An exosome capable of targeting and blocking chemokine receptors and its preparation method and application

A chemokine receptor and exosome technology, applied in the field of cell biology, can solve the problems of poor efficacy and poor targeting of anti-inflammatory drugs, achieve inhibition of chemokine signals, reduce inflammatory cell infiltration, improve The effect of the local microenvironment

Active Publication Date: 2021-12-10
THE FIRST AFFILIATED HOSPITAL OF BENGBU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is that the existing anti-inflammatory drugs have poor curative effect and poor targeting

Method used

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  • An exosome capable of targeting and blocking chemokine receptors and its preparation method and application
  • An exosome capable of targeting and blocking chemokine receptors and its preparation method and application
  • An exosome capable of targeting and blocking chemokine receptors and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of Exo membrane component Lamp2b and broad-spectrum chemokine receptor binding peptide vMIP-II fusion gene expression vector vMIP-II-Lamp2b plasmid

[0044] Referring to the full-length RNA sequence of the chemokine receptor binding peptide vMIP-II in the NCBI gene bank (GeneID: 4961514), the nucleotide sequence encoding the polypeptide was designed, and its amino acid sequence is shown in SEQ ID No.1.

[0045] According to the full-length RNA sequence of the lysosome-associated membrane protein Lamp2b in the NCBI gene bank (GeneID: NM_010685.3), find out the sequence encoding the protein, and insert the nucleotide sequence encoding vMIP-II into the nucleotide encoding Lamp2b In the sequence, the specific position is located after 129 nucleotides downstream of the start codon of the nucleotide sequence of Lamp2b, thereby constituting the vMIP-II-Lamp2b fusion gene. The full sequence length of the fusion gene is 1008bp, and the tail end of the Lamp2...

Embodiment 2

[0069] Example 2 Establishment of a cell line stably expressing vMIP-II-Lamp2b fusion protein

[0070] Main experimental materials and instruments: plasmid extraction kit (Qiagen, cat.no.12263); tissue culture 6-well plate (Nunclon, cat.no.140685); biological safety cabinet (Thermo); cell culture box (Thermo) ; fluorescence microscope (Nikon); ABI PRISM7500 real-time PCR System; human embryonic kidney cells HEK293T; DMEM highglucose (GIBCO, cat.no.11995); FBS, heat inactivated (GIBCO, cat.no.26140-079); Glutamax (GIBCO, cat.no.35050); Penicillin-Streptomycin (GIBCO, cat.no.15140-122); Pancreatin-EDTA, 0.25% (GIBCO, cat.no.3197); Calcium-magnesium-free phosphate Buffer (GIBCO, cat.no.14190); Hank's balanced salt solution, 1× (GIBCO, cat.no.14025-092); HEPES (Sigma, cat.no.H4034); Opti-MEM (GIBCO, cat .no.31985); pCMV-dR8.9, purchased from Addgene; pCMV-VSV-G, purchased from Addgene.

[0071] HEK293T cells (ATCC No.CRL-11268) must be selected to be in the vigorous growth perio...

Embodiment 3

[0077] Example 3 qPCR identification of vMIP-II-Lamp2b gene overexpression effect

[0078] Main reagents and instruments: PureLink TM Genomic DNA Mini Kit (Invitrogen TM , K182001); 2XSG Fast qPCR master mix (BBI company, B639271); Thermo Fisher nanodrop one ultra-trace nucleic acid analyzer; Bio-Rad CFX96 fluorescence quantitative PCR instrument.

[0079] Collect cells, press PureLink TM Genomic DNA Mini Kit manual to extract cellular DNA. Measure the DNA concentration with a Thermo Fisher nanodrop one ultra-micro nucleic acid analyzer; configure the PCR reaction system according to Table 6. The PCR instrument was used for the reaction, and the cycle conditions were as follows: 95°C for 3min; 95°C for 3sec, 60°C for 30sec, 30 cycles; 4°C for storage. The primers used are listed in Table 7. Quantitative values ​​and analysis using 2 -ΔΔ Ct analysis. The result is as Figure 8 As shown, it indicates that the HEK293T cell line stably expressing vMIP-II-Lamp2b highly expr...

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Abstract

The invention belongs to the technical field of cell biology, and in particular relates to an exosome capable of targeting and blocking chemokine receptors and its preparation method and application. The technical problem to be solved by the present invention is that the existing anti-inflammatory drugs have poor curative effect and poor targeting. The present invention provides an exosome capable of targeting and blocking chemokine receptors, and expresses fusion expression of lysosome-associated membrane protein 2b (Lamp2b) and viral macrophage inflammatory protein II (vMIP‑II). The exosomes not only have the characteristics of ordinary Exo, but also target the inflammatory site rich in chemokine receptors through vMIP‑II, thereby inhibiting chemokine signaling, reducing inflammatory cell infiltration, and improving the local microenvironment. It is of great significance to the clinical treatment of central nervous system (CNS) inflammation-related diseases.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to an exosome capable of targeting and blocking chemokine receptors and its preparation method and application. Background technique [0002] Central nervous system (CNS) inflammatory diseases can be caused by infection, trauma, autoimmunity and other factors. Their common features are the activation and increase of local inflammation-related cells and the infiltration of peripheral inflammatory cells, leading to the increase of inflammatory factors, the formation of an inflammatory microenvironment, and ultimately organ dysfunction. Therefore, using appropriate methods to give anti-inflammatory therapy as early as possible and improve the local microenvironment of the central nervous system is an important strategy for the treatment of related diseases. [0003] At present, there are many basic and clinical studies on anti-inflammatory regimens, which can be summa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/65C12N15/62A61K48/00A61K38/19A61P25/00
CPCC12N5/0686C12N15/85C12N15/65C07K14/70596C07K14/522A61K48/0058A61K48/0008A61K38/195A61P25/00C12N2510/00C12N2800/107C12N2509/00C07K2319/00C07K2319/60
Inventor 吕合作胡建国李璟璐陈雨晴丁淑琴
Owner THE FIRST AFFILIATED HOSPITAL OF BENGBU MEDICAL COLLEGE
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