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Purification method of candidate antigen Glu of streptococcus mutans recombinant subunit genetic engineering vaccine

A genetically engineered vaccine and Streptococcus mutans technology, which is applied in the field of purification in the preparation of recombinant Streptococcus mutans caries vaccine candidate antigen Glu, and can solve problems such as body damage and insufficient attenuation.

Active Publication Date: 2014-05-28
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although traditional inactivated vaccines and live attenuated vaccines can inhibit the adhesion of Streptococcus mutans to the tooth surface, the antibodies induced by whole cells have cross-reaction with heart and kidney tissues, and live attenuated vaccines have insufficient attenuation or virulence. Recovery can cause damage to the body and are difficult to apply clinically

Method used

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  • Purification method of candidate antigen Glu of streptococcus mutans recombinant subunit genetic engineering vaccine
  • Purification method of candidate antigen Glu of streptococcus mutans recombinant subunit genetic engineering vaccine
  • Purification method of candidate antigen Glu of streptococcus mutans recombinant subunit genetic engineering vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of Antigen Glu

[0042] (1) Cloning of Streptococcus mutans Glu gene

[0043] 1. Streptococcus mutans UA159 (from the American Type Culture Collection Center, ATCC) (preserved by the Department of Clinical Microbiology and Immunology, Third Military Medical University of the Chinese People's Liberation Army)

[0044] 2. Take out the preserved Streptococcus mutans UA159 strain from the liquid nitrogen tank, spread it on the BHI solid medium, and cultivate it overnight at 37°C. Genome extraction kit to extract the genome of Streptococcus mutans.

[0045] 3. Using the PCR method to amplify the Glu coding gene from the Streptococcus mutans genome respectively.

[0046] 1) The primers were designed and synthesized as follows (the base sequence of the restriction site is underlined)

[0047] According to the gene sequence published by GenBank and the principles of primer design, the corresponding primers were designed and restriction sites were intr...

Embodiment 2

[0145] Example 2 Autoclave, centrifuge

[0146] 1. Ferment the constructed Escherichia coli engineering bacteria expressing the Streptococcus mutans recombinant subunit genetically engineered protein Glu, and the expression rate of the target protein is 26%, and the bacteria are collected by centrifugation for later use.

[0147] 2. Mix and suspend 200-500 g of bacteria in PBS (10-20 mM, pH 7.0-7.5) buffer according to the weight: volume ratio of 1:10, and pre-cool at 4°C.

[0148] 3. High-pressure homogenizer: Use distilled water to flush the pipeline of the high-pressure homogenizer, and turn on the low-temperature circulation system to pre-cool to 1-4°C for standby.

[0149] 4. Add the pre-cooled suspended bacteria into a high-pressure homogenizer, maintain the pressure at 60-80Mpa to break the bacteria 3-5 times, take a smear of the broken bacteria and stain it with crystal violet, and the number of unbroken bacteria in each field of view under the oil microscope is less...

Embodiment 3

[0152] Example 3 Inclusion body washing and lysis

[0153] 1. Resuspend the centrifugal pellet with 9 times the volume of pH 7.0-7.520mM PB buffer containing 1% Triton X-100, and mix thoroughly with a high-speed disperser, then gently stir with a stirrer for 30min, and then Centrifuge at 10,000×g for 30 minutes, collect the precipitate, and repeat once;

[0154] 2. Resuspend the centrifuged pellet with 9 times the volume of pH 7.0-7.520mM PB buffer containing 2M urea, mix well with a high-speed disperser, stir gently with a stirrer for 15min, and then centrifuge at 10000×g for 30min. Collect the precipitate and repeat once;

[0155] 3. Weigh the wet weight of the precipitate, add PB (10-20mM, pH7.0-7.5) buffer solution containing 8M urea according to the weight: volume ratio of 1:10, stir and dissolve overnight at 4°C, centrifuge at 13000g for 30 minutes at high speed, and collect supernatant.

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Abstract

The invention provides a purification method of a candidate antigen Glu of streptococcus mutans recombinant subunit genetic engineering vaccine. The purification method comprises the steps as follows: fermented escherichia coli thalli of Glu protein of the streptococcus mutans recombinant subunit genetic engineering vaccine are collected, high-pressure homogenizing and thallus breaking are performed, a supernatant is collected and centrifuged, and precipitation is collected; inclusion body washing is performed; inclusion body pyrolysis is performed; a PBS (phosphate buffer solution) containing urea is adopted for resuspension and centrifugation, precipitation is removed, and a supernatant is stored; preliminary renaturation: the PB solution containing urea is taken, an inclusion body dissolution solution is dropwise added into the mixed buffer solution slowly for centrifugation, precipitation is removed, and a supernatant is subjected to column purification and affinity chromatography purification: target protein is purified under the condition of the PB solution containing urea, a PB solution containing sodium fluoride is adopted to perform enzyme digestion elution after the target protein is combined with an additive, and a collected eluent is the Glu protein subjected to chromatographic purification. The antigen purified with the method is high in purity, and the technology is simple.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and relates to a purification method in the preparation of recombinant Streptococcus mutans dental caries vaccine candidate antigen Glu. Background technique [0002] Caries is an infectious disease that causes chronic and progressive destruction of tooth hard tissue under the action of various factors mainly including bacteria. It is characterized by high incidence and wide distribution. Generally, the average prevalence rate of dental caries can be around 50%. It is the main common disease of the oral cavity and one of the most common human diseases. The World Health Organization has listed it as one of the three major diseases of human beings, along with cancer and cardiovascular diseases. [0003] At present, it is believed that bacteria are a necessary condition for the occurrence of caries. It is generally believed that there are two types of cariogenic bacteria, one is acid-produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12R1/46C12R1/19
CPCC12N9/1051
Inventor 张卫军刘开云付强邹全明郭刚张怡孙红武付启欢樊绍文卢陆高莺
Owner CHENGDU OLYMVAX BIOPHARM
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