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Construction method and application of bacterial biofilm vesicles (BBV) as vaccine vectors

A technology of bacterial biofilm and construction method, which is applied in the field of construction of bacterial biofilm vesicles as vaccine carriers, can solve the problems of low OMV yield and modification efficiency, difficult membrane assembly technology, and limit the development of bacterial membrane carrier technology, and achieve improved Level of cellular immune response, promotes uptake and maturation, improves safety

Active Publication Date: 2021-04-16
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, bacterial biofilm contains more outer membrane proteins, polysaccharides, peptidoglycan, etc., which brings difficulties to membrane assembly technology. At present, the assembly technology of bacterial biofilm still stays on the basis of using OMV released by bacteria itself, such as using OMV and Tumor cell fusion, using OMV including gold particles, using OMV to carry drugs, etc., but the yield and modification efficiency of OMV are low, which seriously limits the development of bacterial membrane carrier technology

Method used

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  • Construction method and application of bacterial biofilm vesicles (BBV) as vaccine vectors
  • Construction method and application of bacterial biofilm vesicles (BBV) as vaccine vectors
  • Construction method and application of bacterial biofilm vesicles (BBV) as vaccine vectors

Examples

Experimental program
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Effect test

Embodiment 1

[0053] In this example, bacterial biofilm vesicles (BBV) are used as a vaccine carrier to construct a new coronavirus vaccine. The specific method is:

[0054] The DNA sequence encoding the recombinant protein containing SARS-CoV-2 RBD domain Asn331-Val524 (YP_009724390) and ClyA protein (AAL55667) was directly synthesized, and the RBD gene was connected to the 3' end of the ClyA gene by genetic engineering technology and cloned into the pThioHisA plasmid , positive plasmids were subsequently confirmed by restriction endonuclease analysis and sequencing. Transform the recombinant plasmid into E.coli BL21 and culture it in LB medium. When the OD600 of the bacterial solution reaches 0.4-0.6, add 1mmol / L isopropyl β-D-1-thiogalactopyranoside (IPTG , Solarbio), induced the expression of the recombinant protein ClyA-RBD at 30°C overnight; added 2mM EDTA·2Na the next day, continued to cultivate for 2h; centrifuged the culture medium at 10000rpm at 4°C for 30min, and collected the ba...

Embodiment 2

[0060] Embodiment 2——The structure and load capacity of RBD-BBV

[0061] This embodiment compares the genetic modification technology based on BBV and OMV respectively of embodiment 1 and comparative example 1, and sample is stained with rapid silver staining kit (Beyotime), analyzes RBD-BBV (iodixanol density gradient) with Image Lab Before centrifugal purification), RBD load in RBD-OMV; use Bradford kit (Sangon Biotech) to analyze BBV output; as figure 2 The results shown prove that the loading of RBD on BBV is significantly higher than that of OMV, the arrow in the figure marks the position of RBD protein, and the loading of RBD is about 28.16 times higher than that of OMV ( image 3 ), in addition, the yield of RBD-BBV was about 107 times higher than that of RBD-OMV ( Figure 4 ). More importantly, no bacterial nucleic acid components were detected in RBD-BBV, which greatly improved the safety of the vaccine.

[0062] In a word, the genetic engineering modification tec...

Embodiment 3

[0065] Example 3 - Evaluation of RBD-BBV as a vaccine

[0066] 1. RBD-BBV promotes the uptake and maturation of DC cells, and also mediates the lysosomal escape of RBD

[0067] Efficient uptake of antigens by DC cells is the key to the success of the vaccine. Experiments have found that both RBD-BBV and BBV can be efficiently taken up by DC cells ( Figure 10 ), we also see that both RBD-BBV and BBV can effectively stimulate the maturation of DC cells, such as Figure 11 As shown, the specific performance is the high expression of CD11c+CD80+ and CD11c+CD86+ on DC cells. Such as Figure 12 , RBD-BBV and BBV can also stimulate the secretion of DC inflammatory factors IL-6 and IL-1β. In summary, we observed that RBD-BBV and BBV have the same characteristics in DC cell uptake and stimulation maturation, indicating that these effects originate from the nanostructure of BBV, as well as the stimulation and targeting of PAMPs on BBV to DC cells. It is crucial to identify BBV as a...

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Abstract

The invention provides a construction method and application of bacterial biofilm vesicles (BBV) as a vaccine vector, an engineering modified bacterial biofilm is driven to complete high-efficiency self-assembly by utilizing ultrahigh pressure for the first time, the ability of ClyA to assemble pores on a bacterial outer film is used for carrying RBD to construct burred bacterial vesicles (RBD-BBV) in a prokaryotic system, and the RBD-BBV efficiently exposes the correctly folded RBD on the surface of the vesicles and shows the burred vesicle structure. The RBD-BBV can be efficiently taken up by DC cells and stimulates the maturation of DC cells, and meanwhile, the bacterial biofilm mediates the lysosome escape of the RBD. After RBD-BBV immunization of mice, SARS-COV-2 specific neutralizing antibodies and SARS-COV-2 specific CD4+ T and CD8+ T cell responses are induced in the mice. The RBD-BBV also enhances the local stability of the antigen in vivo and induces the generation of memory T cells. The RBDBBV vaccine form disclosed by the invention has the unique characteristics that many other vaccine forms cannot be realized, and a new thought can be possibly provided for the development of SARSCoV2 vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a construction method and application of a bacterial biofilm vesicle (BBV) as a vaccine carrier. Background technique [0002] SARS-CoV-2 is a new type of coronavirus with an obvious spike structure on its surface. The RBD on the spike protein can bind to the ACE2 of the recipient cell, thereby mediating the invasion of the virus. Although some drugs have been found to treat COVID-19 in experimental animal models, the development of SARS-CoV-2 vaccines has placed more expectations than therapeutic drugs. At present, SARS-CoV-2 vaccines such as inactivated vaccines, mRNA vaccines, subunit vaccines, and viral vector vaccines have entered clinical trials. However, the current form of vaccine has many restrictive problems such as high production cost, low yield, and difficult transportation. [0003] RBD is considered to be an effective vaccine target, and vaccines targeting RBD have b...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C12N1/21A61K39/215A61P31/14A61K47/46A61K39/39A61P37/04C12R1/19
Inventor 黄惟巍马雁冰杨忠倩李维冉华良群张启书龙琼白红妹杨旭孙文佳
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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