Bovine rotavirus vp8* subunit recombinant chimeric protein and its application

A bovine rotavirus and chimeric protein technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of increased vaccine production cost, difficulties in vaccine development and production, etc. Effect

Active Publication Date: 2017-11-21
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional vaccine development is mainly to develop bivalent vaccines to prevent and control G6P[5] and G10P[11] rotavirus strains at the same time, which brings certain difficulties to vaccine development and production, and is bound to lead to increased vaccine production costs

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
  • Bovine rotavirus vp8* subunit recombinant chimeric protein and its application
  • Bovine rotavirus vp8* subunit recombinant chimeric protein and its application
  • Bovine rotavirus vp8* subunit recombinant chimeric protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of the expression vector containing the target fragment

[0030] The material used was bovine rotavirus of genotype G5P[6], which was cultured with primary African green monkey kidney (AGMK) cells. The medium used was EMEM, in which pancreatin was added to a final concentration of 0.5 μg / ml, penicillin 100 IU / ml, streptomycin 100 μg / ml, and amphotericin B 2.5 μg / ml. The formula of EMEM medium (high glucose type) is shown in Table 1.

[0031] Table 1 EMEM medium formula

[0032]

[0033] (1) Primer design

[0034] According to the VP4 gene sequence of bovine rotavirus disclosed in GenBank (gene accession number: JF693062.1), primers were designed using Oligo7.0 and DNAStar software, and the primer sequences are shown in Table 2. The annealing of the first pair of primers VP8*-ab-0-F and VP8*-ab-0-R serves as the template for subsequent amplification, a and b represent aa 1-11 and aa 218-235 respectively), the italic underlined part is the re...

Embodiment 2

[0056] Example 2 Expression of bovine rotavirus VP8* subunit recombinant chimeric protein

[0057] Using the heat shock method, the expression plasmid pET28a-P2-VP8*-(ab) 3 , pET28a-P2-VP8*-ab, pET28a-VP8*-(ab) 3 , pET32a-VP8*-ab, pET32a-VP8*-a and pET28a-VP8* were transformed into E. coli BL21(DE3) competent cells. A single clone was picked from the agar plate and inoculated in LB liquid medium containing 50 μg / ml kanamycin (pET28a vector) or 50 μg / ml ampicillin (pET32a vector) (added with 2% glucose, 1% ethanol and 2 %glycerin). When the absorbance value at 600 nm reached 0.5, IPTG was added to a final concentration of 0.5 mM, and the protein expression was induced overnight at 18°C. Then, the recombinant E.coli cells were collected by centrifugation at 10000g at 4°C for 15min, and stored at -80°C for later use (recombinant protein SDS-PAGE electrophoresis results are as follows: Image 6 A and Image 6 Shown in B, P2-VP8*-(ab) 3 The amino acid sequence is shown in SEQ...

Embodiment 3

[0058] Example 3 Western blot detection of recombinant protein

[0059] The expressed recombinant protein was electrophoresed by 15% SDS-PAGE, then transferred to PVDF membrane, blocked with PBST containing 5% skim milk at 37°C for 1 h, washed 3 times with PBST, and diluted 1:2000 times with anti-His-tag mAb 4 Incubate overnight at ℃, wash 3 times with PBST, and incubate with 1:5000-fold dilution of HRP-labeled goat anti-mouse IgG at 37 ℃ for 1 h; wash 3 times with PBST, and develop DAB color ( Figure 7 ).

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 present invention provides a bovine rotavirus VP8* subunit recombinant chimeric protein, which is a recombinant protein epitope vaccine in which T cell epitope polypeptide P2 in tetanus toxin is introduced into bovine rotavirus VP8* subunit multi-copy chimeric gene Among them, it can greatly improve the immune efficacy of epitope vaccines, and can induce cross-neutralizing immune protection, and induce higher neutralizing antibody titers, and can induce high titers of anti-P[5] and P[11] genes Type-specific rotavirus neutralizing antibodies. Utilizing the bovine rotavirus VP8* subunit recombinant chimeric protein of the present invention can simultaneously prevent and treat the G6 and G10 type rotaviruses that are mainly prevalent in the world, and is suitable for preparing a safe and low-cost bovine rotavirus vaccine. The combination of the developed rotavirus vaccine can effectively reduce the economic loss caused by rotavirus gastroenteritis to the cattle industry, and has broad market prospects.

Description

technical field [0001] The invention belongs to the field of molecular biology and genetic engineering, in particular to the recombinant chimeric protein of bovine rotavirus VP8* subunit and its application. Background technique [0002] Rotavirus is a member of the family Reoviridae and the genus Rotavirus, and is the main pathogen that causes gastroenteritis in a variety of newborn animals and infants. Bovine rotavirus diarrhea is mainly characterized by depression, anorexia, diarrhea, and dehydration. Calves within 7 days of age are most susceptible to infection. The incidence rate can be as high as 90% to 100%, and the mortality rate can reach 10% to 50%. If secondary Escherichia coli septicemia can lead to a further increase in mortality, the economic benefits of the cattle industry are seriously jeopardized. [0003] Rotavirus particles consist of three capsids, the outermost capsid consisting of VP7 and the spike protein VP4, both of which can independently induce ne...

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 Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/15A61P31/14
Inventor 闻晓波朱光怡冉旭华苗艳张峣曹思倪宏波朱战波
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap