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Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application

A virus-like particle and hepatitis E technology, which is applied in the field of medical bioengineering, can solve the problems that recombinant proteins cannot be processed and modified, and affect biological activity.

Inactive Publication Date: 2014-04-23
DALIAN HISSEN BIO-PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the recombinant hepatitis E vaccine that has been successfully developed and marketed all over the world uses the expression system of prokaryotic E. coli expression system. The expressed hepatitis E virus antigen can only form hepatitis E virus-like particles through proper assembly conditions in vitro. Prokaryotic expression The disadvantage of the system is that the expressed recombinant protein cannot be processed and modified, which affects its biological activity

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  • Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application
  • Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application
  • Method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] A method for purifying recombinant Hansenula hepatitis E virus-like particles, comprising the following steps:

[0053] (1) Cultivation and fermentation: Inoculate 1% of the working seed batch recombinant hepatitis E vaccine engineered bacteria into 200ml of yeast nitrogen source medium, carry out primary culture at 30°C for 20 hours, and use UV-visible spectrophotometry Detect the absorbance value of primary culture A 600nm When the temperature is 10, transfer the culture to 2L of yeast nitrogen source medium and carry out secondary culture at 30°C for 20 hours, and use UV-visible spectrophotometry to detect the absorbance value of the secondary culture A 600nm At 10 o’clock, the culture was transplanted to glycerol medium, the culture temperature was 30°C, the culture time was 15 h, and the culture volume was 15 L. Then, methanol was added at a rate of 1% final concentration for induction culture for 80 h, and the fermented culture was collected. ;

[0054] (2) The ...

Embodiment 2

[0069] A method for purifying recombinant Hansenula hepatitis E virus-like particles, comprising the following steps:

[0070] (1) Cultivation and fermentation: Inoculate 1% of the working seed batch recombinant hepatitis E vaccine engineered bacteria into 200ml of yeast nitrogen source medium, carry out primary culture at 33°C for 20h, and use UV-visible spectrophotometry Detect the absorbance value of primary culture A 600nm When the temperature is 20, transfer the culture to 2L of yeast nitrogen source medium and carry out secondary culture at 33°C for 20 hours, and use UV-visible spectrophotometry to detect the absorbance value of the secondary culture A 600nm At 18 o'clock, the culture was transplanted into glycerol medium, the culture temperature was 30°C, the culture time was 15 h, and the culture volume was 25 L. Then methanol was added at a rate of 1% final concentration for induction culture for 75 h, and the fermented culture was collected. ;

[0071] (2) The ferm...

Embodiment 3

[0086] A method for purifying recombinant Hansenula hepatitis E virus-like particles, comprising the following steps:

[0087] (1) Cultivation and fermentation: Inoculate the working seed batch of recombinant hepatitis E vaccine engineering strains at 1% in 200ml of yeast nitrogen source medium, carry out primary culture at 31.5°C for 20h, and use UV-visible spectrophotometry Determination of the absorbance value of primary culture by A 600nm At 15 o'clock, transfer the culture to 2L of yeast nitrogen source medium for secondary culture at 31.5°C for 20 hours, and use UV-visible spectrophotometry to detect the absorbance value A of the secondary culture 600nm At 15 o'clock, the culture was transplanted to glycerol medium, the culture temperature was 30°C, the culture time was 15 h, and the culture volume was 20 L. Then, methanol was added at a rate of 1% to induce culture for 78 h, and the fermented culture was collected. ;

[0088] (2) The fermented culture collected in ste...

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Abstract

The invention relates to a method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application. The method comprises the following steps: cultivating and fermenting working seed lot recombined hepatitis E vaccine engineering strains, and collecting a fermented culture; sequentially performing cell disruption, clarification, ultra-filtration, silica gel adsorption, ultra-filtration and concentration liquid change, chromatographic purification, desalination, disinfection and filtration on the culture to obtain the purified recombined hansenula polymorpha hepatitis E virus-like particles. The size of the purified recombined hansenula polymorpha hepatitis E virus-like particles is in accordance with the theoretical size of natural hepatitis E virus particles and is 27-34nm, a gene sequence is in accordance with a theoretical gene sequence, the expressed foreign protein molecular weight is in accordance with the expected target protein size, and the recombined hansenula polymorpha hepatitis E virus-like particles are good in immunogenicity. The invention also discloses the application of the recombined hansenula polymorpha hepatitis E virus-like particles in preparation of vaccine. The recombined hansenula polymorpha hepatitis E virus-like particles are mixed with an aluminum adjuvant to form the vaccine. An animal immunogenicity detection result shows that the vaccine prepared from the recombined hansenula polymorpha hepatitis E virus-like particles is good in immunogenicity.

Description

technical field [0001] The invention belongs to the technical field of medical bioengineering, and in particular relates to a method for purifying recombinant Hansenula hepatitis E virus-like particles and an application for preparing a vaccine. Background technique [0002] Hepatitis E (Hepatitis E, HE) is an acute endemic intestinal disease that is mainly transmitted by the fecal-oral route. Hepatitis E is mostly acute and generally does not develop into chronic hepatitis. Hepatitis E as an intestinal infectious disease can account for 50% of acute viral hepatitis in some developing countries. The clinical symptoms of hepatitis E are basically the same as those of hepatitis A. Patients mainly present with jaundice, anorexia, hepatomegaly, abdominal pain, nausea, vomiting, and fever. The mortality rate is about 1%, which is higher than that of hepatitis A. [0003] The first pathogenic identification of hepatitis E was obtained in 1983 by Balayan et al. through the study o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N7/02A61K39/29A61P31/14
Inventor 苏彩霞李逦金贞姬韩旭东王琳王文雯徐德启
Owner DALIAN HISSEN BIO-PHARM CO LTD
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