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Multi-epitope antigen of porcine epidemic diarrhea virus, and encoding gene, preparation method and application thereof

A porcine epidemic diarrhea and multi-epitope technology, which is applied in the direction of virus antigen components, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of high production cost, long production cycle and inaccurate antigen content of tissue inactivated vaccines To achieve good biological activity and reactogenicity, avoid non-specific immune response, and avoid strong virulence

Active Publication Date: 2019-08-06
ZHONGKAI UNIV OF AGRI & ENG
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

In 2006, the genetic derivation analysis of the PEDV-M gene showed that the PEDV epidemic strains in China were less related to the strains in Europe, South Korea and Japan
In actual production, for pig farms with outbreaks of PED, the intestines of affected piglets are collected to prepare tissue-inactivated vaccines to immunize the whole group of sows by intramuscular injection, which has a definite immune effect on PED, but the production cost of tissue-inactivated vaccines is high , long production cycle, inaccurate antigen content, complex antigen components and other shortcomings are not suitable for routine pig farm immunization
In addition, in view of the unsatisfactory immune effect of the cell-inactivated vaccine, and the attenuated live vaccine may transform into a wild strain and cause the virulence to become stronger, so a more stable, efficient and affordable new vaccine is urgently needed in the pig industry

Method used

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  • Multi-epitope antigen of porcine epidemic diarrhea virus, and encoding gene, preparation method and application thereof
  • Multi-epitope antigen of porcine epidemic diarrhea virus, and encoding gene, preparation method and application thereof
  • Multi-epitope antigen of porcine epidemic diarrhea virus, and encoding gene, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the design of PEDV-S1 epitope gene

[0037] According to the epitope region aa499-638 (namely the CO-26K equivalent, COE) possessed by PEDV-S1, linear epitopes S1D5, S1D6 can induce the body to produce neutralizing antibodies, then PEDV-S1 published by GenBank (GenBank: JX501322.1) sequence, selected PEDV-S1 COE, S1D5 and S1D6 three epitope genes combined with two flexible polypeptide genes as the main epitope gene of this multi-epitope antigen (referred to as PEDV-SE), and then by Shanghai Sangon Bioengineering (Shanghai) Co., Ltd. synthesized PEDV-SE-PUC57 glycerol bacteria.

Embodiment 2

[0038] Embodiment two, the purification of target gene PEDV-SE and expression vector ppGEX-4T-1

[0039] 1. PEDV-SE-PUC57 glycerol bacteria and ppGEX-4T-1 glycerol bacteria amplification and plasmid extraction

[0040] (1) Streak PEDV-SE-PUC57 glycerol bacteria and ppGEX-4T-1 glycerol bacteria on an Amp medium plate, and culture them in a 37°C incubator for 12-16 hours;

[0041] (2) Pick a single colony and inoculate them in 5 mL LB broth containing 100 μg / mL Amp, and culture them in a constant temperature shaker at 37°C at 200 rpm for 16-20 hours;

[0042] (3) Plasmid extraction of PEDV-SE-PUC57 glycerol bacteria and ppGEX-4T-1 glycerol bacteria, the steps are as follows:

[0043] (A) Divide 5 mL of bacterial liquid into 5 centrifuge tubes, and centrifuge at 10,000×g for 1 min.

[0044] (B) Pour out the culture medium, add 250 μL Solution I each, and mix well by pipetting.

[0045] (C) After adding 250 μL Solution II, shake the tube gently for 2-3 minutes; avoid vigorous m...

Embodiment 3

[0080] Embodiment three, construction of PEDV-SE chimeric ppGEX-4T-1 expression vector

[0081] 1. Ligate the digestion products of PEDV-SE and ppGEX-4T-1

[0082]Get the digestion product of the PEDV-SE target gene fragment (510bp) and ppGEX-4T-1 expression vector (5915bp) in Example 2, the connection system is as shown in Table 2; wherein the amount of target gene material: ppGEX-4T-1 material amount=10:1, the amount of target gene material is 0.3 pmol, and the amount of expression vector material is 0.03 pmol. The measured target fragment concentration of PEDV-SE was 40.12 μg / mL, and the measured concentration of ppGEX-4T-1 vector was 42.58 μg / mL. Since 1pmol1000bpDNA is 0.66μg. Then 1 pmol 510bp target fragment is 0.337 μg, and 1 pmol 5915bp or so vector is 3.90 μg.

[0083] Table 2 connection system

[0084]

[0085] The volume of the PEDV-SE target gene that needs to be added is x, which can be listed as follows

[0086] 40.12μg / mL·x=0.337μg / pmol×0.3pmol

[0087...

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Abstract

The invention provides a gene encoding a multi-epitope antigen of porcine epidemic diarrhea virus, the multi-epitope antigen of the porcine epidemic diarrhea virus, and a preparation method of the multi-epitope antigen of the porcine epidemic diarrhea virus. The invention has the following advantages and beneficial effects: 1) the multi-epitope antigen of the porcine epidemic diarrhea virus is a multi-epitope antigen using norovirus P particles chimeric with the porcine epidemic diarrhea virus, natural structural proteins of pathogens are fully reduced, only main viral surface antigen proteinsare expressed, and nonspecific immunoreaction, strong virulence reversion and other problems induced by many irrelevant antigens are solved obviously; 2) the preparation method is low in preparationcost, short in time consuming and safety; and 3) the prepared multi-epitope antigen of the porcine epidemic diarrhea virus has good bioactivity and reactogenicity.

Description

technical field [0001] The present invention relates to the field of biotechnology. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is an acute intestinal infectious disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), characterized by vomiting and watery diarrhea. All breeds of pigs are susceptible, and have a high lethal rate to young piglets; PEDV belongs to the genus Coronaviridae of the family Coronaviridae of the order Nidoviridae. The nucleic acid of the virus is a positive-sense single-stranded RNA. The total length of the genome is about 28kb, and the main structural protein is the spike sugar protein (S protein), small membrane protein (E protein) and nucleocapsid protein (N protein). [0003] The disease was first reported in the UK in 1978, and has since been found in countries all over the world. The disease was first reported in China in 1980. In the second half of 2010, PED broke...

Claims

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Application Information

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IPC IPC(8): C12N15/50C12N15/70C12N15/66C07K14/165A61K39/215A61P31/14
CPCC07K14/005C12N15/70C12N15/66A61K39/12A61P31/14C12N2770/20022C12N2770/20034
Inventor 刘文俊杨培洁邓汝森周德荣黄运茂田允波
Owner ZHONGKAI UNIV OF AGRI & ENG
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