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

Rabbit hemorrhagic disease virus (RHDV) type II VLP vaccine

A rabbit virus hemorrhage, virus-like technology, applied in the direction of viruses, vaccines, virus peptides, etc., to achieve the effect of preventing infection, high safety, and good immune effect

Pending Publication Date: 2020-08-25
YEBIO BIOENG OF QINGDAO
View PDF7 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no RHDV2 vaccine actually approved for marketing on the market

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
  • Rabbit hemorrhagic disease virus (RHDV) type II VLP vaccine
  • Rabbit hemorrhagic disease virus (RHDV) type II VLP vaccine
  • Rabbit hemorrhagic disease virus (RHDV) type II VLP vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Cutting, optimization of VP60 gene

[0020] The structural protein gene VP60 of RHDV2 with the amino acid sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 1 was synthesized by Shanghai Bioengineering Co., Ltd. and linked into the PUC57 vector.

[0021] At the same time, the antigenic site of VP60 was analyzed, and the 25 hydrophobic amino acids at the C-terminal that were not involved in the structural domain were deleted, which was conducive to the soluble expression of the protein. At the same time, it was optimized according to the codon preference of insect cells; the modified amino acid sequence was optimized. for SEQ ID NO:4( figure 1 ). The optimized and modified sequence is capable of expressing antigenic sites well in insect cells, and can be automatically assembled into VLPS.

[0022] The selected genes were codon-optimized, and the nucleotide sequences of the optimized genes were respectively SEQ ID NO: 3, synthesized by Shan...

Embodiment 2

[0023] Embodiment 2, the construction of the recombinant baculovirus expressing VP60 gene

[0024] 2.1 Enzyme digestion reaction

[0025] 2.1.1 Mark the 1.5mL EP tube to be used, and add and mix the sample according to the following table in the 1.5mL EP tube: the reaction system is 50 μL, and the sample addition is shown in the table below:

[0026]

[0027] 2.1.2 Place the 1.5mL EP tube in step 2.2.1 in a constant temperature water bath at 37°C for 2-3 hours.

[0028] 2.1.3 Gel recovery of double enzyme digestion products

[0029] Take out the above-mentioned double digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein.

[0030] (1) Mark the sample collection EP tube, adsorption column and collection tube.

[0031] (2) Weigh the marked empty EP tube and record the value.

[0032] (3) Carefully cut out a single target DNA band from the agarose gel on a gel cutter with a scalpel and put it into a clean 1.5mL centrifuge tube.

[0...

Embodiment 3

[0094] Embodiment 3: SF9 cell transfection

[0095] (1) Preparation: UV sterilization in a biosafety cabinet for 30 minutes; TNM-FH culture solution was placed in a 27°C water bath and preheated to 27°C.

[0096] (2) Add 2 μg of recombinant DNA to 100 μl of TNM-FH medium without serum and double antibody, and mix well. Add 9 μl Cellfectin Reagent to 100 μl TNM-FH medium without serum and double antibody, and mix well. The liposomes were mixed with the recombinant DNA and allowed to stand at room temperature for 40 min.

[0097] (3) Take out the 6-well plate cells from the incubator at 27°C, discard the supernatant medium, wash the cells three times with pre-warmed TNM-FH culture medium, and discard the TNM-FH culture medium.

[0098] (4) Add 2 ml of 10% fetal bovine serum TNM-FH culture solution to each cell well.

[0099] (5) Gently add the mixture of recombinant DNA and liposomes into each well of cells, mix gently, and culture statically at 27°C for 5-6h.

[0100] (6) D...

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 provides a virus-like particle of a rabbit hemorrhagic virus (RHDV) and a preparation method thereof. The provided virus-like particle is obtained by culturing and collecting after a cell of an insect is infected by a recombinant baculovirus; the recombinant baculovirus comprises a nucleotide fragment with an encoding VP60 protein; and an amino acid sequence of the VP60 protein is SEQ ID NO: 4. The invention also provides a RHDV subunit vaccine, the RHDV subunit vaccine comprises an antigen and a vaccine adjuvant, and the antigen is the virus-like particle prepared by the invention. The VLP is more similar to a natural virus in spatial conception, can stimulate a body to produce cellular immunity and humoral immunity at the same time, has a good immunity effect, and furthermore, has high security because the VLP is free of viral nucleic acid and a potential viral pathogenic gene.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, in particular to a rabbit viral hemorrhagic disease virus type II VLP subunit vaccine. Background technique [0002] Rabbit plague, also known as rabbit hemorrhagic disease (RHD), is an acute and highly fatal infectious disease of rabbits caused by rabbit hemorrhagic disease virus (RHDV). After the first report of RHD in my country in 1984, many countries in the world have successively reported the occurrence and prevalence of RHD. A new strain of RHDV was first reported in France in 2010, and researchers named it RHDV2. Compared with classic RHDV, rabbits and wild rabbits of all ages are susceptible to RHDV2, which is also the main reason why RHDV2 quickly replaced classic RHDV as the popular strain after outbreaks in other countries. RHDV2 seriously affects the development of the rabbit industry, causing huge economic losses, and also leads to a sharp decrease in the number of glo...

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): C12N15/866C12N15/40C07K14/08A61K39/12A61P31/14
CPCC12N15/86C07K14/005A61K39/12A61P31/14C12N2710/14043C12N2800/105C12N2770/16022C12N2770/16052C12N2770/16023C12N2770/16034A61K2039/5258A61K2039/552
Inventor 郭伟伟向银辉刘大卫郭玉广楚电峰范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO