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Purification of recombinant coxsackievirus A16 (CA16) virus-like particles, application thereof in vaccine and vaccine

A virus-like, particle-based technology, applied in the direction of positive-sense single-stranded RNA viruses, viruses, viral peptides, etc., can solve the problems of limiting large-scale production requirements, separation of baculovirus particles, and impact on vaccine quality, achieving strong immunogenicity , good immunogenicity, beneficial to large-scale industrial production

Active Publication Date: 2019-04-26
深圳鑫泰康生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using insect cells to prepare VLP requires high culture conditions and complicated purification process, which limits the large-scale production requirements; in addition, the baculovirus-insect cell expression system produces baculovirus particles and other pollutants that affect the vaccine effect. Baculovirus particles are difficult to separate from the prepared VLP, and measures such as inactivation treatment are required, so it has a great impact on the quality of the vaccine

Method used

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  • Purification of recombinant coxsackievirus A16 (CA16) virus-like particles, application thereof in vaccine and vaccine
  • Purification of recombinant coxsackievirus A16 (CA16) virus-like particles, application thereof in vaccine and vaccine
  • Purification of recombinant coxsackievirus A16 (CA16) virus-like particles, application thereof in vaccine and vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1 Recombinant CA16 yeast expression strain is fermented and cultivated in a 30L fermenter

[0084] The recombinant Hansenula strain was inoculated into 100 mL primary seed medium (0.67% yeast nitrogen source medium, purchased from SIGMA Company, 0.5% ammonium sulfate, 2% glucose), and cultured at 33° C. on a shaking table at 200 rpm for 20 ~24 hours. Then the whole amount was inoculated in 1000mL secondary seed medium (0.67% yeast nitrogen source medium, 0.5% ammonium sulfate, 2% glycerol), at 33 ° C, 200 rpm shaking culture for 20 to 24 hours (OD 600nm up to 8-10). Then inoculate the whole amount into a 30L fermenter, which contains 12L fermentation medium, adjust the pH value of the fermentation liquid with ammonia water to maintain at 5.0+0.5, the fermentation temperature is 30°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 cont...

Embodiment 2

[0091] The separation and purification of embodiment 2CA16 virus-like particles

[0092] Cell collection: the fermentation broth was collected, the precipitate was collected by centrifugation at 6500 rpm, and the cells were washed twice with cell washing buffer.

[0093] Breaking: The collected Hansenula cells were resuspended in cell lysis buffer (20mM NaH 2 PO 4 , 2mM EDTA-Na 2 , 0.2-1.0M NaCl, 2mM PMSF, 0.5% Tween-80, pH6.8-7.4), use a high-pressure homogenizer to break the cells twice under the condition of a pressure of 1200bar, and the cell breakage rate reaches more than 80%.

[0094] Clarification: Pour the crushed cell solution into a centrifuge tube, centrifuge at 7000rpm for 40min, and collect the clarified solution. Or filter the crushed cell fluid through a depth filter at a flow rate of 1300mL / min / m 2 , to collect the clarified solution overnight.

[0095] Ultrafiltration: Use 50mM Tris, 0.25M NaCl and 5% glycerol to form a buffer solution of pH 8.0 to form ...

Embodiment 3

[0109] Preparation of Example 3 Recombinant CA16 VLP Vaccine

[0110] Control of main indicators: Antigen (CA16 virus-like particles) content is 10 μg / mL; aluminum content is controlled at 0.40-0.60 mg / mL; pH value is controlled at 6.6-7.4.

[0111] Preparation method: Dilute the self-made aluminum hydroxide adjuvant to 0.40-0.60 mg / mL with sterile 0.9% sodium chloride solution. Slowly add the purified CA16 stock solution to the adjuvant to make it fully adsorbed, and the vaccine preparation is completed. The prepared vaccine testing standards and testing results are shown in Table 4.

[0112] Table 4 Test results of recombinant CA16 virus-like particle vaccine

[0113] Test items

[0114] The results in Table 4 show that the detection indicators of recombinant CA16 virus-like particle vaccines all meet the detection standards, especially the antigen content is much higher than the minimum standard.

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Abstract

The present invention provides a method for purifying recombinant coxsackievirus A16 (CA16) virus-like particles, an application thereof in an vaccine, and the vaccine. The method comprises carrying out high density fermentation culture of recombinantly expressed engineering bacteria and methanol-induced expression of CA16 viroid granulin, carrying out centrifugation for collecting thalli, performing high-pressure homogenization and breaking, and purifying the supernatant by ultrafiltration, ion exchange chromatography, hydroxyapatite chromatography and molecular sieve chromatography and the like. The CA16 viroid particle vaccine provided by the invention has good immunogenicity, safety, immunological characteristics and biological activity. The process is simple, and the purification is performed by chromatographic methods. The purification method is more conducive to linear amplification compared with density gradient centrifugation, and can be used in large-scale preparation and purification, and a high-purity (more than 99%) virus-like particle (VLP) stock solution can be obtained and can be used to prepare a vaccine for preventing CA16 infection, and has good economic value and application prospects.

Description

technical field [0001] The invention relates to the field of biological products, in particular to a method for purifying recombinant CA16 virus-like particles and a vaccine prepared by using the purified CA16 virus-like particles. Background technique [0002] Hand, foot and mouth disease (HFMD) is a common infectious disease in children. The main clinical manifestations are herpes on the hands and feet and oral mucosal eruption. In severe cases, aseptic meningoencephalitis, brainstem encephalitis, neurogenic pulmonary edema, and heart damage may occur in a small number of cases. [0003] Hand, foot and mouth disease is a global infectious disease, and there are reports of the prevalence of this disease in most parts of the world. In 1957, the first outbreak occurred in New Zealand. In 1958, Robinson in Canada isolated Coxsackie virus group A type 16 (CA16) from patient stool and throat swab. In the mid-1970s, Bulgaria and Hungary successively broke out the epidemic of C...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/085C07K1/14C07K1/34C07K1/18C07K1/16A61K39/125A61K39/39A61P31/14C12R1/78
CPCA61K39/12A61K39/39A61P31/14C07K14/005C12N2770/32334C12N2770/32323A61K2039/55505A61K2039/5258Y02A50/30
Inventor 李国顺顾美荣张改梅刘司航刘俊杰马廷涛郭林简伟肖海峰刘建凯朱征宇甘建辉郑海发
Owner 深圳鑫泰康生物科技有限公司
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