Unlock instant, AI-driven research and patent intelligence for your innovation.

Recombinant reovirus, preparation method and application thereof

A reovirus and coding technology, applied in the application field of preparing HBV vaccine, can solve problems such as failure to become a mature product, and achieve the effect of facilitating promotion

Inactive Publication Date: 2015-05-27
孙逸武
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in the end, it failed to pass the test of clinical trials and failed to become a mature product

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
  • Recombinant reovirus, preparation method and application thereof
  • Recombinant reovirus, preparation method and application thereof
  • Recombinant reovirus, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] This example describes the construction method of the recombinant reovirus strain.

[0024] 1. Reovirus genome construction

[0025] (1) Reovirus gene cloning: 10 gene fragments of reovirus were generated by RT-PCR, and these PCR amplified products were cloned into pBluescriptII vector, and then cloned into plasmid pT7 controlled by T7RNA green enzyme promoter. The cloned products were all transfected with competent bacteria DH5α. After solid culture in agarose, a single colony was selected and cultured in liquid to obtain the cloned gene, which was further sequenced, analyzed and identified.

[0026] Table 3. Reovirus genome PCR primers

[0027]

[0028]

[0029] (2) Construction of Sigma3 / HBsAg fusion gene

[0030] The above cDNA plasmid of reovirus type III Dearing was used as a template for PCR, and the primers in the following list 4 were used to generate the 5' and 3' end fragments of Sigma3 protein cDNA (1; 3). The PCR primers of the generated reovirus c...

Embodiment 2

[0045] This example describes the detection of anti-HBsAg antibodies.

[0046] WesternBlot was used to detect HBsAg and reovirus protein. L929 cells infected by wild genotype and reovirus variants were dissolved in RIPALysisBuffer (NaCl150mM, NP-401%, Sodium deoxycholate0.5%, SDS0.1%, 3.1M Tris, pH8.0). After separation by SDS gel electrophoresis, transfer to PVDF membrane. The PVDF membrane was blocked with TBS-T solution containing 3% BSA (20mM Tris-HCl, pH7.5, 150mmNaCl, and0.05%Tween20), and then anti-HBsAg polyclonal antibody (Biosciences) or anti-HBsAg was added at 1:500 to 1:1000. Reovirus Sigma3 monoclonal antibody 4F2 (MPBiomedicals). After 2 h, incubation was continued for 1 h with HRP antibody (1:1000) in TBS-T. Its test results are as image 3 .

Embodiment 3

[0048] This example describes the results of animal experiments.

[0049] C57BL / 6J mice were managed by an animal room approved by the scientific research management agency. All mice were housed at 23°C with normal diurnal intervals. Mice at 6-8 weeks will be selected for the experiment and divided into control (virus expressing wild-type Sigma3) and vaccine (virus expressing fusion Sigma3 / HBsAg) groups, with 8 mice in each group. Will 1X10 6 PFU of virus was inoculated into the mouth of mice. Serum and feces samples were taken at 0; 2; 4; 6; 8; 10; 12; 14 weeks, the feces were prepared into 20% suspension with PBS, and the centrifuged supernatant was collected. All samples were stored at -20°C. Anti-HBsAg IgG and IgA antibodies in mouse serum were detected by ELISA (HBsAbELISA Kit, Biocare), and the specific method was according to the operating instructions provided by the Biocare. in Figure 4 The histogram of the detection results of anti-HBsAg-IgG in mouse serum is ...

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 relates to recombinant reovirus, and especially relates to the recombinant reovirus capable of expressing hepatitis B surface antigen or antigen epitope, a preparation method and an application of recombinant reovirus in HBV vaccine. The recombinant reovirus uses a gene engineering technology for inserting DNA neutralizing epitope coding with HBsAg having strong immunogenicity and antigenicity into external layer of reovirus or non-structural protein genome, recombinant virus strain capable of copying and expressing HBV antigen in a stable mode can be established through screening, compared with currently used intramuscular injection-type hepatitis b virus (HBV) vaccine, the oral vaccine has the advantages of convenience, economy and easy popularization.

Description

technical field [0001] The invention belongs to the field of biomedicine, and particularly relates to a recombinant reovirus capable of expressing hepatitis B surface antigen or epitope, its preparation method and its application in the preparation of HBV vaccine. Background technique [0002] my country is a hepatitis B endemic area. The average carrier rate of hepatitis B surface antigen (HBsAg) is about 10%, and the infection rate of hepatitis B in the national population is as high as 60%. Liver cirrhosis and liver cancer caused by it are even more unsolved problems in the world. The currently clinically used hepatitis B vaccine uses HBsAg produced by bioengineering as the antigen, and must be injected intramuscularly to activate the body's humoral immune system. Neither convenient nor economical, there has been a long-awaited oral hepatitis B vaccine. American scientists first successfully expressed HBsAg in potatoes, and domestic scholars followed up by expressing HBs...

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
IPC IPC(8): C12N7/01A61K39/15A61P31/20
Inventor 孙逸武马洪宝
Owner 孙逸武
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com