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Fusion protein, fusion protein preparation method and porcine oral vaccine or medicine

A fusion protein and protein technology, applied in biochemical equipment and methods, fusion peptides, drug combinations, etc., can solve problems such as limited vaccine effects and achieve the effect of protecting diseases

Active Publication Date: 2016-12-07
CHANGSHA LVYE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But such vaccines still have limited effectiveness

Method used

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  • Fusion protein, fusion protein preparation method and porcine oral vaccine or medicine
  • Fusion protein, fusion protein preparation method and porcine oral vaccine or medicine
  • Fusion protein, fusion protein preparation method and porcine oral vaccine or medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] This embodiment illustrates the preparation method of recombinant Omp16-PEDVS / ATCC393 Lactobacillus lactis (oral vaccine used in Example 3), comprising the following steps:

[0047] 1. Synthesize the OMP16-PEDVS gene, add a Sal I restriction endonuclease site at the 5' end, and add an EcoRV restriction endonuclease site at the 3' end. The synthetic gene is cloned on the pUC57 vector and named as pUC57-Omp16-PEDVS (the sequence of its fusion protein is shown in sequence SEQ.No.2), the Sal I and Eco RV double enzyme digestion identification results of pUC57-Omp16-PEDVS plasmid are as follows figure 2 shown.

[0048] 2. Transformation of ATCC393 Lactobacillus lactis with pVE5523-Omp16-PEDVS plasmid

[0049] The correctly sequenced pUC57-Omp16-PEDVS plasmid and the pVE5523 expression vector plasmid were digested with Sal I and Eco RV, the target fragment and the pVE5523 vector fragment were recovered from the gel, ligated with T4Ligase (T4DNA ligase), and the ligated prod...

Embodiment 2

[0058] This embodiment illustrates the preparation method of recombinant Omp16-PEDVS and PEDVS prokaryotic expression protein (two kinds of injection vaccines used in embodiment 3), comprising the following steps:

[0059] 1. Construction of pET28a-OMP16-PEDVS expression vector

[0060] Using the pVE5523-OMP16-PEDVS gene as a template, design the upstream primer F1 (see sequence SEQ.No.3) and downstream primer R1 (see sequence SEQ.No.4) for amplifying OMP16-PEDVS, and add Nco I restriction endonuclease site, and 3' plus Xho I restriction endonuclease site, the gene was subcloned into pET28a(+) prokaryotic expression vector, named pET28a-OMP16-PEDVS. The result is as Figure 4 1, and transform the correct plasmid into BL21(DE3) E. coli.

[0061] 2. Construction of pET28a-PEDVS expression vector

[0062] Using the pVE5523-OMP16-PEDVS gene as a template, design the upstream primer F2 (see sequence SEQ.No.5) and downstream primer R2 (see sequence SEQ.No.6) for amplifying PEDVS,...

Embodiment 3

[0066] This example illustrates the comparison of the immune effects of OMP16-PEDVS lactic acid bacteria (the oral vaccine prepared in Example 1), OMP16-PEDVS protein and PEDVS protein (two injection vaccines prepared in Example 2).

[0067] 1. Animal immunity

[0068] Take 50 healthy female SPF grade (specific pathogen-free animals) C57 / B6 mice and divide them into OMP16-PEDVS lactic acid bacteria, OMP16-PEDVS protein group, OMP16-PEDVS protein+Freund's adjuvant group (OMP16-PEDVS+F' ), PEDVS protein group and control group. Those skilled in the art know that Freund's complete adjuvant generally plays a role in enhancing the immune effect in injection vaccines. The concentration used for protein immunization was 1 mg / ml, and the Freund's complete adjuvant group was added with protein:Freund's complete adjuvant for emulsification at a ratio of 1:1, and 200 μl of each mouse was injected into the leg muscles for immunization; 14 days after the first immunization, A booster imm...

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Abstract

The invention provides a fusion protein and a porcine oral vaccine or medicine. The fusion protein is obtained by fusion of all or part of fragments of brucella outer membrane protein (OMP16) and porcine epidemic diarrhea virus S protein (PEDV-S). The porcine oral vaccine or medicine is obtained by steps: loading the fusion protein into a lactobacillus expression vector, transforming the lactobacillus expression vector into a Lactobacillus casei standard strain, and culturing the strain. The porcine oral vaccine is capable of effectively stimulating intestinal local immune cells to generate secretory IgA and is applicable to elimination of intestinal mucosal pathogens such as bacteria, viruses and parasites, and defects that conventional vaccination causes stress and antibodies fail to reach positions of the pathogens are avoided. In addition, due to adoption of an active lactobacillus vector system, possibility of degradation and ineffectiveness of inactivated vaccines before reaching small intestine mucosa is avoided. The porcine oral vaccine and the medicine are effective in prevention and treatment of porcine epidemic diarrhea virus infection.

Description

technical field [0001] The invention relates to the field of virus prevention and control, in particular to a fusion protein, a preparation method thereof and an oral vaccine for pigs. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by Coronaviridae porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) and is characterized by diarrhea, vomiting, dehydration and highly lethal sucking piglets of all ages. Highly contagious enteric infectious disease. The infection of this disease is very serious in pig farms in Asia, especially in my country, and its infection rate is significantly higher than that of the other two porcine viral diarrhea pathogens --- porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus ( PRV). Due to the serious harm of PED to piglets, domestic and foreign scholars have successively developed PEDV vaccines and applied them to the immune prevention of PED in pig farms. H...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70A61K39/215A61K39/39A61P1/12
CPCA61K39/12A61K39/39A61K2039/542A61K2039/55516C07K14/005C07K14/23C07K2319/00C12N2770/20022C12N2770/20034A61K2300/00
Inventor 张大生滕清梅谭力
Owner CHANGSHA LVYE BIOTECHNOLOGY CO LTD
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