Cryptocaryon irritans recombinant protein vaccine as well as preparation method and application

A technology for stimulating Cryptonucleus and proteins, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of the difficulty in preventing the infection of marine fish by stimulating Cryptonucleus and inhibiting the DNA sequence of antigens. and other problems, to achieve the effect of excellent expression effect, large amount of protein expression, and maximum amount of protein expression

Active Publication Date: 2018-03-20
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention has three purposes of the invention: (1) In order to overcome the disadvantages that the existing inhibitory antigen DNA sequence is difficult to express in bacteria and that there is no effective genetic recombinant vaccine for preventing seawater fish stimulating Cryptocaryon infection on the market, a kind of Can be expressed in Escherichia coli, has immunogenicity, and can prevent the Cryptocaryon stimulator recombinant protein vaccine from the Cryptocaryon stimulator infection of seawater fish; There is no effective gene recombination vaccine to prevent the infection of C

Method used

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  • Cryptocaryon irritans recombinant protein vaccine as well as preparation method and application
  • Cryptocaryon irritans recombinant protein vaccine as well as preparation method and application
  • Cryptocaryon irritans recombinant protein vaccine as well as preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0045] Example 1

[0046] 1. Collection and cultivation of parasites

[0047] Fish infected with Cryptocaryon stimuli were collected from fish rafts in Ningde and Putian and raised in an aquarium with a water temperature of 25°C. After the cystic body (tomont) was collected from the bottom of the aquarium, it produced ciliated larvae and infected pomfret (Trachinotus ovatus) or pearl gentian (E. fuscoguttatus X gentian E. Lanceolatus).

[0048] The process of parasite culture used in this application is as follows: use a brush or a small paint brush to gently sweep and collect cysts that stimulate Cryptocaryon from the bottom of the aquarium, and use seawater to wash off excess residue or mucous membranes , the cyst body is cultured on a petri dish with filtered sea water, and cultured at room temperature for 3-7 days. The ciliated larva (theront) that stimulates Cryptocaryonia will be produced from the cyst body. At this time, the solution containing the ciliated larvae ca...

Example Embodiment

[0086] Example 2

[0087]The difference between this example and Example 1 is that the constructed recombinant plasmid pFNCJ was transformed into the competent cells of BL21DE3, and then evenly spread on the LB plate containing 48ug / mL kanamycin sulfate, and then placed upside down at 38 ℃ incubator overnight, select single clones from the transformed plates, inoculate into LB medium containing 48ug / mL of kanamycin sulfate, cultivate at 38 ℃ shaker at 580rpm / mim, until the OD600 is 0.5-0.8, IPTG was added to the culture medium in the test tube to a final concentration of 0.5 mM, and then placed at 28°C to induce expression.

[0088] The C. stimuli recombinant protein vaccine is mixed into the feed or bait of the vaccine-use object, so as to prevent the vaccine-use object from being infected with C. stimuli.

Example Embodiment

[0089] Example 3

[0090] The difference between this example and Example 1 is that the constructed recombinant plasmid pFNCJ was transformed into BL21DE3 competent cells, and then evenly spread on the LB plate containing 52ug / mL kanamycin sulfate, and then placed upside down at 36 ℃ incubator overnight, select single clones from the transformed plates, inoculate into LB medium containing 52ug / mL kanamycin sulfate, cultivate at 36 ℃ shaker at 620rpm / mim, until the OD600 is 0.5-0.8, IPTG was added to the culture medium in the test tube to a final concentration of 0.5 mM, and then placed at 37°C to induce expression.

[0091] The C. stimuli recombinant protein vaccine is mixed into water to soak the object of use of the vaccine, so as to prevent the object of use of the vaccine from being infected with C. stimuli.

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Abstract

The invention relates to a cryptocaryon irritans recombinant protein vaccine of which the amino acid sequences are as follows: NWVEKTAAADWKGTFVVTSSSCLASCGWKIGTTVVIADKASDATKVTWVTWVGTAHTTDSTNVDVASGSCKYVSSVANAGTAGTAAEVLNNNDECVFATGMCTIMGQKQKSPAKVTFNRDTTLDTKPFQILYKQLAMVPKAQTTVQAAADQATDCDTKASLVDTTTDAKSIVGTLKLSKATCDKCSWDTTKDLKITQDATKKYMVTLAGTIKETTSGDCKNKLTASEACYVTKKDDKTFILVSCTTLDTTNKGIPIAITTANSKTTLTLTRTDSSSQACNVVGEIT. By adopting the vaccine, the defects that an immobilization antigen DNA (Deoxyribonucleic Acid) sequence is hard to express in bacteria, and an effective gene recombinant vaccine for preventing cryptocaryon irritans infections of seawater fishes is not available in the market, are overcome, and the vaccine has the advantages of expression in escherichia coli, immunogenicity and prevention of cryptocaryon irritans infections of seawater fishes.

Description

technical field [0001] The invention relates to a recombinant protein vaccine that stimulates cryptocleidia and its preparation method and application, and is applied in the field of seawater fish disease prevention. Background technique [0002] Cryptocaryon irritans is a pathogenic organism that causes a variety of marine fish (including wild fish, ornamental fish or farmed fish) to be infected with seawater white spot disease. It is considered to be one of the most economically damaging parasites in fisheries, causing huge economic losses to aquaculture fisheries in all major marine aquaculture areas around the world. Although chemicals can be used to prevent or treat fish infected with Cryptocaryonia irritatus, they cause serious marine pollution and the fish produced are also contaminated, which poses a health hazard to those who eat them. [0003] The life cycle of Cryptocaryon stimuli consists of four stages: trophont, protomont, tomonts cyst and theront. Studies on...

Claims

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Application Information

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IPC IPC(8): C07K14/44C12N15/30C12N15/70A61K39/002A61P33/02
CPCA61K39/00C07K14/44C12N15/70C12N2800/22
Inventor 张子平王艺磊冯建军邹志华张鑫何亮华
Owner FUJIAN AGRI & FORESTRY UNIV
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