Hansenula polymorpha engineering bacteria for efficiently expressing CA10 virus-like particles and application thereof

A high-efficiency expression, Hansenula yeast technology, applied in the field of biomedicine, can solve the problems of difficult proportion of solid virus particles, virulence recovery, incomplete inactivation, etc., and achieve easy large-scale production, simple process operation, and high antigen recovery rate. Effect

Active Publication Date: 2020-10-16
BEIJING MINHAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inactivated vaccines have the risk of incomplete inactivation or virulence recovery, and the epitopes of inactivated vaccines are easily destroyed during inactivation, and the proportion of hollow and solid virus particles in inactivated vaccines is difficult to control, resulting in large differences between batches, and the Problems such as low production

Method used

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  • Hansenula polymorpha engineering bacteria for efficiently expressing CA10 virus-like particles and application thereof
  • Hansenula polymorpha engineering bacteria for efficiently expressing CA10 virus-like particles and application thereof
  • Hansenula polymorpha engineering bacteria for efficiently expressing CA10 virus-like particles and application thereof

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preparation example Construction

[0068] The invention provides a method for preparing and purifying CA10 virus-like particles, and an application in the field of vaccines. Through the high-density fermentation culture of recombinantly expressed Hansenula engineering bacteria and the expression of CA10 virus-like particle protein induced by methanol, the bacteria were collected by centrifugation and subjected to high-pressure homogenization and crushing. The supernatant was subjected to ultrafiltration, ion exchange chromatography, and hydroxyapatite. Obtained after purification by chromatography and molecular sieve chromatography. The CA10 virus-like particle provided by the present invention and the vaccine prepared therefrom have good immunogenicity, safety, immune characteristics and biological activity, simple process, and chromatography method is adopted for purification, which is more conducive to linear amplification than density gradient centrifugation. It can be prepared and purified on a large scale...

Embodiment 1

[0083] Example 1 The acquisition of CA10 recombinant Hansenula engineering bacteria

[0084] According to the nucleotide sequences of the P1 and 3CD proteins of the recently popular Coxsackievirus A10 strain, Vector software was used to optimize the design of the P1 and 3CD gene sequences according to the preferred codons of Hansenula to increase their expression.

[0085] The sequence of the yeast secretion signal peptide or the sequence of the transcription termination signal recognized by yeast was removed during the optimization process. In order to avoid the GC content of the translated mRNA being too high, the secondary structure of the mRNA affecting the translation efficiency, and considering the enzyme cutting site, the gene sequence at certain positions was adjusted appropriately to ensure that the amino acid remained unchanged. The nucleotide sequences of the optimized CA10 virus P1 and 3CD genes are shown in SEQ ID NO: 1 and 2, respectively.

[0086] In the presen...

Embodiment 2

[0091] Embodiment 2 Recombinant CA10 yeast expression strain is fermented and cultivated in a 30L fermenter

[0092] Strain CA10-W was inoculated into 100ml primary seed medium (0.67% yeast nitrogen source medium, 0.5% ammonium sulfate, 2% glucose), and cultured at 33°C and 200rpm on a shaker for 18-22h. Take the primary seed culture solution and inoculate it into 1000ml of the secondary seed medium, and incubate at 33°C and 200rpm on a shaker for 21 to 23 hours. Inoculate the secondary seed culture liquid into a 30L fermenter, adjust the pH value of the fermentation liquid with ammonia water to maintain at 5.0+0.5, the fermentation temperature is 30±1°C, the rotation speed is controlled at 350-750rpm, and the air flow rate is 0.5-1.0m 3 / h, high-density fermentation requires pure oxygen supplementation, dissolved oxygen is controlled at 20-60%, the carbon source in the fermentation medium is exhausted in 16-18 hours, and a total of 2.0L of glycerin is added, and the total gro...

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Abstract

The invention provides hansenula polymorpha engineering bacteria for efficiently expressing CA10 virus-like particles and application thereof. The engineering bacteria comprise a recombinant vector carrying CA10 virus P1 and 3CD genes, and an original strain of the engineering bacteria is uracil-deficient hansenula polymorpha AU 0501; and the P1 and 3CD genes are optimized according to hansenula polymorpha preferred codons. The invention further provides a preparation method of the CA10 virus-like particles. The preparation method comprises the steps that the engineering bacteria are cultured,the CA10 virus-like particles are expressed, and the virus-like particles are separated and purified by adopting ultrafiltration and three-step chromatography processes. The CA10 virus-like particlesand a vaccine prepared from the CA10 virus-like particles have good immunogenicity, safety and biological activity, the process is simple, purification is conducted by adopting a chromatography method, large-scale preparation and purification can be achieved, a high-purity (greater than 99%) VLP protein stock solution is obtained, and the engineering bacteria can be used for preparing the vaccinefor preventing CA10 infection and have good economic value and application prospects.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to Hansenula engineering bacteria highly expressing CA10 virus-like particles and applications thereof. Background technique [0002] Hand, foot and mouth disease (HFMD) is a common infectious disease in children, and it is a class C infectious disease caused by a variety of viruses. Coxsackievirus A10 (CA10) is one of the pathogens causing HFMD. Infects children under 5 years of age. Coxsackievirus A group 10 is one of the main pathogens of hand-foot-to-foot disease in my country in recent years. CA10 can cause herpetic angina, CA10 can cause herpetic angina, and a small number of patients show symptoms of the nervous system and even convulsions or complications of cardiopulmonary function. [0003] Since 2008, Coxsackievirus A10 has been circulating in some parts of China. In 2010, hand, foot and mouth disease caused by CA10 broke out in France, and then hand, foot and mouth disease c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C07K14/085C07K1/34C07K1/18C07K1/16C07K1/36C07K1/14A61K39/125A61P31/14C12R1/78
CPCC07K14/005C12N15/815A61K39/12A61P31/14C12N2770/32022C12N2770/32023C12N2800/22A61K2039/5258A61K2039/55505C12N2770/32334C12N2770/32123C12N2770/32134C12N2770/32151C12N2770/32122C12N15/81C12N2770/32323A61K2039/575A61K39/135C07K14/09C12N7/00
Inventor 李国顺顾美荣郭林简伟刘俊杰肖海峰张改梅赵丽丽谢学超陈磊徐颖之刘建凯
Owner BEIJING MINHAI BIOTECH
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