Expression vector of fusion protein for enhancing target RNA loading into exosome and establishment method and application thereof

An expression vector and fusion protein technology, applied in the field of molecular biology, can solve the problem of lack of effective means, and achieve the effect of reducing undesired RNA entry into exosomes, efficient loading, and reducing loading

Inactive Publication Date: 2018-11-06
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of effective means for how to efficiently load targ

Method used

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  • Expression vector of fusion protein for enhancing target RNA loading into exosome and establishment method and application thereof
  • Expression vector of fusion protein for enhancing target RNA loading into exosome and establishment method and application thereof
  • Expression vector of fusion protein for enhancing target RNA loading into exosome and establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Construction of RNA-binding protein exosomes

[0048] The specific steps to build are as follows:

[0049] (1) Trizol extracts total RNA from mouse fibroblasts;

[0050] (2) Using Promega M-MLV reverse transcriptase to reverse transcribe RNA to obtain mouse fibroblast cDNA;

[0051] The reaction system is shown in Table 1:

[0052] Table 1 reaction system

[0053] RNA template

2μg

5×RT buffer

4μl

dNTP Mixture (10mM)

2μl

0.1M DTT

2μl

M-MLV reverse transcriptase

1μl

overall system

20μl

[0054] Mix the above reagents evenly, centrifuge at low speed for a short time, place in a 37°C incubator for 1 hour, then inactivate at 80°C for 5 minutes, and store at -20°C.

[0055] (3) Using the reverse transcription cDNA as a template, clone the CDS region of the RNA binding protein HuR and the CDS region of the membrane molecule CD9;

[0056] The primers and PCR reaction system are shown in Tab...

Embodiment 2

[0074] Example 2: Verification test of physicochemical properties of modified exosomes

[0075] like Figure 4 As shown, the viral packaging cell HEK293T was infected with the virus expressing the fusion protein CD9-HuR constructed in Example 1 to obtain virus particles expressing the fusion protein. Bone marrow mesenchymal cells MSC were infected with the virus, and then cultured under serum-free conditions, exosomes were collected between 48 and 72 hours, and exosomes were extracted through the exosome isolation and extraction kit ExoQuick. Identify the number, size, and fusion expression efficiency of exosomes.

[0076] like Figure 5 As shown, the collected exosomes were diluted with PBS at 1:1000, and then the number and size of exosomes were identified by Nanosight, and it was found that the size of exosomes was within the range of 40-200nm.

[0077] like Image 6 As shown, the shape of the exosomes was further clarified by electron microscopy, and the modified exoso...

Embodiment 4

[0079] Example 4: Nucleic acid loading test of modified exosomes

[0080] Infect the exosome packaging cells with the constructed virus expressing the fusion protein CD9-HuR, then transfect the exosome packaging cells with miR155 or dCas9-ARE to be loaded, and then culture them under serum-free conditions for 48 to 72 hours. Exosomes were collected in between, and exosomes were extracted by exosome separation and extraction method.

[0081] (1) Loading and delivery of miR155

[0082] miR155 was directly synthesized.

[0083] The constructed virus expressing the fusion protein CD9-HuR was used to infect the exosome packaging cells, and then the exosome packaging cells were simultaneously transfected with miR155 to be loaded, then cultured under serum-free conditions, and the exosome packaging cells were collected between 48 and 72 hours. Exosomes are extracted by exosome isolation and extraction methods.

[0084] The loading, delivery and intervention efficiency of exosomes ...

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Abstract

The invention discloses an expression vector of a fusion protein for enhancing target RNA loading into an exosome and an establishment method and application thereof. The expression vector comprises atarget RNA binding protein sequence and an exosome membrane targeting protein sequence. The target RNA binding protein sequence comprises an RNA binding protein complete sequence or an RNA binding structural domain sequence. The exosome membrane targeting protein sequence can be localized in the exosome. In addition, the establishment method of the expression vector of the fusion protein for enhancing target RNA loading into the exosome is designed. The exosome containing the expression vector of the fusion protein for enhancing target RNA loading is prepared. The expression vector or the exosome is applied to targeting delivery of gene treatment and RNA regulated relevant disease treatment. Efficient candidate RNA molecule loading can be achieved, and meanwhile cellular endogenous RNA loading of the exosome is decreased. The expression vector can serve as an optimized vector for targeting delivery of gene treatment.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an expression vector for a fusion protein that enhances loading of target RNA into exosomes and its construction method and application. Background technique [0002] Exosomes refer to small membrane vesicles containing complex RNA and proteins, which are mainly derived from the multivesicular bodies formed by the invagination of intracellular lysosomal particles, and are released into the extracellular matrix after the fusion of the multivesicular outer membrane and the cell membrane middle. CD9 is a four-time transmembrane molecule, which is the hallmark membrane molecule of exosomes. At present, many studies have reported that the tracking of exosomes can be achieved by modifying the CD9 molecule, such as by fusing with EGFP, and the targeting of exosomes can be enhanced by fusing the extracellular segment of CD9 with a targeting peptide. [0003] RNA-binding prote...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/85A61K48/00
CPCA61K48/00C12N15/62C12N15/85
Inventor 袁丽君李者龙杨国栋周雪莹赵联璧韦梦影孙汶齐
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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