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Mutant Escherichia coli heat liable toxin, preparation method thereof, adjuvant and vaccine increasing immunoreaction

A technology of Escherichia coli and thermotoxin, applied in the directions of biochemical equipment and methods, antiviral agents, chemical instruments and methods, etc., can solve the problems of low toxin production and yield, Escherichia coli infection, toxicity, etc., and achieve good immune effect. , lower cost, less toxic effect

Inactive Publication Date: 2014-05-07
AGRI TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Although LTI has the effect of an adjuvant, there are many disadvantages of using enterotoxin-producing E. coli to produce LTI as an adjuvant, including: (1) There is a risk of infection when manipulating enterotoxin-producing E. coli
(2) The toxin production of different strains is different and the yield is low (about 60μg-2.5mg) (Lasaro et al., 2006)
(3) Although natural toxins can enhance the immune effect, they are toxic and not suitable for direct use as vaccine adjuvants
However, none of these inventions can mass-produce mutant Escherichia coli heat-resistant toxin in large quantities; therefore, the main purpose of the present invention is to increase the yield of mutants to reduce the production cost of protein adjuvants

Method used

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  • Mutant Escherichia coli heat liable toxin, preparation method thereof, adjuvant and vaccine increasing immunoreaction
  • Mutant Escherichia coli heat liable toxin, preparation method thereof, adjuvant and vaccine increasing immunoreaction
  • Mutant Escherichia coli heat liable toxin, preparation method thereof, adjuvant and vaccine increasing immunoreaction

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[0063] A kind of mutant Escherichia coli heat avoiding toxin and its preparation method of the present invention, especially about a method of improving the yield of mutant Escherichia coli heat avoiding toxin to reduce the production cost of protein adjuvant and its experimental method steps and experimental results , which will be described in the form of specific examples, and the process and results are only illustrative, and are not intended to limit the scope of the patent application that the present invention intends to claim.

example 1

[0065] Isolation of strains

[0066] Pig intestines were collected from pig farms where pig diarrhea occurred. The porcine intestinal fluid was collected with a sterilized inoculation loop and streaked on MacConkey agar medium. After culturing overnight, a single red colony was selected for biochemical tests, including indole test, methyl red test, Voges-Prokauer reaction and citric acid test. After the bacterial strains are tested, if the indole test is positive, the methyl red test is positive, the Fopp's reaction is negative and the citric acid test is negative, then it is determined to be Escherichia coli. Further, carry out serum agglutination test with flagellar antigen K88 antiserum, if the agglutination test is positive, it means that the strain is K88 + Escherichia coli. The strains from different farms were numbered 171, 196 and 286, respectively.

example 2

[0068] Extraction of Enterotoxigenic Escherichia coli Plasmid

[0069] Plasmid Miniprep Purification Kit II (GeneMark, Taiwan) was used to extract the Escherichia coli plasmid, and the operation procedure was carried out according to the method provided by the manufacturer. Solution I, solution II, solution III, washing solution A, washing solution B and eluting solution mentioned in the process are all reagents attached to the kit. Take 1.5 mL of overnight cultured bacterial solution to a microcentrifuge tube, centrifuge (21,000×g, 3 minutes, room temperature) to collect the bacterial cells, discard the supernatant, and repeat this step twice. Resuspend the cells in 200 μL of solution I. Then add 200 μL of Solution II and mix gently several times. Next, 200 μL of solution III was added and mixed gently several times. The supernatant was collected by centrifugation (21,000 xg, 10 minutes, room temperature). Place the purification column (spin column) on the collection tube...

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Abstract

The invention provides mutant Escherichia coli heat liable toxin, a preparation method thereof, an adjuvant and a vaccine increasing immunoreactions. The preparation method and products are realized by site-specific mutagenesis of Escherichia coli heat liable toxin genes, replacing signal peptide of LT-A and LT-B of the Escherichia coli heat liable toxin to signal peptide of outer membrane proteins of Escherichia coli, and transforming the signal peptide of the outer membrane proteins of Escherichia coli. In addition, the mutant Escherichia coli heat liable toxin and the preparation method are advantaged by being capable of applying in adjuvant and vaccines, and the mutant Escherichia coli heat liable toxin can be produced in a mass manner, and cost is reduced.

Description

technical field [0001] The invention relates to a mutant Escherichia coli heat-resistant toxin and a preparation method thereof, in particular to a method for increasing the production of mutant Escherichia coli heat-resistant toxin to reduce the production cost of protein adjuvants and vaccines. Background technique [0002] Escherichia coli belongs to Enterobacteriaceae in taxonomy, and is a facultative anaerobic, non-spore-forming Gram-negative bacteria. Most Escherichia coli are harmless to humans and animals, but some serotypes of Escherichia coli can cause symptoms such as diarrhea, food poisoning and extraintestinal infection. Pathogenic Escherichia coli can be divided into two types: Diarrheagenic E.coli and extraintestinal pathogenic E.coli (ExPEC) according to different pathogenic patterns. Among them, diarrheal E. coli can be further divided into enteropathogenic E. coli (EPEC), enterotoxigeic E. coli (ETEC), enteroinvasive E. coli (enteroinvasive E. coli) ; EIE...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/245A61K39/39A61P31/04A61P31/12C12R1/19
Inventor 林俊宏王志鹏谢明伟方健宇曾皓真杜清富
Owner AGRI TECH RES INST