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Protein recombinant lactococcus lactis for secretory expression of core antigen COE of PEDV (Porcine Epidemic Diarrhea Virus) as well as preparation method and application of protein recombinant lactococcus lactis

A Lactococcus lactis, core antigen technology, applied in the field of animal biomedical engineering, can solve problems such as poor immune effect, and achieve the effect of promoting healthy development and reducing harm

Active Publication Date: 2014-10-29
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defects in the prior art that the immune effect of the PEDV vaccine is not good, and to provide a preparation method for recombinant Lactococcus lactis that secretes and expresses the PEDV core antigen COE protein

Method used

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  • Protein recombinant lactococcus lactis for secretory expression of core antigen COE of PEDV (Porcine Epidemic Diarrhea Virus) as well as preparation method and application of protein recombinant lactococcus lactis
  • Protein recombinant lactococcus lactis for secretory expression of core antigen COE of PEDV (Porcine Epidemic Diarrhea Virus) as well as preparation method and application of protein recombinant lactococcus lactis
  • Protein recombinant lactococcus lactis for secretory expression of core antigen COE of PEDV (Porcine Epidemic Diarrhea Virus) as well as preparation method and application of protein recombinant lactococcus lactis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Construction of secreted recombinant Lactococcus lactis containing COE gene

[0052] S1. Construction of secretory recombinant plasmid pNZ8048-COE

[0053] S11. Codon preference optimization and synthesis of related gene sequences: according to the sequence of the target gene PEDV COE gene and the characteristics of the expression vector pNZ8048, as well as the signal peptide sequence added for the purpose of efficient secretion and expression SP Usp45 And the enhanced sequence [(amino acid sequence is: LEISSTCDA, the insertion position is after the Usp45 signal peptide, before the target gene COE, in the middle, and the positional relationship is: ( PnisA:: SP Usp45 :: LEISSTCDA :: COE ]), and the His (histidine) tag sequence added for easy detection, the entire expected expression sequence is available on the dedicated website ( http: / / genomes.urv.es / OPTIMIZER ) to optimize the codon preference for expression of lactic acid bacteria, and send the 42...

Embodiment 2

[0064] Application of embodiment 2 Lactococcus lactis in the preparation of PEDV vaccine

[0065] Prepared by Example 1 L. lactis NZ9000 / pNZ8048-COE recombinant Lactococcus lactis is made into an oral vaccine for preventing porcine epidemic diarrhea, and the immune effect of the vaccine is studied, including the following steps:

[0066] S1. L. lactis Preparation of NZ9000 / pNZ8048-COE recombinant Lactococcus lactis oral vaccine: the L. lactis NZ9000 / pNZ8048-COE recombinant expression bacteria were inoculated in G-M17C liquid medium at a ratio of 1:100, cultured overnight at 30°C, and the overnight culture was inoculated in 10 mL of G-M17 liquid medium at a ratio of 1:50. Continue to culture for about 2.5 hours until the bacteria enter the logarithmic growth phase, add 1 μg / mL inducer (Nisin) to a final concentration of 2 ng / mL, stop the culture after 6 hours of induction, and the whole culture at this time is directly used as an oral vaccine , using the gradient diluti...

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Abstract

The invention belongs to the field of animal biomedicine engineering, and in particular discloses protein recombinant lactococcus lactis for secretory expression of a core antigen COE of a PEDV (Porcine Epidemic Diarrhea Virus) as well as a preparation method and an application of the protein recombinant lactococcus lactis. The method comprises the following steps: connecting a signal peptide and other sequences onto a gene of the core antigen COE of the PEDV via an overlap extension PCR method by designing multiple primers, connecting the gene to a lactococcus lactis expression vector pNZ8048 and introducing the vector with the gene into a cell of the lactococcus lactis NZ9000 in an electrotransformation manner so as to obtain recombinant bacteria; and inducing the recombinant bacteria with nisin so as to obtain an expression, and directly taking all induced cultures as oral vaccines capable of stimulating mice and inducing strong cellular immune responses. Thus, the protein recombinant lactococcus lactis can serve as a novel oral vaccine product with a good industrial prospect and has a positive effect for reducing harms of the PEDV to pig industry, thereby playing a great practical significance for promoting the healthy development of the pig industry.

Description

technical field [0001] The invention relates to the field of animal biomedical engineering, more specifically, to a recombinant Lactococcus lactis that secretes and expresses the core antigen COE protein of porcine epidemic diarrhea virus and its preparation method and application. Background technique [0002] Porcine Epidemic Diarrhea (PED) is one of the world's porcine infectious diseases. It was first reported in the UK in 1978, and then broke out in many parts of the world, including my country, causing serious damage to the pig industry in various countries. huge loss. The outbreak of PED in Asia caused extremely high mortality. Studies have shown that when PED broke out in Japan from 1993 to 1994, more than 10,000 piglets died in 5 pig farms, and the mortality rate was 30% to 60%. According to reports, from 2010 From February to November 2011, the number of piglet deaths caused by the outbreak of PED in my country was more than 50,000, and the mortality rate was as hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21A61K39/215A61P31/14C12R1/01
Inventor 马静云李智丽曾喜多魏钟艳孙宝丽谢青梅毕英佐
Owner SOUTH CHINA AGRI UNIV
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