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Purifying process for oral recombinant helicobacter pylori vaccine

A technology for Helicobacter pylori and vaccines, which can be used in antibacterial drugs, bacterial antigen components, chemical instruments and methods, etc., and can solve problems such as difficult preparations

Inactive Publication Date: 2015-10-07
CHONGQING KANG WEI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinant developed by Orovax in 1999 H. pylori The urease therapeutic vaccine has entered phase II clinical trials. Although it has a good conservative antigen and tolerance, it requires adjuvants and multiple doses; the supervised Salmonella expressing urease studied by Oravax has entered phase I clinical trials In the test, it was easy to take and did not produce a clear mucosal immune response; the inactivated whole bacterial antigen vaccine developed by Antex Biologics entered Phase II clinical trials, and it has relatively complete immunity, but it is difficult to prepare, and further investigation is needed for safety (Zou Quanming. Research progress of Helicobacter pylori vaccine. Gastroenterology [J], 2007, 12 (9): 567-570)

Method used

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  • Purifying process for oral recombinant helicobacter pylori vaccine
  • Purifying process for oral recombinant helicobacter pylori vaccine
  • Purifying process for oral recombinant helicobacter pylori vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Cell fragmentation

[0030] Weigh 2000g of engineering bacteria, add 10mmol / L pH 8.0 Tris-HCl buffer solution at a ratio of 1:10, mix well, break the bacteria with a high-pressure homogenizer, break the bacteria three times at 700bar, and use a tubular centrifuge at 9000rpm Centrifuge at a flow rate of 1L / min, and the precipitate is the crude inclusion body. Microscopic examination of bacterial fragmentation rate ≥ 99%.

[0031] Washing and lysis of inclusion bodies

[0032] (1) Weigh 300g of inclusion bodies, add washing solution I at a ratio of 1:10, after the high-speed disperser is completely dispersed, mix and stir for 20min, centrifuge at 5000rpm for 40min, and collect the precipitate.

[0033] (2) Add washing liquid II to the precipitate, after the high-speed disperser is completely dispersed, mix and stir evenly, centrifuge at 5000rpm for 40min, and collect the precipitate.

[0034] (3) Add inclusion body lysate to the precipitate at a ratio of 1:10, stir overn...

Embodiment 2

[0042] Cell fragmentation

[0043] Weigh 2000g of engineering bacteria, add 10mmol / L pH 6.0 Tris-HCl buffer solution at a ratio of 1:10, mix well, break the bacteria with a high-pressure homogenizer, break the bacteria twice at 600bar, and use a tubular centrifuge at 8000rpm Centrifuge at a flow rate of 1L / min, and the precipitate is the crude inclusion body. Microscopic examination of bacterial fragmentation rate ≥ 99%.

[0044] Washing and lysis of inclusion bodies

[0045] (1) Weigh 300g of inclusion bodies, add washing solution I at a ratio of 1:10, after the high-speed disperser is completely dispersed, mix and stir for 15min, centrifuge at 4000rpm for 20min, and collect the precipitate.

[0046] (2) Add washing liquid II to the precipitate, after the high-speed disperser is completely dispersed, mix and stir evenly, centrifuge at 4000rpm for 20min, and collect the precipitate.

[0047] (3) Add inclusion body lysate to the precipitate at a ratio of 1:10, stir overnight...

Embodiment 3

[0055] Cell fragmentation

[0056] Weigh 3000g of engineering bacteria, add 10mmol / L pH 9.0 Tris-HCl buffer solution at a ratio of 1:10, mix well and break the bacteria with a high-pressure homogenizer, break the bacteria three times at 800bar, and use a tubular centrifuge at 10000rpm Centrifuge at a flow rate of 2L / min, and the precipitate is the crude inclusion body. Microscopic examination of bacterial fragmentation rate ≥ 99%.

[0057] Washing and lysis of inclusion bodies

[0058] (1) Weigh 400g of inclusion bodies, add washing solution Ⅰ at a ratio of 1:10, after the high-speed disperser is completely dispersed, mix and stir for 30min, centrifuge at 6000rpm for 40min, and collect the precipitate.

[0059] (2) Add washing liquid II to the precipitate, after the high-speed disperser is completely dispersed, mix and stir evenly, centrifuge at 6000rpm for 40min, and collect the precipitate.

[0060] (3) Add inclusion body lysate to the precipitate at a ratio of 1:10, stir...

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Abstract

The invention discloses a purifying process for an oral recombinant helicobacter pylori vaccine. The effective component of the vaccine is fusion protein of Escherichia coli heat labile enterotoxin B subunit (LTB) and urase subunit B (UreB). LTB-UreB vaccine protein with high purity is obtained by subjecting a collected fermented genetically-engineered bacterium expressing the LTB-UreB fusion protein to high pressure bacterium breaking, washing, cleavage, anion-exchange column chromatography, ultrafiltration, gel filtration chromatography, etc. The purifying process has the advantages of simplicity, high efficiency, good repeatability, high yield and easy realization of industrial production; the obtained target protein has a purity of more than 98% according to detection results of high performance liquid chromatography; and animal test proves that the oral recombinant helicobacter pylori vaccine protein prepared by using the purifying process has good immunocompetence and immunoprotectivity.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a purification process of oral recombinant Helicobacter pylori vaccine. technical background [0002] Helicobacter pylori ( Helicobacter pylori , H. pylori ) is a Gram-negative, microaerophilic bacterium that is closely associated with chronic gastritis, peptic ulcer, and gastric adenocarcinoma. H. pylori Infection is one of the most common chronic infectious diseases, which has caused more than 50% of the world's population to be infected. In China, H. pylori More than 600 million people have been infected. H. pylori The toxins and toxic substances in the stomach can not only damage the gastric mucosa, but also cause the body to produce inflammation and immune responses, affect the secretion of gastric acid, and eventually lead to the formation of a series of diseases. [0003] In 1994 the World Health Organization has H. pylori Listed as a class I carcinogen (grade c...

Claims

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Application Information

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IPC IPC(8): C12N9/80C07K14/245A61K39/02A61K39/39A61P31/04
Inventor 刘丹刘亚龙翁樑邹雪潘安龙徐增晟李建旭苏亚南
Owner CHONGQING KANG WEI BIOTECH
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