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Use of protein TolC in preparing immunological formulation and vaccine

A protein and use technology, applied in the field of medical use of protein TolC, can solve problems such as no related reports, and achieve the effect of strong immunogenicity and good application prospects

Inactive Publication Date: 2006-12-13
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no relevant reports on the serological study of this protein, and the possibility that this protein may be used as a neutralizing antigen for the preparation of immune preparations or vaccine development

Method used

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  • Use of protein TolC in preparing immunological formulation and vaccine
  • Use of protein TolC in preparing immunological formulation and vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of Immune Serum

[0028] 1 Materials and methods

[0029] Shigella flexneri 2a 2457T, an international standard strain, was purchased from the market; LB medium was prepared in accordance with the "Molecular Cloning Experiment Guide"; big-eared white rabbits were purchased from the Animal Center of the Academy of Military Medical Sciences, and the animal grade was grade 1.

[0030] 1.1 Strains and culture conditions

[0031] Cultivate overnight at 37°C in LB medium, transfer to fresh medium at a ratio of 1:100, and culture at 37°C at 250 rpm until the early stationary phase, OD 600 Stop culture when it reaches 3.3.

[0032] 1.2 Preparation of serum

[0033] Bacterial culture conditions were the same as above. The big-eared white rabbits were used in the experiment, and the live bacterial immunogen was injected into the ear vein, with an interval of 5 days, and immunized 6 times, the first time was 500 million, the second time w...

Embodiment 2

[0035] Example 2 Two-dimensional electrophoresis and Western Blotting of the outer membrane protein of Shigella flexneri

[0036] 1 Materials and methods

[0037] 1.1 The reagents used are products of Amrsco Company, analytically pure. The electrophoresis apparatus is a product of Amersia Company, and the rest are the same as in Example 1.

[0038] 1.2 Preparation of the outer membrane protein of Shigella flexneri:

[0039] Shake Shigella flexneri 2a 2457T 37°C overnight, collect the cells by centrifugation at 2000g for 15 minutes, wash with PBS three times, resuspend the cells in 50mmol / L Tris-HCl (pH7.5) solution, sonicate, 6000g Centrifuge for 10 minutes to remove the precipitate, and use 10 times the volume of pre-cooled O.1mmol / L Na for the supernatant 2 CO 3 (pH11) dissolved, ice-bathed for 1h, 100000g ultracentrifuged for 1h, the precipitate was washed once with 50mmol / L Tris-HCl (pH7.5), centrifuged at 100000g for 1h, and the precipitate was the outer membrane prot...

Embodiment 3

[0045] Example 3 In-gel digestion and MALDI-TOF mass spectrometry detection

[0046] 1 Material method

[0047] 1.1 The mass spectrometer is Reflex.III MALDI-TOF-MS from Bruker, Germany, and the rest of the materials are the same as above.

[0048] 1.2 Cut corresponding protein spots from Coomassie Brilliant Blue-stained gel, and refer to the literature [Liao Xiang, Ying Tianyi, Wang Hengliang, etc. for the method of in-gel enzyme digestion. Coomassie Brilliant Blue-stained two-dimensional electrophoresis gel in-gel enzyme digestion method improvement. Biotechnology Communications 2003;14:509-511]. Mass spectrometry was performed at the National Biomedical Analysis Center using a Reflex.III MALDI-TOF-MS (Bruker, Germany) mass spectrometer.

[0049] 1.3 Database query

[0050] Peptide mass fingerprint searches were performed in the corresponding databases using Mascot (http: / / www.matrixscience.com). At the same time, the Mascot search was run locally using the geno...

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Abstract

The invention discloses the usage of TolC protein. The TolC has strong serum reaction, is used to detect and diagnose dysentery, and is used as protective antigens to prepare vaccine.

Description

technical field [0001] The invention relates to the medical application of a protein TolC, in particular to the application of the protein in the preparation of immune preparations and vaccines. Background technique [0002] Dysentery is an intestinal infectious disease that is prevalent all over the world. It breaks out occasionally in developed countries, but it is the main cause of diarrhea in developing countries. The annual number of cases of dysentery in the world is about 164.7 million, and the annual death toll exceeds 1.1 million (Kotloff KL, Winickoff JP, Ivanoff B, et al. Global burden of shigella infextions: implications for vaccine development and implementation of control strategies. Bull World Health Organ 1999; 77:651). The epidemic in our country is mainly Freund's dysentery, so it is of great significance to carry out in-depth research on it. Considering the mechanism of bacterial infection and immunity, it is recognized as an ideal way to find good antig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/112A61P31/04G01N33/68G01N33/563G01N33/569
CPCY02A50/30
Inventor 王恒樑黄留玉应天翼冯尔玲苏国富
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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