the fliC protein and GAPDH protein microcapsule vaccine can be used for preventing and treating the Edwardsiella tarda disease of the fish.

Description

technical field [0001] The invention belongs to the technical field of aquaculture vaccine preparation, in particular to a fliC protein and GAPDH protein microcapsule vaccine. Background technique [0002] Edwardsiella tarda (Edwardsiella tarda) is an important pathogenic bacterium of aquatic animals, which belongs to Enterobacteriaceae, Edwardsiella genus. pathogenic bacteria. The bacterium can infect many kinds of fish including freshwater and marine culture, and can cause a large number of deaths in a short period of time, which has brought huge economic losses to the aquaculture industry in our country. [0003] The use of vaccines can improve the specific immunity level of farmed fish, so that fish can develop immunity against infection by specific pathogenic microorganisms, without polluting the environment, and without drug residues. It is the mainstream development direction of fish disease prevention and control in the future. Therefore, screening antigens with go...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61K9/50A61K47/36A61P31/04A23K20/147A23K50/80
CPCA61K9/5036A61K39/025A61K2039/542A61K2039/552
Inventor 类延乐张铭李明亮沈彤孙翠彦
Owner 佳纬生物技术有限公司
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