Porcine rotavirus (PRV) VP fusion protein reconstruction body as well as preparation method and application thereof

A porcine rotavirus and fusion protein technology, which is applied in the field of fusion proteins composed of porcine rotavirus proteins VP8 and VP7 and TAT and its preparation, can solve the problems of strong virulence and not being the mainstream of vaccine research, and achieve expression Large quantity, low cost, good safety effect

Active Publication Date: 2018-08-03
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the potential danger of virulence reversion in live attenuated vaccines, it is no longer the mainstream of vaccine research.

Method used

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  • Porcine rotavirus (PRV) VP fusion protein reconstruction body as well as preparation method and application thereof
  • Porcine rotavirus (PRV) VP fusion protein reconstruction body as well as preparation method and application thereof
  • Porcine rotavirus (PRV) VP fusion protein reconstruction body as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] [Example 1] Preparation of fusion gene VP8-VP7-TAT

[0060] The amino acid sequences of VP8 [sequence number: AY523636.1] and VP7 [sequence number: AER25320.1] of mature PRV were obtained from the gene bank of the American Center for Biotechnology (NCBI), and the VP8 and VP7 protein sequences were intercepted with The main structural functional region of immunogenicity is converted into a nucleotide sequence containing E. coli preferred codons, and according to the characteristics of fusion expression between the gene encoded by the protein transduction domain polypeptide TAT and the foreign protein gene, after optimization The 3' end of the gene sequence is connected to the nucleotide sequence of TAT, that is, VP8-VP7-TAT, the sequence of which is the nucleotide sequence shown in SEQ ID NO:1. The VP8-VP7-TAT sequence was artificially synthesized by Tianyi Huiyuan Company, and connected to the expression vector pGEX-6p-1 to construct the recombinant expression plasmid p...

Embodiment 2

[0061] [Example 2] Construction of recombinant plasmids pGEX-VP8-TAT and pGEX-VP7-TAT

[0062] Using the recombinant expression plasmid pGEX-VP8-VP7-TAT as a template, the VP8-TAT and VP7-TAT sequences were respectively cloned by recombinant PCR method, digested by EcoR I and Xho I, electrophoresed on agarose gel, and Cut off the band to be recovered quickly under ultraviolet light, purify it with Kangwei Century Agarose Gel DNA Recovery Kit, put a single target DNA band into a clean Eppendorf tube, and weigh it. Add three times the volume of sol solution PG to the gel block (the weight of the gel is 0.1 g, its volume can be regarded as 100 μl, and so on). Water bath at 60°C for 10 minutes, during which time the Eppendorf tube was gently turned up and down every 2 minutes to ensure that the gel was fully dissolved. Add 250 μl of Buffer PS to the adsorption column, let it stand for 2 minutes, then centrifuge at 12000 rpm for 2 minutes, and discard the liquid in the collection ...

Embodiment 3

[0089] [Example 3] Genetically engineered bacteria E.coli BL21(DE3)(pGEX-VP8-VP7-TAT), E.coli BL21(DE3)(pGEX-VP8-TAT), E.coli BL21(DE3)(pGEX- Construction of VP7-TAT)

[0090] Take 1 μl of plasmids pGEX-VP8-VP7-TAT, pGEX-VP8-TAT, and pGEX-VP7-TAT to transform Escherichia coli BL21 (DE3) competent cells. Positive transformants were screened out by PCR identification. The positive clones obtained were genetically engineered strains E.coli BL21(DE3)(pGEX-VP8-VP7-TAT), E.coli BL21(DE3)(pGEX-VP8-TAT), E.coli BL21(DE3)(pGEX- VP7-TAT).

[0091] The genetically engineered strain E.coli BL21(DE3)(pGEX-VP8-VP7-TAT) was sent to the China Center for Type Culture Collection on January 19, 2018 for preservation. The taxonomic name: Escherichia coli BL21(DE3)(pGEX-VP8 -VP7-TAT), Escherichia coli BL21(DE3)(pGEX-VP8-VP7-TAT), deposit number: CCTCC NO: 2018047, address: China. Wuhan. Wuhan University.

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Abstract

The invention discloses a porcine rotavirus (PRV) VP fusion protein reconstruction body as well as a preparation method and application thereof. The fusion protein VP8-VP7-TAT provided by the invention comprises truncation fragments VP8 and VP7 of a PRV coat shell structure protein VP4 and eleven core amino acids bonded to a TAT protein transduction peptide basic amino acid enrichment region at anend C. A mouse is immunized with the fusion protein VP8-VP7-TAT by means of intraperitoneal injection or oral intragastric administration, so that the organism can be effectively inducted to generatea humoral immune response and a mucosal immune response, and high immunogenicity is achieved. Through fusion expression of PRV proteins VP8, VP7 and TAT, a novel method is provided for the preventionof PRV infection, and a basis is laid for the development of a novel PRV oral vaccine.

Description

technical field [0001] The invention belongs to the technical field of genetic bioengineering, and specifically relates to a fusion protein composed of porcine rotavirus proteins VP8 and VP7 and TAT, a preparation method and application thereof. Background technique [0002] Porcine rotavirus is one of the important pathogens causing viral diarrhea in piglets. Piglet diarrhea caused by porcine rotavirus infection is common in every pig-raising country in the world. It has been reported that the incidence of piglets in the UK has exceeded 80%, and the mortality rate is 0-50%. The survey of PRV infection in piglets from 10 days old to pre-weaning in Fuzhou and Nanping, Fujian Province, my country showed that the infection rates reached 82.3% and 91.7% respectively (Zhang Lei, Wei Guangsen. Research progress on porcine rotavirus infection[J].2005( 4), 42-44), the virus is ubiquitous in pig herds whether it is simple infection or mixed infection, thus causing huge economic loss...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/22C12N15/62C12N1/21A61K39/15A61P31/14C12R1/19
CPCA61K39/12A61K2039/552A61K2039/57A61K2039/575A61P31/14C07K14/005C07K2319/10C12N2720/12322C12N2720/12334
Inventor 徐进平姚帅
Owner WUHAN UNIV
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