Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Bifunctional antigen as well as preparation method and application thereof

A dual-function, antigenic technology, applied in the field of biomedicine, can solve the problem of no new coronavirus and pneumococcal vaccine antigen and its form, and achieve the effect of preventing and/or pneumococcus, low cost, and improving the level of neutralizing antibodies

Pending Publication Date: 2021-07-27
江苏坤力生物制药有限责任公司
View PDF6 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no vaccine antigen and its form on the market that can target both novel coronavirus and pneumococcus

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Bifunctional antigen as well as preparation method and application thereof
  • Bifunctional antigen as well as preparation method and application thereof
  • Bifunctional antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The preparation of the Escherichia coli engineering bacteria expressing SARS-CoV-2 recombinant RBD protein of embodiment 1

[0064] Entrusted Suzhou Jinweizhi to synthesize pET-28a plasmids containing SEQ ID NO:5 and SEQ ID NO:6 respectively, and the sequencing verification was correct.

[0065] The correct plasmid containing SEQ ID NO:5 was transformed into BL21(DE3) competent, and the single clone was inoculated into 3mL liquid LB medium containing 100ng / mL kanamycin, 220rpm, 30°C shaking overnight culture, as seeds.

[0066] Inoculate the seeds into TB medium containing 100ng / mL kanamycin at a ratio of 1:500, culture at 220rpm, shake at 37°C until OD 600 =2, add final concentration of 0.5mM IPTG, continue culturing for 4h, and collect the cells by centrifugation for purification.

[0067] Take 10OD cells and resuspend them with 1mL lysate (10mM Tris-HCl, pH 8.0), sonicate the bacteria, and conduct SDS-PAGE electrophoresis analysis of the total protein, supernatant a...

Embodiment 2

[0068] Example 2 Preparation of Expi293 recombinant cells expressing SARS-CoV-2 recombinant RBD protein (sequence is SEQ ID NO: 3), using QIAGEN plasmid extraction kit to extract the plasmid of the correct target gene for sequencing

[0069] According to the volume required for transfection, prepare a freshly subcultured Expi293 cell suspension (purchased from ATCC CRL-1573) to 2.0-2.5×10E6 cells / mL.

[0070] Prepare the transfection reagent-plasmid complex, namely liquid A (plasmid containing SEQ ID NO:6 synthesized in Example 1 / Opti-MEM=1 μg / 50 μl) and liquid B (PEI / Opti-MEM=5 μl / 50 μl) , Solution A and solution B were placed independently at room temperature for 10 minutes.

[0071] Add solution B to solution A dropwise, incubate at room temperature for 10 minutes, add cell suspension dropwise, and culture with shaking (115rpm, 36.8°C, 5% CO 2 ), 0.4mL KT Feed (Zhuhai Kairui) can be added after 24 hours, and the shaking culture is continued for 5 days to 7 days under the s...

Embodiment 3

[0073] Example 3 Purification and identification of SARS-CoV-2 recombinant RBD protein (sequence is SEQ ID NO: 2) Escherichia coli or cell expression

[0074] The Escherichia coli expression bacterium containing SEQ ID NO: 5 in Example 1 was resuspended in the Tris buffer solution of pH 8.0 with 1:20 (W / V), and was broken for 3 times with a high-pressure homogenizer 900bar* Inclusion bodies were obtained by centrifugation. Dissolve and denature the inclusion bodies with a solution (20mM ethylene glycol, 8M Urea, 50mM DTT pH8.0) and add refolding solution (20mM Tris, pH8.0, 3M urea, 0.05mM cystamine, 1mM cysteamine) for refolding 20 hours. Adjust the pH value of the renaturation supernatant to 7, centrifuge at 10000×g for 1 hour, take the supernatant and purify it through Cytiva SP Sepharose Fast Flow at a flow rate of 1-100cm / h to remove the adsorbed HCP (host cell residual protein, Host cell protein) Purify the target protein with polymers; collect the peak of the target RB...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a bifunctional antigen as well as a preparation method and application thereof. The bifunctional antigen provided by the invention is formed by covalent coupling of pneumococcal capsular polysaccharide and SARS-CoV-2 spike S protein or a fragment thereof; in the bifunctional antigen, the mass ratio of the pneumococcus capsular polysaccharide of each serotype to the spike S protein or the fragment thereof is 0.2 to 2: 1. Compared with the spike S protein or the fragment antigen of the spike S protein, the bifunctional antigen provided by the invention has the advantages that the neutralizing antibody level aiming at the new coronavirus can be obviously improved; and meanwhile, a high-level specific antibody aiming at pneumococcal capsular polysaccharide can be generated.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a bifunctional antigen, its preparation method and application. Background technique [0002] The disease caused by pathogenic microorganisms invading the respiratory tract and multiplying is called respiratory tract infection. According to its location, it is divided into upper respiratory tract infection and lower respiratory tract infection. The former includes rhinitis, pharyngitis, and laryngitis; the latter includes tracheitis, bronchitis, and pneumonia. Respiratory tract infection is an infectious disease caused by a variety of microorganisms including bacteria, viruses, mycoplasma, fungi, and parasites. Respiratory tract infections are mostly caused by viruses, and bacterial infections are often secondary to viral infections, forming bacterial and viral co-infection and aggravating the condition. This type of disease can occur in four seasons and at any age (infan...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50C07K1/107C12N15/70A61K39/295A61K39/09A61K39/215A61P31/04A61P31/14G01N33/68G01N33/569
CPCC07K14/005C12N15/70A61K39/12A61K39/092A61P31/04A61P31/14G01N33/6854G01N33/56983G01N33/56944C12N2770/20022C12N2770/20034C12N2770/20051A61K2039/70A61K2039/575G01N2333/165G01N2469/20Y02A50/30
Inventor 方红春张银川孙祖勇赵志强
Owner 江苏坤力生物制药有限责任公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com