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Recombinant staphylococcus aureus for preparing bacterial vesicle multi-combined vaccine as well as preparation method and application of recombinant staphylococcus aureus

A staphylococcus and multiple vaccine technology, applied in the field of biomedicine, can solve the problems of complex preparation procedure, pathogenicity and high cost, and achieve the effects of improving preparation efficiency, simplifying purification process and improving safety

Pending Publication Date: 2022-05-10
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some of these multiple vaccine preparation processes are to prepare each vaccine according to the standard process, and then mix them in a certain ratio; risk of pathogenicity

Method used

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  • Recombinant staphylococcus aureus for preparing bacterial vesicle multi-combined vaccine as well as preparation method and application of recombinant staphylococcus aureus
  • Recombinant staphylococcus aureus for preparing bacterial vesicle multi-combined vaccine as well as preparation method and application of recombinant staphylococcus aureus
  • Recombinant staphylococcus aureus for preparing bacterial vesicle multi-combined vaccine as well as preparation method and application of recombinant staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Knockout and identification of agrA gene in Staphylococcus aureus

[0077]S. aureus is the first Gram-positive bacterium that has been confirmed to produce MVs (Lee et al., Proteomics, 2009; 9:5425–36), but the MVs produced by wild-type S. aureus are virulent and affect MVs Safety of use (Yuan et al., Nano Lett, 2018; 18:725-33), this application knocks out the agrA gene of the quorum sensing system in Staphylococcus aureus RN4220 to construct a safe strain of Staphylococcus aureus;

[0078] 1. Construction of knockout vector

[0079] The agrA gene sequence on the genome of Staphylococcus aureus RN4220 strain was analyzed as SEQ ID NO: 6, and the amino acid sequence of the AgrA protein was SEQ ID NO: 1. Design primers at about 900bp upstream and downstream of the knockout sequence site. For the left homology arm amplification primers, see the sequence listing SEQ ID NO: 19 (primer P1) and 20 (primer P2), and for the right homology arm amplification primers,...

Embodiment 2

[0095] Example 2 Virulence Detection of Membrane Vesicles of Staphylococcus aureus RN4220-ΔagrA Knockout Strain

[0096] The quorum sensing system Agr is an important virulence regulation system of S. aureus, which controls the expression of hundreds of virulence factors. The loss of this system function will lead to a significant decrease in the virulence of the strain (Reye et al., JBacteriol, 2011; 193: 6020-31); In order to observe the safety performance of bacterial membrane vesicles after agrA knockout, the applicant prepared membrane vesicles of RN4220-ΔagrA knockout strain and its wild strain, and used Balb / c mouse animal experiment to detect the toxicity of membrane vesicles. To verify the safety of membrane vesicles of RN4220-ΔagrA knockout strain.

[0097] 1. Cultivation of Staphylococcus aureus RN4220 and RN4220-ΔagrA

[0098] Pick a single colony from the BHI solid plate, inoculate it in 3ml of BHI liquid medium, culture it with shaking at 37°C for 18 hours, in...

Embodiment 3

[0105] Example 3 Construction of Staphylococcus aureus RN4220-ΔagrA / lcrV engineering bacteria

[0106] It has been reported that Eno of Staphylococcus aureus can be presented in membrane vesicles (Yuan et al., Nano Lett, 2018; 18:725-33). This application uses enolase Eno (48kDa) as the fusion target molecule in Staphylococcus aureus, The fusion protein of Yersinia pestis protective antigen LcrV and Eno was constructed by genetic engineering technology, and the fusion protein was presented in MVs by using the membrane vesicle localization function of Eno.

[0107] 1. Selection of LcrV molecules

[0108] Yersinia pestis (Yersinia pestis) is a bacillus in the genus Yersinia. It is the pathogen of bubonic plague, pneumonic plague and septicemic plague. Deadly disease; the main clinical manifestations are high fever, swollen and painful lymph nodes, bleeding tendency, special inflammation of the lungs, etc.; it has been recorded as far back as more than 2,000 years ago, and three...

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Abstract

The invention provides a recombinant staphylococcus aureus for preparing a bacterial vesicle multi-combined vaccine, the agr system function of the recombinant staphylococcus aureus is inactivated, and the recombinant staphylococcus aureus contains two or more than two antigen peptide fragment coding genes from heterologous pathogens. The antigen peptide fragment coding genes are respectively inserted into a coding gene of a staphylococcus aureus fusion target molecule to obtain a fusion protein coding sequence, the staphylococcus aureus fusion target molecule is selected from staphylococcus aureus protein presented on vesicles, and the generated vesicles can present antigen peptide fragments from two or more than two heterologous pathogens. The invention also provides a preparation method and application of the compound. The safe staphylococcus aureus provided by the invention can be used for preparing a bacterial vesicle multi-combined vaccine, and has important practical significance for preventing infection of pathogens corresponding to exogenous target molecules.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a bacterial membrane vesicle multiple vaccine and a preparation method thereof. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, referred to as Staphylococcus aureus) is a Gram-positive coccus, often arranged in a grape shape, and widely distributed in nature. Korean researchers took the lead in confirming that Staphylococcus aureus can produce and secrete bacterial membrane vesicles (MVs) (Lee et al., Proteomics, 2009; 9:5425-36). [0003] MVs are a membranous structure naturally produced by bacteria during their growth and reproduction and secreted into the extracellular environment, with a diameter of 20-400nm; the secreted MVs can carry various important antigens from bacteria, including engineered fusion proteins Composition (Qiao et al., Front Microbiol, 2021; 12:729369). Genetic engineering technology can be used to fuse the protective exog...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/62C12N15/74A61K39/116A61K39/085A61K39/02A61P31/04C12R1/445
CPCC07K14/31C07K14/24C07K14/195C12N9/88C12N9/0008C12N15/74A61K39/085A61K39/0291A61K39/0208A61P31/04C12Y402/01011C12Y102/01051C07K2319/00A61K2039/70
Inventor 饶贤才朱柯亭陈娟胡珍周人杰尚伟龙杨裔饶一凡彭华刚胡启文王玉亭
Owner ARMY MEDICAL UNIV
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