Mucosal vaccine adjuvants containing bacterial flegellins as an active component

一种疫苗佐剂、鞭毛蛋白的技术，应用在含有效成分的医用配制品、细菌抗原成分、抗体医疗成分等方向，能够解决不适人类、高肠毒素毒性等问题

Active Publication Date: 2007-02-07
李浚行 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these adjuvants are exotoxins with high enterotoxin toxicity and are therefore not suitable for direct use in humans

Method used

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  • Mucosal vaccine adjuvants containing bacterial flegellins as an active component
  • Mucosal vaccine adjuvants containing bacterial flegellins as an active component
  • Mucosal vaccine adjuvants containing bacterial flegellins as an active component

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of transposon library

[0043] Vibrio vulnificus MO6-24 / O type strain (obtained from J. Glenn Morris, Department of Epidemiology, University of Maryland Medical College Hospital) and mini-Tn5 lacZ1 strain including E.coli SM10λpir (from Braunschweig, GBF National Research, Germany) Center for Biotechnology, obtained from Kenneth N. Timmis) were cultured in a shaker at 37°C and 210 revolutions per minute. The two bacteria were cultured in 10 ml of 2.5HI (2.5% NaCl heart infusion) broth and 20 ml of A single colony was inoculated in LB broth (containing 100 μg / ml ampicillin and 100 μg / ml kanamycin).

[0044] The inoculated culture was centrifuged the next day, washed and centrifuged twice with antibiotic-free LB broth medium, and then suspended in 100 microliters of new LB broth medium. A single bacterial suspension of E. coli and Vibrio vulnificus was mixed together and dropped on an LB agar plate. After culturing overnight at 37°C, 800 microliters of ...

Embodiment 2

[0046] Example 2 Screening of Transposon Mutant Colonies Losing Motility

[0047] Each clone of the Vibrio vulnificus MO6-24 / O transposon library prepared in Example 1 was cultured overnight at 37°C, and then inoculated into semi-solid HI containing 0.3% agar using a sterilized toothpick (Heart Extract) agar plate and incubate at 37°C for 6 hours. Then by measuring the range of movement of the bacteria after growth, the degree of motility of the bacteria is determined.

[0048] Three transposon mutant clones that almost completely lost motility were selected through the screening step, and an experiment was performed to identify the mutant genes inserted into the transposon.

Embodiment 3

[0049] Example 3 Identification of flagellin operon gene

[0050] The cosmid gene library was screened to clone genes near the transposon insertion region, and DNA fragments were used as primers for random polymerase chain reaction (Polymerase Chain Reaction, PCR) amplification. A two-step PCR amplification method was used to amplify the DNA fragments near the insertion site of the transposon. In the first step of PCR, random primer 1 (5-GGCCACGCGTCGACTAGTCANNNNNNNNNNACGCCC-3) of sequence number 13 and mini-Tn5 lacZ1 specific primer 1 of sequence number 14 (5-TTCTTCACGAGGCAGACCTCAGCGC-3) were used. The first PCR settings are as follows: denaturation at 94°C for 30 seconds, annealing at 30°C for 30 seconds, then extension at 72°C for 1 minute and 30 seconds, 5 cycles; then denaturation at 94°C for 30 seconds, annealing at 45°C for 30 seconds, and extension at 72°C 2 minutes, 30 cycles, and then 30 cycles of PCR reaction. Use the product of the first PCR as a template for the second...

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Abstract

The present invention relates to mucosal vaccine adjuvants containing flagellins, the structural component of flagella, originated from Vibrio vulnificus, Salmonella typhimurium, and Listeria monocytogenes as an active component.

Description

Technical field [0001] The present invention relates to a mucosal vaccine adjuvant containing flagellin as an active ingredient. Flagellin is a structural component of bacterial flagella, which is derived from Vibrio vulnificus, Salmonella typhimurium and monocytic proliferation Listeria monocytogenes. Background technique [0002] The infectious disease caused by Vibrio vulnificus or its abbreviation "V.vulnificus" has a short history, but its clinical cases have been continuously reported worldwide, and this disease is one of the newly observed diseases. Although the absolute number of clinical cases of the disease is less than the number of cases of cholera or salmonella food poisoning, it has become an important social problem due to its high mortality rate and catastrophic clinical manifestations. [0003] Hollis et al. of the Centers for Disease Control in USA (CDC) first reported on Vibrio vulnificus in 1976 after 11 years of studying the bacteriological characteristics of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K39/08
CPCA61K39/08A61K39/39A61K2039/541A61K2039/55516A61K2039/55544A61P15/16A61P31/00A61P35/00Y02A50/30G02F1/1309G02F2203/69
Inventor 李浚行李施恩金守永
Owner 李浚行
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