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Multi-tissue restriction type coxsackie group B type III virus inserted with miRNA complementary sequence and application thereof

A technology of complementary sequence and insertion sequence, which is applied in the field of medicine and biology, can solve the problems of virus replication, damage to the body, and reduction, and achieve the effect of avoiding fatal damage and reducing recovery pressure

Pending Publication Date: 2021-10-22
首都儿科研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in addition to myocardial tissue damage in patients with viral myocarditis, CVB3 infection can also cause a large number of viruses to replicate in the pancreas, brain tissue, etc., leading to pancreatitis, meningitis, and serious damage to the body.
We also found that after myocardial-restricted CVB3 infected mice, the virus titer in the heart was significantly lower than that of the control group, but the pancreas still had a large number of virus replication, which indicated that our construction of myocardial-restricted CVB3 still had limitations

Method used

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  • Multi-tissue restriction type coxsackie group B type III virus inserted with miRNA complementary sequence and application thereof
  • Multi-tissue restriction type coxsackie group B type III virus inserted with miRNA complementary sequence and application thereof
  • Multi-tissue restriction type coxsackie group B type III virus inserted with miRNA complementary sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Construction of CVB-miRT*3.

[0043] 1. The source and sequence structure of CVB3-WT strain

[0044] The virus used in the present invention is CVB3m strain (GenBank: M33854.1).

[0045] 2. CVB-miRT*3 insertion sequence (complementary sequence structure of 3 miRNAs), insertion position, construction method

[0046] The plasmid carrying the sequence of CVB3m strain was constructed by reverse genetics method. According to the tissue specificity of microRNA, we selected miR-206, miRNA-29a-3p, and miRNA-124-3p with muscle, pancreas, and brain tissue specificity. According to databases and literature reports, the sequences of these three miRNAs are completely consistent between humans and mice. Mature miRNAs with core sequences were selected in the miRbase database.

[0047]

[0048]

[0049] A synthetic insert sequence was constructed, as shown in SEQ ID NO.1 below:

[0050] 5'CCACACACTTCCTTACATTCCACGATTAACCGATTTCAGATGGTGCTAACCGGTGGCATTCACCGCGTGCCTTA ...

Embodiment 2

[0055] Example 2. Functional verification of CVB-miRT*3 by in vitro experiments

[0056] Firstly, the transfection of the modified plasmid and the quantitative experiment of virus production were carried out, and the amount of virus produced at different time points after transfection with different concentrations of plasmids was measured by using the plaque formation experiment. The quantification curve was made according to the calculation of PFU / ml, which became the key point of this experiment. Positive quality control.

[0057] Synthesize miRNA-206, miRNA-29a-3p, miRNA-124-3p in vitro, observe the effect and dose-effect relationship of exogenous miRNA on CVB-miRT*3 specificity in HeLa cells: set the concentration of 100nM to synthesize the above miRNA and the corresponding control miRNAcon were transfected into HeLa cells separately or mixed, and the miRNA-con control transfection group was set at the same time. After 4 hours, CVB-miRT*3 was applied, MOI=1.0, and the prot...

Embodiment 3

[0065] Example 3. Functional verification of CVB-miRT*3 by in vivo experiments

[0066] 6-8 week-old BALB / c male mice (18-22 g) were to be purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences. (The animal experiment protocol passed the animal ethics review of the Capital Institute of Pediatrics). The mice were randomly divided into 4 groups with 15 mice in each group.

[0067] (1) To assess the virulence of the virus in combination with the death rate of the animals after inoculation:

[0068] Press 1*10 4 , 1*10 5 , 1*10 6 , 1*10 7 , 1*10 8 , 1*10 9 Virus titer of PFU / mouse The mice were inoculated intraperitoneally, and the median lethal dose (LD50) of the virus to mice was calculated according to the method of Reed-Muench.

[0069] (2) Assess the pathogenic pathology of modified CVB3:

[0070] ① Mice were grouped and intraperitoneally injected according to the above, and the inoculation amount was the half-lethal dose (LD50) of ...

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Abstract

The invention relates to a multi-tissue restriction type coxsackie group B type III virus inserted with a miRNA complementary sequence and an application thereof. The miRNA comprises the following components: miR-206, miRNA-29a-3p, and miRNA-124-3p. The application means that the multi-tissue restricted coxsackie group B type III virus can be used for preparing heart, pancreas and brain tissue restricted vaccines.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and in particular relates to a multi-tissue-restricted Coxsackie B group III virus inserted with a miRNA complementary sequence and an application thereof. Background technique [0002] Coxsackie B group virus (CVB) has been identified as the main pathogen causing severe acute and chronic myocarditis, pancreatitis, encephalitis and other related diseases, among which Coxsackie B group III virus (CVB3) is common pathogenic microorganisms. The massive replication of the virus in the target organ will directly lyse the cells, and at the same time cause severe immune inflammatory damage in the damaged area. For example, activated T cells will destroy virus-infected cells through cell-mediated cytotoxicity, and cytokines in The generation, chemotaxis and infiltration of the lesion will aggravate the damage to the target organ. Therefore, effectively inhibiting virus replication in targe...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/113A61K39/125A61P31/14
CPCC12N7/00C12N15/113A61K39/12A61P31/14C12N2310/141C12N2770/32021C12N2770/32034
Inventor 何峰肖宗慧姚海兰刘哲伟吴建新冯淼刘卓欧阳胜荣李媛媛
Owner 首都儿科研究所
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