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Recombinant Enterococcus faecium vaccine strain labeled with eGFP and having resistance to porcine transmissible gastroenteritis and porcine epidemic diarrhea, and application thereof

A technology for porcine epidemic diarrhea and Enterococcus faecium, which is applied in vaccines, multivalent vaccines, recombinant DNA technology, etc., can solve the problems that restrict the popularization and application of recombinant lactic acid bacteria preparations, unfavorable large-scale production, and hidden dangers of biological safety, so as to avoid unsafe Hidden danger, inherent immune adjuvant effect, good immune effect

Inactive Publication Date: 2018-03-23
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2016, Zhang et al. orally immunized pigs with recombinant Salmonella typhimurium expressing TGEV and PEDV S proteins. Although the pigs could be induced to produce neutralizing antibodies, there were biological safety risks in using pathogenic bacteria as vaccine antigen delivery carriers.
However, the recombinant lactic acid bacteria system uses the chloramphenicol resistance gene as a selection marker. With the application of the lactic acid bacteria preparation in practice, the transfer of resistance factors will bring serious biosafety consequences. In the absence of inducers, the recombinant lactic acid bacteria will stop the secretion of vaccine antigens, which is not conducive to large-scale production, and further restricts the popularization and application of the recombinant lactic acid bacteria preparation

Method used

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  • Recombinant Enterococcus faecium vaccine strain labeled with eGFP and having resistance to porcine transmissible gastroenteritis and porcine epidemic diarrhea, and application thereof
  • Recombinant Enterococcus faecium vaccine strain labeled with eGFP and having resistance to porcine transmissible gastroenteritis and porcine epidemic diarrhea, and application thereof
  • Recombinant Enterococcus faecium vaccine strain labeled with eGFP and having resistance to porcine transmissible gastroenteritis and porcine epidemic diarrhea, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Comparison of probiotics between Enterococcus faecium E.faecium N-16 strain and Lactobacillus casei L.casei 393

[0072] The present invention compares and analyzes the probiotic properties of Enterococcus faecium N-16 strain (CCTCC NO: M 2017516) isolated from pig intestines and Lactobacillus casei L. casei 393, a commonly used strain for constructing genetically engineered lactic acid bacteria. The specific method is as follows:

[0073] The experimental pigs were divided into 3 groups, 10 pigs in each group. The experimental group I was fed the common diet supplemented with Enterococcus faecium N-16 strain, and the experimental group II was fed the normal diet supplemented with Lactobacillus casei L. casei 393 , the experimental group III was the normal diet control group. After feeding for 21 days, the growth of the piglets was observed. It can be seen that the weaned piglets fed with Enterococcus faecium N-16 had a better overall mental state than the oth...

Embodiment 2

[0081] Example 2 Construction of eGFP-marked, fusion-expressed TGEV 6Ds and PEDV ps420 recombinant Enterococcus faecium

[0082] 1. Construction of pMD18-T-6Ds plasmid

[0083] The nucleotide sequence encoding the D antigen site of TGEV TH98 strain (SCYTVSDSSFFSYGEIPFGVTDGPRYCY) was repeated six times, and a linker sequence (GGCGGTGGCGGAGGCGGA) was inserted between each segment. A total of 5 linkers were inserted, and the resulting repeated sequence was named 6Ds. The 6Ds gene sequence was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO.1. The obtained gene sequence was connected with pMD18-T, transformed into TG1, and the plasmid extracted from the recombinant bacteria was identified by PCR, enzyme digestion and sequencing, and the recombinant plasmid was named pMD18-T-6Ds.

[0084] 2. Construction of pMD18-T-ps420 plasmid

[0085] The PEDV strain is the PEDV HLJ-2012 strain isolated and cultivate...

Embodiment 3

[0106] Example 3 Immunogenicity evaluation of recombinant Enterococcus faecium pLXeno-eGFP-6Ds-ps420 / E.faecium N-16

[0107] The present invention systematically evaluates the immunogenicity of the recombinant Enterococcus faecium pLXeno-eGFP-6Ds-ps420 / E.faecium N-16 using BALB / c mice as animal models. Eight-week-old BALB / c mice were divided into 4 groups, 10 mice in each group, and were orally inoculated with recombinant Enterococcus faecium pLXeno-eGFP-6Ds-ps420 / E.faecium N-16, pLXeno-6Ds-ps420 / E. faecium N-16 and pLXeno / E.faecium N-16, oral dose is 200 μL 2×10 9 CFU / mL recombinant Enterococcus faecium, oral administration of 200 μL PBS was used as control. The immunization program was once every 2 weeks, and a total of 3 times of immunization. ELISA method was used to detect the level of sIgA antibody in feces and vaginal mucus of BALB / c mice and the level of IgG antibody in serum, antibody neutralization titer detection and lymphocyte proliferation experiment. The speci...

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Abstract

The invention discloses a recombinant Enterococcus faecium vaccine strain labeled with eGFP and having resistance to porcine transmissible gastroenteritis and porcine epidemic diarrhea, and an application thereof. The vaccine strain adopts Enterococcus faecium isolated from a porcine intestinal colon segment as a vaccine antigen delivery live vector, and contains an eGFP-labeled constitutive-expression TGEV S protein D antigen site and PEDV S protein neutralizing epitope region ps420 Protein recombinant expression vector. A bivalent oral vaccine having resistance to the porcine transmissible gastroenteritis and porcine epidemic diarrhea, prepared from the recombinant Enterococcus faecium vaccine strain, can achieve the purpose of preventing two-disease through one-time immunization, and has the advantages of environmental protection, high efficiency, safety and good probiotics.

Description

technical field [0001] The present invention relates to a vaccine strain and a construction method thereof, in particular to an Enterococcus faecium vaccine strain capable of simultaneously resisting porcine transmissible gastroenteritis and porcine epidemic diarrhea and a construction method thereof, and also relates to the preparation of the vaccine strain against porcine Application of a bivalent oral vaccine against transmissible gastroenteritis and porcine epidemic diarrhea. The invention belongs to the technical field of veterinary medicine. Background technique [0002] Porcine transmissible gastroenteritis of swine virus (TGEV) and porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV) are viruses of the genus Coronaviridae, which cause acute enteritis, diarrhea and death in pigs after infection. The main symptoms of porcine transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED). The outbreaks of TGE and PED show obvious seasonal c...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/62C12N15/66A61K39/225A61K39/215A61P1/00A61P1/04A61P31/14A61P1/12C12R1/46
CPCA61K39/12A61K2039/542A61K2039/70C07K14/005C07K2319/60C12N15/66C12N15/74C12N2770/20022C12N2770/20034A61K2300/00
Inventor 徐义刚王丽乔薪瑗唐丽杰李一经于美玲
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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