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Recombinant lactococcus lactis expressing S318 gene of porcine epidemic diarrhea virus and application thereof

A technology for porcine epidemic diarrhea and Lactococcus lactis, which is applied to recombinant Lactococcus lactis and application fields, and can solve problems such as unsatisfactory effects of inactivated vaccines

Inactive Publication Date: 2017-05-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the oral vaccine has no early interference effect, active immunity can be induced only 5 days after infection with PEDV, so we cannot hope to vaccinate newborn piglets, but focus on how to quickly protect suckling piglets within the first few days; therefore, oral , intranasal or intramuscular injection of inactivated vaccines are not ideal

Method used

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  • Recombinant lactococcus lactis expressing S318 gene of porcine epidemic diarrhea virus and application thereof
  • Recombinant lactococcus lactis expressing S318 gene of porcine epidemic diarrhea virus and application thereof
  • Recombinant lactococcus lactis expressing S318 gene of porcine epidemic diarrhea virus and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Synthesis of target genes and primers

[0077] 1. Synthesis of the target gene

[0078] According to the PEDV S gene sequence (ACESSION: AFV59239) published on NCBI, the 1422-2376bp gene sequence of the full length of the S gene (see Sequence Table 3) was obtained, and codon optimization was carried out through the codon preference table of MG1363 Lactococcus lactis ( See sequence listing 2)

[0079]Codon preference table for Lactococcus lactis MG1363

[0080] Codon Frequency Table Used Lactococcus lactis subsp.Cremoris MG1363

[0081]

[0082] 2. Primer Synthesis

[0083] 2.1. Gene synthesis primers

[0084] A pair of primers were designed according to the gene sequence synthesized by codon optimization (see Sequence Table 1) and the pMG36e vector multiple cloning site, the upstream and downstream were respectively:

[0085] PEDV-F: GG GTCGAC CATGCAAGTTGCTTTTGAT,

[0086] Sal I

[0087] PEDV-R: GG GGTACC TTAAATTGACATTGAA;

[0088] Kpn I

[0089] Restric...

Embodiment 2

[0100] recombinant vector PEDV-S 318 - Construction of pMG36e

[0101] 1. Amplification of the target gene fragment

[0102] According to the instructions of the plasmid mini-extraction kit, the synthetic S 318 Rapid extraction of gene plasmids and pMG36e vectors

[0103] Amplification of codon-optimized S with Prime-STAR 318 The gene sequence was amplified in 6 tubes in total, 50 μl in each tube, Prime-STAR amplification system:

[0104]

[0105] Prime-STAR amplification reaction procedure:

[0106] Pre-denaturation: 98°C, 2min; denaturation: 98°C, 10sec; annealing: 55°C, 15sec; extension: 72°C, 1min; post-extension: 72°C, 10min; cycle: 30

[0107] After the amplification is completed, add 5.5 μl of 10×loading buffer, electrophoresis with 1% agarose gel, cut out the target band and recover the target fragment with a gel recovery kit.

[0108] 2. S 318 Double digestion of gene and pMG36e vector

[0109] Simultaneous digestion of S with two restriction enzymes, Sal Ⅰ...

Embodiment 3

[0136] Recombinant strain PEDV-S 318 - Construction of MG1363 recombinant bacteria

[0137] 1. Preparation of related media and reagents

[0138] SGM17 medium: 10ml M17 medium, 1562μl 50% sucrose, 200μl 50% glucose, 100μl 2mol / LMgCl 2 , 40μl 0.5mol / LCaCl 2 .

[0139] Solution Ⅰ: 84ml M17 medium, 1ml 50% glucose, 10ml 10% glycine, 5ml ddH 2 O

[0140] Solution Ⅱ: 34ml 50% sucrose, 10ml glycerin, 56ml ddH 2 O

[0141] 2. Preparation of MG1363 Lactococcus lactis Competence (Preparation and use now)

[0142] The preparation steps of MG1363 Lactococcus lactis competent are as follows:

[0143] 2.1 Take 50 μl of MG1363 seed solution with a pipette gun, add it to 5ml of sterilized M17 medium, and culture at 30°C for 10 hours;

[0144] 2.2 Inoculate the MG1363 seed solution cultivated for 10 hours into sterilized 15ml Solution I medium with a 2% inoculation amount, and culture it statically at 30°C;

[0145] 2.3 Measure the absorbance of the bacterial solution at a wavelengt...

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Abstract

The invention discloses a recombinant lactococcus lactis expressing an S318 gene of a porcine epidemic diarrhea virus and an application thereof. A constitutive expression vector pMG36e of lactococcus lactis MG1363 is applied to expressing. The lactococcus lactis is the generally recognized as safe (GARS) microorganism and has a beneficial adjusting effect on growth of an animal; a lactic acid bacteria constitutive expression system expresses a target protein without an inducer and synthesizes an exogenous target protein in a growing and splitting process of the lactic acid bacteria, so that toxic and side effects caused by the inducer in a product can be eliminated. A porcine epidemic diarrhea new mucosal immunity vaccine and a feed are mixed to feed pregnant sows and piglets for a while to prevent and control diseases such as porcine virus diarrhea caused by porcine epidemic diarrhea virus.

Description

technical field [0001] The invention relates to the field of livestock genetic engineering vaccines, in particular to a vaccine expressing porcine epidemic diarrhea virus S 318 Gene recombinant Lactococcus lactis and its application. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) of the Coronaviridae family. Vomiting, dehydration and high lethality to suckling piglets are the main features. PED first appeared in British farms in 1971, and then spread throughout the European continent, characterized by sporadic outbreaks in winter. Porcine epidemic diarrhea virus causes frequent watery diarrhea in pigs of all ages, especially newborn piglets with high mortality. Many countries, including the Americas and Asia, have detected positive antibodies to PEDV, and isolated the virus through in vitro culture...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C07K14/165C12N15/74C12N15/66C12N1/21A61K39/215A61P31/14C12R1/46
CPCC07K14/005A61K39/12A61K2039/523A61K2039/552C12N15/66C12N15/746C12N2770/20022C12N2770/20034C12N2800/101C12N2800/22
Inventor 金梅林蔡承志康超吴超孙小美
Owner HUAZHONG AGRI UNIV
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