Recombinant lactococcus lactis expressing S600 gene of transmissible gastroenteritis virus of pigs and application

A technology of Lactococcus lactis, tgev-s600-pmg36e, applied in recombinant Lactococcus lactis and application fields, can solve the problems of vaccines that cannot reach virus strain attack, waste of manpower, material and financial resources, and unsatisfactory immune effects.

Inactive Publication Date: 2017-06-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pigs of various ages and breeds are susceptible to TGEV, and the mortality rate of piglets within 2 weeks of age is as high as 100%. Although adult pigs can generally tolerate TGEV infection, they lose fat and reduce feed remuneration, resulting in a waste of manpower, material resources and Financial resources, causing great economic losses to the pig industry
[0003] The prevention and control against TGEV currently mainly relies on vaccination, and the currently used tissue or cell inactivated vaccines and attenuated vaccines have various defects to varying degrees, including unsatisfactory immune effects, high costs, recombination and resurrection, or detoxification and loosening.
In particular, the virulence and biological characteristics of viruses that have been circulating in recent years have mutated, and traditional vaccines often fail to achieve the effect of resisting natural epidemic strains.

Method used

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  • Recombinant lactococcus lactis expressing S600 gene of transmissible gastroenteritis virus of pigs and application
  • Recombinant lactococcus lactis expressing S600 gene of transmissible gastroenteritis virus of pigs and application
  • Recombinant lactococcus lactis expressing S600 gene of transmissible gastroenteritis virus of pigs and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Synthesis of target genes and primers

[0075] 1. Synthesis of the target gene

[0076] According to the TGEV S gene sequence (ACCESSION: AF494337) published on NCBI, take the gene sequence of 1-1800bp (as shown in SEQ ID NO. 1)

[0077] Codon preference table for Lactococcus lactis MG1363

[0078] Codon Frequency Table Used Lactococcus lactis subsp.Cremoris MG1363

[0079]

[0080] 2. Primer Synthesis

[0081] 2.1. Gene synthesis primers

[0082] 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:

[0083] S 600 -P1: GC GTC GAC TAAAAAATTATTTGTTG,

[0084] Sal I

[0085] S 600 -P2:GG GGTACC TTAATCTAAAACATCTG;

[0086] Kpn I

[0087] Restriction endonucleases are Sal Ⅰ and Kpn Ⅰ, respectively. Due to the frame shift phenomenon in the upstream, a base T is added after the Sal Ⅰ restriction site, a...

Embodiment 2

[0096] recombinant vector TGEV-S 600 - Construction of pMG36e

[0097] 1. Amplification of the target gene fragment

[0098] According to the instructions of the plasmid mini-extraction kit, the synthetic S 600 Rapid extraction of gene plasmids and pMG36e vectors

[0099]Amplification of codon-optimized S with Prime-STAR 600 The gene sequence was amplified in 6 tubes in total, 50 μl in each tube, Prime-STAR amplification system:

[0100]

[0101] Prime-STAR amplification reaction procedure:

[0102] Pre-denaturation: 98°C, 2min; denaturation: 98°C, 10sec; annealing: 55°C, 15sec; extension: 72°C, 2min; post-extension: 72°C, 10min; cycle: 30

[0103] After the amplification is completed, add 5.5 μl 10×loading buffer, electrophoresis with 1% agarose gel, cut out the target band and recover the target fragment with a gel recovery kit.

[0104] 2. S 600 Double digestion of gene and pMG36e vector

[0105] Simultaneous digestion of S with two restriction enzymes, Sal Ⅰ and...

Embodiment 3

[0134] recombinant strain TGEV-S 600 - Construction of MG1363 recombinant bacteria

[0135] 1. Preparation of related media and reagents

[0136] SGM17 medium: 10ml M17 medium, 1562μl 50% sucrose, 200μl 50% glucose, 100μl 2mol / LMgCl 2 , 40μl 0.5mol / LCaCl 2 .

[0137] Solution Ⅰ: 84ml M17 medium, 1ml 50% glucose, 10ml 10% glycine, 5ml ddH 2 o

[0138] Solution Ⅱ: 34ml 50% sucrose, 10ml glycerin, 56ml ddH 2 o

[0139] 2. Preparation of MG1363 Lactococcus lactis Competence (Preparation and use now)

[0140] The preparation steps of MG1363 Lactococcus lactis competent are as follows:

[0141] 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;

[0142] 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;

[0143] 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 the S600 gene of a transmissible gastroenteritis virus of pigs and application. A constitutive expression vector pMG36e of lactococcus lactis MG1363 is utilized for expression. Lactococcus lactis is a generally regarded as safe (GARS) microorganism, and has a beneficial regulating effect on the growth of an animal. When a constitutive expression system of lactic acid bacteria expresses target protein, no inducer is required. In the process of self growth division, foreign target protein is synthesized, and toxicity and side effects in products caused by the inducer can be eliminated. A new mucosal immunity vaccine of infectious gastroenteritis of pigs is mixed with feed to obtain a mixture, and then the mixture is fed to pregnant sows and piglets for a period of time to prevent and control diseases such as viral diarrhea of pigs caused by the transmissible gastroenteritis virus of pigs.

Description

technical field [0001] The invention relates to the field of livestock genetic engineering vaccines, in particular to a vaccine expressing porcine transmissible gastroenteritis virus S 600 Gene recombinant Lactococcus lactis and its application. Background technique [0002] Transmissible gastroenteritis of pigs (TGE) is a kind of porcine transmissible gastroenteritis virus of pigs (TGEV) caused by Coronaviridae Coronaviridae virus (Transmissible gastroenteritis virus of pigs, TGEV) with vomiting, dehydration, severe Diarrhea is an acute, highly contagious infectious disease characterized by diarrhea. Pigs of various ages and breeds are susceptible to TGEV, and the mortality rate of piglets within 2 weeks of age is as high as 100%. Although adult pigs can generally tolerate TGEV infection, they lose fat and reduce feed remuneration, resulting in a waste of manpower, material resources and Financial resources cause great economic losses to the pig industry. [0003] The pr...

Claims

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Application Information

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IPC IPC(8): C12N15/50C07K14/17C12N15/74C12N15/66C12N1/21A61K39/225A61P31/14C12R1/46
CPCA61K39/12A61K2039/523A61K2039/552C07K14/005C12N15/746C12N2770/20022C12N2770/20034C12N2800/101C12N2800/22
Inventor 金梅林蔡承志康超吴超孙小美
Owner HUAZHONG AGRI UNIV
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