Constructing and expression method of recombinant lactobacillus for hog cholera virus E2 gene

A technology of recombinant lactic acid bacteria and swine fever virus, which is applied in the direction of virus/phage, virus, virus peptide, etc., can solve the problems of the construction of recombinant lactic acid bacteria of swine fever virus E2 gene and the immunogenicity research has not yet been reported, and achieve the protection of swine fever infection. , maintain the balance of intestinal flora, improve the effect of immunity

Inactive Publication Date: 2016-11-09
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the construction of recombinant lactic acid bacteria with the E2 gene of classical swine fever virus and the study of its immunogenicity have not been reported yet.

Method used

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  • Constructing and expression method of recombinant lactobacillus for hog cholera virus E2 gene
  • Constructing and expression method of recombinant lactobacillus for hog cholera virus E2 gene
  • Constructing and expression method of recombinant lactobacillus for hog cholera virus E2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Cloning and sequence analysis of the E2 gene of classical swine fever virus F114 strain

[0050] 1.1 Virus cultivation

[0051] 1.1.1 Recovery and culture of PK-15 cells

[0052] Take out the PK-15 cells frozen at -80°C, thaw them in a water bath at 37°C immediately, suspend the cells with 10ml DMEM complete medium, centrifuge at 1000rpm for 5min, discard the supernatant, then resuspend the cells with 10ml DMEM complete medium, move to In the cell culture flask, place at 37°C 5% CO 2 Cultured in an incubator. After subculture for 3 generations, the cells grow stably and can be expanded for culture. 1.1.2 Infection

[0053] Get cultured 24h, well-growth 80% single-layer PK-15 cells, suck out the old culture medium, wash twice with Hank's solution, get the CSFV F114 strain blood virus stored in freezer, use DMEM incomplete medium to press 0.1MOI (infect ratio) diluted and inoculated in the cells, the amount added is just enough to cover the bottom of the b...

Embodiment 2

[0099] Example 2: Construction of recombinant plasmid PNZ44-E2

[0100] 2.1. E2 gene PCR amplification of PMD-18-E2

[0101] 2.1.1 Primer design

[0102] According to the gene sequence of the classical swine fever virus E2 protein and referring to the multiple cloning site of the plasmid pNZ44, two primers were designed to synthesize and clone the E2 gene, and the Nco I restriction site was introduced upstream of the gene, and the Hind III restriction site was introduced downstream site. Primers were synthesized by Takara Company and diluted to 10 pmol / μL with ultrapure water.

[0103] The designed primer sequences and names are as follows:

[0104] pNZE2f: 5'-CATGCCATGGTGCGCTTAGCCTGCAAG-3' (primer at the 5' end, containing the Nco I restriction site CCATGG) pNZE2r: 5'-CCCAAGCTTGGGCTGCAGAATTCCTAGTCAAACC-3' (primer at the 3' end, containing the HindIII restriction site AAGCTT)

[0105] 2.1.2 PCR reaction system and reaction conditions

[0106] The PCR reaction system is: 1...

Embodiment 3

[0117] Example 3: Electrotransformation of lactic acid bacteria with recombinant plasmid pNZ0844-E2 and its expression

[0118] 3.1. Preparation of Lactobacillus casei electroporation competent cells

[0119] Lactobacillus casei LV glycerol was streaked on the MRS agar plate without antibiotics, and cultured anaerobically at 37°C for 24 hours. Inoculate a single colony in 3mL MRS medium, and culture overnight at 37°C; the next day, transfer the overnight culture to 50mL MRS medium at a ratio of 1:50, and culture statically at 37°C until the culture grows to the effective growth stage when (OD 600 =0.6~0.8), centrifuge (2500rpm.4°C, 10min) to collect the cells, wash the cells with pre-cooled electroporation wash buffer (EPWB) for 3 times (1mL / time), and wash the cells with pre-cooled electroporation wash buffer ( Electroporation buffer, EPB) wash cells once, wash cells once with 1mL pre-cooled 30% PEG (polyethylene glycol) 1500 + 100% EPWB, resuspend cells in 500 μL 30% PEG15...

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Abstract

The invention provides a method of recombinant lactobacillus for expressing hog cholera virus E2 protein. The method comprises the following steps of 1, cloning the gene of a strain 2 of hog cholera virus F114, and analyzing a sequence; 2, constructing a recombinant expression carrier PNZ44-E2 of the hog cholera virus; 3, performing electrotransformation and expression on the recombinant plasmid PNZ44-E2 in lactobacillus casei; 4, analyzing the immunogenicity of the recombinant lactobacillus. The method has the beneficial effects that the recombinant lactobacillus has the good property of probiotics and dual properties of expressing foreign protein; after an animal orally takes viable organism, the health-care function of lactobacillus as probiotics and the properties of lactobacillus expressing the hog cholera E2 protein in intestinal tracts and reaching immunity are realized; the specific humoral immunity and cell immunity capabilities of body are improved, and the resistance of body to hog cholera virus is improved by the immunity of intestinal mucosa; by utilizing the platform, a scientific foundation is laid for the modification of engineering strain of probiotics and the function of using the recombinant lactobacillus expressing the hog cholera virus E2 protein as the oral administration vaccine to prevent and control hog cholera.

Description

technical field [0001] The invention provides the construction and immunogenicity analysis of recombinant lactic acid bacteria with the E2 gene of swine fever virus, and belongs to the field of biotechnology. Background technique [0002] Classical swine fever virus (CSFV) is a highly contagious infectious disease caused by classical swine fever virus (CSFV). Based on the huge economic impact of CSF on the pig industry, the International Office of Epizootics listed it as one of the 16 legal infectious diseases in category A One of them, my country has also listed it as a first-class animal infectious disease, as one of the main infectious diseases for strict detection and quarantine. CSFV mainly invades the host through the digestive tract of susceptible pigs, such as swallowing pollutants, through the oral cavity or intestinal mucosa; once a pig is infected with CSFV, tonsils, mucous membranes, and immune system damage will occur, causing continuous high fever, leading to ac...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/11C12N15/74
CPCC07K14/005C12N15/746C12N2770/24322C12N2800/101
Inventor 张以芳童亮张绍美杨洁马志亮周玉照张辰宇徐佳柴俊刘旭川
Owner YUNNAN AGRICULTURAL UNIVERSITY
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