Staphylococcus aureus mutant strain, and preparation method and applications thereof

A staphylococcus and aureus technology, applied in the biological field, can solve the problems of poor infection protection effect, weak protective effect, and low yield of specific epidemic strains, and achieve the goals of improving vaccine safety, stable biological properties, and simplifying the purification process Effect

Active Publication Date: 2013-10-02
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation of pseudovirus vaccines is mostly carried out by mammalian cells, with low yield and high cost
[0006] US Schein CH et al. (BMC Bioinformatics.2012; 13Suppl13:S9) disclosed a method for preparing EDIII protein with consistent physical and chemical properties designed by computer for the protein sequence of 671 strains of DV envelope E protein III region (EDIII). Although the antibodies produced by protein immunization have a wide range of protection, their specificity is poor, and the protective effect against infection by specific epidemic strains may not be good, which is not conducive to popularization
[0007] The first dengue vaccine reported by French Sabchareon A et al. (Lancet.2012;380(9853):1559-67) - CYD-TDV recombinant attenuated tetravalent dengue virus vaccine can be immune to type 1, 3 and 4 dengue virus strains effect, but its overall effect is not satisfactory, and its protection against the type 2 dengue virus strain most common in Thailand is weak
[0008] Singapore Sim AC et al. (Vaccine.2008;26(9):1145-54) reported a recombinant Streptococcus lactis preparation expressing dengue virus EDIII, but the protective ability to mice of the same or different species varied greatly. The effects and immune pathways need to be further evaluated

Method used

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  • Staphylococcus aureus mutant strain, and preparation method and applications thereof
  • Staphylococcus aureus mutant strain, and preparation method and applications thereof
  • Staphylococcus aureus mutant strain, and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Construction of Staphylococcus aureus RN4220Δagr

[0071] The virulence expression of Staphylococcus aureus is strictly regulated, and the global regulatory factor (accessory gene regulator, agr) plays an important role in regulating the virulence of S. aureus, and the mutation or deletion of agr will lead to a decrease in virulence (Rudkin JK, et al. J Infect Dis, 2012;205:798–806). In order to improve the safety of vaccine preparation engineering bacteria, the inventor knocked out the agr gene of Staphylococcus aureus RN4220, the steps are as follows:

[0072]1. Construction of linear gene targeting fragments

[0073] (1) Primer design: According to the DNA sequence of agr in the genome sequence of Staphylococcus aureus RN4220 (download address: http: / / jb.asm.org / content / 193 / 9 / 2332 / suppl / DC1[Supplemental file1]) (Its sequence is shown in SEQ ID NO: 1) Design gene targeting left and right arm PCR primers, the base sequence is as follows:

[0074] Linear ...

Embodiment 2

[0108] Example 2 Identification of major antigens carried by membrane vesicles of Staphylococcus aureus RN4220Δagr

[0109] The membrane vesicles secreted by bacteria can carry various components of bacteria, including major antigens, LPS, DNA, etc., and the components carried by different bacteria are significantly different. Coccus RN4220Δagr membrane vesicles were prepared, and the protein antigens carried by the membrane vesicles were analyzed and identified;

[0110] 1. Culture of Staphylococcus aureus RN4220Δagr

[0111] Pick a single colony from the TSB solid plate, inoculate in 2ml of TSB medium, culture with shaking at 37°C for 20h, inoculate 300ml of fresh TSB medium at a ratio of 1:1000 the next day, and culture with shaking at 37°C, respectively, at 6h and 24h after culture Collect the culture supernatant.

[0112] 2. Preparation of Membrane Bubbles

[0113] (1) The collected bacterial culture supernatant was filtered with a 0.45 μm filter, and the obtained fi...

Embodiment 3

[0119] Example 3 Construction, screening and identification of Staphylococcus aureus RN4220Δagr / EDIII(+) mutant

[0120]Among the two main antigens carried by the membrane vesicles of Staphylococcus aureus RN4220Δagr identified by mass spectrometry, the pyruvate dehydrogenase β subunit (37kDa) encoded by the pdhB gene was adopted in Lee EY et al. (Proteomics2009;9:5425-36) The membrane vesicles produced by Staphylococcus aureus ATCC14458 identified by proteomic technology also contain them, so the pdhB gene was selected as the target gene for exogenous gene knock-in.

[0121] 1. Construction of linear knock-in fragments

[0122] (1) Primer design, see the corresponding position of each primer figure 1 Shown:

[0123] ① According to the DNA sequence of pdhB (its sequence is shown in SEQ ID NO: 3) in the genome of Staphylococcus aureus RN4220, design gene-targeting left and right arm PCR primers. The base sequences are as follows: respectively, as shown in SEQ ID NO: 17, 18 ...

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Abstract

The invention discloses a staphylococcus aureus mutant strain, and a preparation method and applications thereof in general. The preservation number of the mutant strain is CGMCC No.7583. The preparation method comprises following steps: performing gene knockout of agr gene, which is used for encoding a global regulatory factor (Agr) and is in staphylococcus aureus RN 4220 strain chromosomes, by application of homologous recombination; implanting a degenerate sequence used for encoding dengue virus envelope E protein EDIII region to 3' terminal of RN4220 pdhB gene, which is used for encoding pyruvate dehydrogenase beta-subnit ,by application of homologous recombination; implanting chloramphenicol resistant encoding gene at the back of EDIII; and then performing nucleotide sequencing for identification. The mutant strain of the invention is taken as engineering bacteria, and after fermentation, bacterial membrane vesicles containing dengue virus EDIII recombinant protein are purified from obtained fermentation supernatant. The dengue virus membrane vesicles produced by bacteria are used as a vaccine in prevention of infectious disease caused by dengue virus.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular, the invention relates to the construction of recombinant staphylococcus aureus containing dengue virus protective antigen gene by DNA recombination technology, and the method and application of using the recombinant bacteria to prepare dengue fever membrane vesicles. Background technique [0002] Dengue virus (DV) is a single-stranded positive-sense RNA virus of the Flaviviridae family, which is transmitted by mosquitoes. There are four serotypes of DV, which are called DV-1, DV-2, DV-3, and DV-4. After being infected with any of the serotypes, humans can produce protective immunity against this serotype virus, but the cross-protective immunity between different types is relatively short-lived, and non-neutralizing antibodies produced by virus infection can also mediate Antibody-dependent enhancement (ADE), leading to severe dengue hemorrhagic fever and dengue shock syndrome (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09A61K39/12A61P31/14C12R1/445
CPCY02A50/30
Inventor 饶贤才袁吉振杨杰胡珍胡晓梅陈炜
Owner ARMY MEDICAL UNIV
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