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Engineering yeast strain for preventing porcine circovirus disease

A technology of porcine circovirus disease and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of unfavorable immunization, short residence time of recombinant strains, and high production costs, so as to improve immunogenicity and immune efficacy, and improve non-specific immunity , Improve the effect of specific immunity

Pending Publication Date: 2016-07-20
GUANGZHOU WISDOM BIO TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the subunit vaccine of Cap protein has a very wide application prospect in the prevention and control of PCV-2 related diseases, and its clinical immune effect has also been affirmed. The current problem is that its production cost is high, which is not conducive to large-scale production. Immunization
[0009] At present, the main problems in the construction of vaccines by microbial surface display technology are that the residence time of recombinant strains on the mucosa is short, and the expression of foreign proteins is too low, which cannot effectively stimulate mucosal immunity.

Method used

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  • Engineering yeast strain for preventing porcine circovirus disease
  • Engineering yeast strain for preventing porcine circovirus disease
  • Engineering yeast strain for preventing porcine circovirus disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 shows the construction of the yeast engineering strain of preventing porcine circovirus disease of antigen LTB, SLT-IIeB and ORF2 fusion protein

[0043] 1) According to the yeast codon preference, transform the LTB gene, SLT-IIeB gene in pathogenic porcine Escherichia coli and the ORF2 of porcine circovirus type 2 virus-encoded viral nucleocapsid protein Cap; design and synthesize the recombinant gene LSO primers;

[0044] 2) LTB, SLT-IIeB and ORF2 after transformation were amplified by overlappingPCR technology, and the full length of the LSO fusion gene was obtained by connecting the linker sequence; the nucleic acid sequence of LSO is shown in SEQ ID NO: 2;

[0045] 3) Saccharomyces cerevisiae display expression vector pHBM368AC was treated with CpoI and NotI double enzymes;

[0046] 4) GTCA was introduced at the 5' end of the fusion gene LSO, and two nucleotide sequences of GGCCA were introduced at the 3' end; the amplified product was treated with T4...

Embodiment 2

[0049] Embodiment 2: the acquisition of S-SP3 recombinant strain

[0050] The recombinant transformants obtained in Example 1 were extracted from the genome, and the positive clones that had been correctly inserted into the LSO gene were identified by PCR; the correct recombinant transformants verified by inoculation were cultured in 50ml YNB-HLAD liquid medium, 28°C, 200rpm for 12-16 hours, then collect the bacteria and transfer them to 100ml YNB-HLAG liquid medium, culture at 28°C, 200rpm for 48 hours, and then centrifuge at 5000rpm for 5 minutes at 4°C to collect the bacteria. The collected bacteria were then subjected to immunofluorescence detection and westernblotting detection, the results are shown in figure 2 .

[0051] figure 2 It is the indirect immunofluorescence detection of the recombinant brewer's yeast INVScl / pHBM368AC-LSO (named S-SP3) of the present invention; where A is the blank control brewer's yeast, and B is the recombinant brewer's yeast, respectivel...

Embodiment 3

[0052] The biological characteristic of embodiment 3S-SP3 recombinant bacterial strain

[0053] 1. Analysis of growth characteristics of S-SP3 recombinant strain

[0054] Streak culture of S-SP3 recombinant strain on solid YEPD plate, pick a single colony into liquid YNB-HLAD medium, culture at 28C, 200r / min for 16 hours, insert 3% inoculum amount into the yeast culture that has been sterilized before Continue to culture in the medium, the temperature of the medium is 28°C, the pH is 4.6, and the rotation speed is 170r / min. Experiments have proved that the genetic engineering strain of the present invention can be fermented at a high density, with an OD600 of over 40, no inducer needs to be added during the whole fermentation process, and it can be strictly expressed at the end of the logarithm. Finally, the biomass of Saccharomyces cerevisiae reached 8.124g / L.

[0055] 2. Genetic stability of S-SP3 recombinant strain

[0056] Streak culture the S-SP3 recombinant strain pre...

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Abstract

The invention discloses an engineering yeast strain for preventing a porcine circovirus disease. A gene engineering bacteria comprise genes for coding LSO fusion protein; the LSO fusion protein has a sequence shown as SEQ ID NO:1. A high-copying yeast integrating type plasmid surface is used for showing the fusion protein of a multiple-epitope antigen of the porcine circovirus PCV2 and a protective antigen LTB SLT IIeB of pathogenic swine colibacillosis; meanwhile, the recombinant fusion protein LTB SLT IIeB per se has the effect of an immunologic adjuvant, is used as both the antigen and an adjuvant, and achieves the dual functions; the immunogenicity and the immune efficiency of CAP protein coded by ORF2 of the porcine circovirus PCV2 shown by recombinant beer yeast strains are improved. The genetic engineering yeast strain can be applied to the prevention and treatment of the porcine circovirus disease.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a yeast engineering bacterium for preventing porcine circovirus disease. Background technique [0002] Porcine circovirus type 2 (PCV-2) is the main pathogen that causes postweaning multisystemic wasting syndrome (postweaningmultisystemicwastingsyndrome, PMWS). It is widely prevalent in the herd and has caused huge economic losses to the pig industry in the world. PCV-2 is often associated with porcine reproductive and respiratory syndrome virus (porcinereproductiveandrespiratorysyndromevirus, PRRSV), porcine parvovirus (porcineparvovirus, PPV), porcine pseudorabies virus (pseudorabiesvirus, PRV), Haemophilus parasuis (Haemophilus parasuis, HPs), pig pleura Actinobacillus pleuropneumoniae (App) and Pasteurella swine (swine pasteullosis, Sp) and many other pathogens are concurrently or secondary infected, which increases the difficulty of their control. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/62A61K36/064A61K48/00A61K39/12A61P31/20C12R1/865
CPCC07K14/245A61K36/064A61K39/12A61K48/00C07K14/005C07K2319/00C12N15/81C12N2750/10022C12N2750/10034C12N2800/102C12N2800/22
Inventor 刘国平吴世林廖生荣郭利伟胡利群蒋思靖周康李宗明吕勇江华峰
Owner GUANGZHOU WISDOM BIO TECH
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