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Recombinant baculovirus with surface displaying porcine epidemic diarrhea virus S protein

A technology for porcine epidemic diarrhea and recombinant baculovirus, which is applied in the directions of viruses, viral peptides, antiviral agents, etc., can solve the problems of mutagenicity, low expression level, poor vaccine persistence, etc., and achieves improved display effect, low cost, and preparation. simple craftsmanship

Inactive Publication Date: 2016-11-09
杭州洪晟生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these vaccines have many problems such as poor persistence, low expression level, poor stability, easy digestion by the gastrointestinal tract, integration of host chromosomes, and mutagenicity.

Method used

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  • Recombinant baculovirus with surface displaying porcine epidemic diarrhea virus S protein
  • Recombinant baculovirus with surface displaying porcine epidemic diarrhea virus S protein
  • Recombinant baculovirus with surface displaying porcine epidemic diarrhea virus S protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Construction of recombinant baculovirus

[0042] According to the amino acid sequence of S protein of porcine epidemic diarrhea virus CV777 strain (Genbank Accession Number: AF353511), the nucleic acid sequence of 21-789 amino acids in the S1 region was optimized, and Hangzhou Qingke Zixi Biotechnology Co., Ltd. was entrusted to synthesize the S1 gene containing DNA: SP-S1-TMD, as shown in SEQ ID NO.1. Among them, the 4-60 base sequence is the signal peptide coding sequence (SP) of the baculovirus AcNPV GP64 protein; the 61-2367 base sequence is the PEDV S1 protein coding sequence; the 2368-2577 base sequence is the herpes virus G protein The coding sequence of the transmembrane domain (TMD).

[0043] According to the optimized nucleic acid sequence, DNAstar software was used to design amplification primers P1 (5'-CGCGGATCCATGGTGTCAGCCATCGTG-3') and P2 (5'-CCCAAGCTTTTACTTTCCCAGTCTGTTC-3'), respectively introducing BamHI and HindIII restriction sites. The...

Embodiment 2

[0048] Example 2: Expression and immune identification of S1 protein

[0049] After the recombinant virus sample obtained in Example 1 was titrated by the terminal dilution method, the dose of MOI=1 was used to infect the pre-plated Sf9 cells in the six-well plate. After 72 hours of infection, centrifuge at 1000rpm for 5 minutes, collect the Sf9 cells precipitated from the bottom of the tube, wash the cells twice with PBS, take the cell pellet, prepare protein samples, and perform SDS-PAGE and Western Blotting analysis to verify whether the S1 protein is expressed. Use PEDV S1 antiserum (self-made, 1:100 dilution) as the primary antibody, and the secondary antibody is HRP-goat anti-rabbit IgG antibody (1:10000 dilution), and ECL chemiluminescence is used to detect S1 western blot, and the detection results are as follows image 3 .

[0050] The expected size of the S1 recombinant protein is about 92kD, from image 3 It can be seen that the S1 protein expressed by the Sf9 cel...

Embodiment 3

[0051] Example 3: Fluorescent observation and subcellular localization

[0052] In order to further confirm the expression localization of PEDV S1 protein in cells, the rvAc-S1 infected Sf9 cells were observed by indirect immunofluorescence. Sf9 cells were inoculated in a 35mm laser confocal special culture dish (Corning). When the cell confluence reached 50%, the Sf9 cells in the culture dish were inoculated with recombinant baculovirus (MOI=1), and immunofluorescence detection was performed 48 hours after infection. The specific operation is as follows: fix the cells with 4% paraformaldehyde at room temperature for 10 min; permeabilize the cells with 0.2% Triton X-100 for 10 min; overnight at ℃; Cy3-goat anti-rabbit polyclonal antibody (Abcam, 1:500) was incubated at 37°C in the dark for 1 hour; DAPI (Sigma) was used to stain the nucleus for 3 minutes; , pH 7.4) and washed 3 times for 5 min each time, the results were observed and photographed under a confocal laser scannin...

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Abstract

The invention provides a recombinant baculovirus with the surface displaying a porcine epidemic diarrhea virus S protein, and a preparation method thereof. The virus adopts PEDV spike protein S1 gene as antigen gene, the recombinant virus is constructed by using an insect baculovirus vector expression system, and an S1 protein is successfully expressed and displayed on the surface of the virus. The recombinant virus is used to immunize and inoculate mice as a PEDV pseudo-virus vaccine, and serum neutralization test and lymphocyte propagation experiment analysis shows that the recombinant virus can arouse an effective immune protection effect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering vaccines, in particular to a recombinant baculovirus displaying porcine epidemic diarrhea virus S protein on its surface and a preparation method for the recombinant baculovirus. Background technique [0002] Porcine epidemic diarrhea (PED) is caused by porcine epidemic diarrhea virus (PEDV) and is characterized by watery diarrhea, dehydration, vomiting and high lethality in suckling piglets. sexually transmitted intestinal diseases. The disease broke out for the first time in the UK in 1971. In 1978, the pathogen was confirmed and reported in Belgium for the first time. Since then, it has occurred in Hungary, Germany, Japan, China, South Korea, Vietnam and other countries and regions. From 2013 to 2014, PED outbreaks also occurred in the United States, Canada, Cuba, the Midwest of Mexico and Japan, and the mortality rate was abnormally high, causing serious economic losses to the swi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/50C12N15/866A61K39/215A61P31/14
CPCC07K14/005A61K39/12A61K2039/5256C12N15/86C12N2710/14043C12N2770/20022C12N2770/20034C12N2800/105
Inventor 陈健史翠萍舒建洪陈琴杨芳
Owner 杭州洪晟生物技术股份有限公司
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