Lactobacillus gene engineering subunit vaccine strain capable of stably expressing porcine rotavirus VP4 protein and preparation method of lactobacillus gene engineering subunit vaccine strain

A porcine rotavirus and subunit vaccine technology, applied in the field of medicine, can solve problems such as biological safety hazards, instability of plasmid expression, loss of plasmids, etc., and achieve good immunogenicity

Inactive Publication Date: 2018-05-04
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are many defects in the expression of exogenous antigens by using the plasmid system. For example, the expression amount of exogenous antigens in the lactic acid bacteria plasmid expression system is significantly lower than that of the E. coli expression system; the plasmid expression is unstable; the plasmid may be lost during bacterial proliferation. The transmission of plasmids between bacteria, especially the presence of antibiotic resistance genes in plasmids, makes lactic acid bacteria a major biological safety hazard as a human and animal vaccine

Method used

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  • Lactobacillus gene engineering subunit vaccine strain capable of stably expressing porcine rotavirus VP4 protein and preparation method of lactobacillus gene engineering subunit vaccine strain
  • Lactobacillus gene engineering subunit vaccine strain capable of stably expressing porcine rotavirus VP4 protein and preparation method of lactobacillus gene engineering subunit vaccine strain
  • Lactobacillus gene engineering subunit vaccine strain capable of stably expressing porcine rotavirus VP4 protein and preparation method of lactobacillus gene engineering subunit vaccine strain

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Experimental program
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Effect test

Embodiment 1

[0053] The selection of embodiment 1 porcine rotavirus VP4 gene insertion site

[0054] Lactobacillus casei were respectively inoculated in MRS liquid medium containing 1% of different carbon sources (glucose, fructose, sucrose, galactose, lactose, trehalose, raffinose, polyfructose), and the total RNA of the bacteria was extracted for cheese Microarray analysis of the whole genome of Lactobacillus, and hierarchical clustering analysis of the microarray detection results to find genes that can be efficiently and stably expressed in different carbon source media. It has been detected that pyruvate hydratase gene RNA can be stably transcribed at a high level in different carbon sources. In order to obtain the recombinant Lactobacillus casei that expresses porcine rotavirus VP4 protein constitutively and efficiently in the genome, the present invention uses the pyruvate hydratase gene site as the recombination site of the VP4 gene, and inserts the stop codon of the pyruvate hydra...

Embodiment 2

[0055] Embodiment 2 Determination of the SD sequence containing the signal peptide sequence

[0056] The signal peptide sequence and ribosome binding site of peptidoglycan hydrolase were predicted by bioinformatics software SignalP-4.0. Using the Lactobacillus casei genome as a template to amplify the SD sequence containing the signal peptide sequence of the peptidoglycan hydrolase, the results are shown in figure 1 As shown, a fragment with a size of about 100 bp was obtained, the nucleotide sequence of which was shown in SEQ ID NO.1, and the fragment was ligated into the cloning vector pUC-57.

Embodiment 3

[0057] Example 3 Preparation of Recombinant Lactobacillus casei with Genome Constitutive High Expression of Porcine Rotavirus VP4 Protein

[0058] 1. Construction of gene targeting plasmid backbone

[0059] (1) Cloning of porcine rotavirus VP4 gene

[0060] Using the cDNA of the porcine rotavirus VP4 gene as a template and using VP4-F / VP4-R as primers, the porcine rotavirus VP4 gene fragment was amplified by PCR, and its nucleotide sequence was shown in SEQ ID NO.2. And at the two ends of the VP4 gene, restriction enzyme sites Pst I and Spe I were added, and the gene was connected to the pMD18-T vector. The primer sequences are shown in Table 1, and the PCR identification results are shown in figure 2 shown.

[0061] Table 1 Primer Sequence

[0062]

[0063]

[0064] (2) Subcloning of upstream and downstream recombinant homology arms

[0065] Using the genome of Lactobacillus casei as a template and using EB-F / EB-R and EA-F / EA-R as primers respectively, the upper an...

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Abstract

The invention discloses a lactobacillus gene engineering subunit vaccine strain capable of stably expressing porcine rotavirus VP4 protein and a preparation method of the lactobacillus gene engineering subunit vaccine strain. The vaccine strain is obtained through the following steps: on the basis of lactobacillus casei with uracil phosphoribosyl transferase, UPP gene deleted, the porcine rotavirus VP4 gene is inserted between a termination codon and a terminator of the neuronspecificenoluse gene of lactobacillus casei through homologous recombination, and the vaccine strain is not provided with an antibiotic selection marker. The experiment proves that after the constructed lactobacillus gene engineering subunit vaccine strain capable of stably expressing the porcine rotavirus VP4 proteinis used for immunizing animals through oral administration, the local mucosal immune response can be induced, the mucous membrane antibody IgA is generated, the body is induced to generate humoral immune response, the serum antibody IgG is then generated, good immunogenicity is displayed, and the constructed lactobacillus gene engineering vaccine strain not labelled by antibiotic resistance accords with the development concept of 'no pollution and environmental protection' of veterinary vaccines.

Description

technical field [0001] The invention relates to a genetically engineered subunit vaccine using lactic acid bacteria as a carrier and a preparation method thereof, in particular to a lactic acid bacteria genetically engineered subunit vaccine whose genome integrates porcine rotavirus VP4 gene and a preparation method thereof. The invention belongs to the technical field of medicine. Background technique [0002] Porcine Rotavirus (Porcine Rotavirus, PRV) belongs to the genus Rotavirus in the Reoviridae family, and is one of the main pathogens that cause viral diarrhea in piglets. It mainly infects piglets aged 1-4 weeks, and has the characteristics of high incidence and wide distribution. . Porcine rotavirus disease is a typical type of infectious disease transmitted by fecal-oral route. At present, the main method to prevent porcine rotavirus infection is vaccination, and common vaccines include strong-toxic inactivated vaccines and attenuated live vaccines. [0003] The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/46C12N15/74A61K39/15A61P31/14C12R1/245
CPCA61K39/12A61K2039/523A61K2039/542A61K2039/552C07K14/005C12N15/746C12N2720/12322C12N2720/12334
Inventor 李一经徐义刚王丽唐丽杰尹纪元施雯
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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