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Polysaccharide and protein conjugate

A protein conjugation and conjugate technology, applied in the field of polysaccharide and protein conjugates, can solve the problems of weak immunogenicity, small molecular weight of LPS, and inability to produce immune boosting effect.

Inactive Publication Date: 2012-07-18
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, LPS has a small molecular weight, weak immunogenicity, and cannot produce immune boosting effect after re-immunization

Method used

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  • Polysaccharide and protein conjugate
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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, the preparation of the conjugate of polysaccharide and protein

[0060] I, extraction and purification of lipopolysaccharide (LPS)

[0061] 1. Extraction of LPS

[0062] The extraction method of LPS is recorded in Qin Min, Yang Guoyou, Zhou Fuchang, etc., Salmonella paratyphi A LPS extraction and O-SP purification. Progress in Microbiology and Immunology, 2005, 33 (01), 27-31, as follows:

[0063] Each time about 30 g of Vibrio cholerae 0139 (purchased from China National Institutes for Food and Drug Control) was added to 300 mL of 95% phenol aqueous solution, and then water was added until the total volume of the mixture was 600 mL. The mixed solution was shaken in a water bath at 68°C for 30 minutes, cooled in an ice bath, and then centrifuged at 7300×g and 10°C for 1 hour. Take the supernatant, add water to the remaining solution to a total volume of 600 mL, and repeat the 68°C water bath and centrifugation steps as described above. The operation was...

Embodiment 2

[0113] Embodiment 2, animal immunity experiment

[0114] 1. Immunity

[0115] Select 5-week-old BALB / c mice (BALB / C mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., female.) were randomly divided into two groups, namely the intraperitoneal injection group and nasal instillation group. Every big group is divided into 5 subgroups again, i.e. normal saline control group (normal saline concentration is 0.9% (mass percentage composition)), LPS (obtained by embodiment 1) immune group, CTB (obtained by embodiment 1) immune group , rBS-WC vaccine immunization group (rBS-WC vaccine was purchased from Shanghai United Cell Bioengineering Co., Ltd.) and LPS-CTB3 (obtained by Example 1, denoted as L-C) immunization group, with 6 mice in each group.

[0116] Mice in each group were immunized three times in total, and immunized on days 1, 15 and 29 (the first immunization was recorded as day 1). In the intraperitoneal injection group, the volume of e...

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Abstract

The invention discloses a polysaccharide and protein conjugate. The polysaccharide and protein conjugate is formed by coupling vibrio cholerae lipopolysaccharide and cholera toxin non-toxic subunit protein, wherein the vibrio cholerae lipopolysaccharide is from vibrio cholerae 0139, the protein content in the vibrio cholerae lipopolysaccharide is not more than 2 percent, the nucleic acid content is not more than 5 percent, and the molecular weight of the vibrio cholerae lipopolysaccharide is 8Kd. The immune effect of the polysaccharide and protein conjugate is higher than that of common rBS-WC (recombinant B subunit-killed whole-cell cholera) vaccine, single LPS (lipopolysaccharide) vaccine or single CTB (cholera toxin B-subunit) vaccine, the conjugate can enhance the immunogenicity of LPS to make an induced immunity reaction have immunologic memory and potentiation effect to generate massive bactericidin, is suitable for various kinds of people, and can initiate mucosal immunity of a body to realize IgA (immunoglobulin A) antibodies massively exist in intestinal secretion fluid and bile.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a conjugate of polysaccharide and protein. Background technique [0002] Cholera is a severe intestinal infection caused by the Gram-negative Vibrio cholerae bacterium. Humans are the natural host of Vibrio cholerae and are infected by ingesting water or food contaminated by Vibrio cholerae. The case fatality rate among infected people can be over 20%, and about 120,000 people die from cholera every year in the world. Its popular feature is explosive, and there have been 7 cholera pandemics in history. Initially occurring simultaneously in several different locations, then rapidly spreading epidemics can affect many countries and persist for many years, especially in some developing countries such as Africa, Asia and Latin America. One of the effective ways to control it is immunization and vaccination, so it is extremely important to seek to develop an effective cholera vaccine s...

Claims

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Application Information

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IPC IPC(8): A61K39/106A61P31/04
Inventor 曹诚王远靳彦文刘萱
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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