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DNA vaccine adjuvant using Micro RNA-155 and construction method thereof

A technology of nucleic acid vaccine and construction method, which is applied in the fields of medical biology and immune activity enhancement of nucleic acid vaccines, and can solve the problems of not using MicroRNA-155 plasmid carrier vaccine adjuvant research reports, etc.

Inactive Publication Date: 2012-12-19
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the existing nucleic acid vaccine adjuvants, there are no research reports and related patents using the MicroRNA-155 plasmid vector as a vaccine adjuvant

Method used

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  • DNA vaccine adjuvant using Micro RNA-155 and construction method thereof
  • DNA vaccine adjuvant using Micro RNA-155 and construction method thereof
  • DNA vaccine adjuvant using Micro RNA-155 and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Construction of pcDNA3.1-pre-miR-155 plasmid

[0027] We first searched the genome sequence encoding the human microRNA-155 precursor molecule (GENBANK: NR_030784, shown in SEQ ID NO: 1) according to the Internet database information (http: / / www.mirbase.org), and designed PCR for its flanking sequence Primers (BamH I and EcoR I restriction sites are added to the upstream and downstream, respectively), using genomic DNA extracted from human peripheral blood mononuclear cells for PCR, the microRNA-155 precursor molecular sequence and PCR primers for cloning are shown in the table below. Thus, a DNA fragment containing the coding sequence of the microRNA-155 precursor molecule was obtained.

[0028]

[0029] The DNA fragment comprising the pre-miR-155 coding sequence of the above clone (such as figure 1 and the following sequences), using BamH I and EcoR I restriction sites. The polyclonal restriction sites flanking the insertion sequence are still retai...

Embodiment 2

[0031] Example 2: In vitro expression verification of pcDNA3.1-pre-miR-155 plasmid

[0032] Using 293T cells (purchased from ATCC, USA) as an in vitro model, cells were transfected with pcDNA3.1-pre-miR-155 plasmid (pMiR-155) using Lipofectamine2000 transfection reagent (Invitrogene, USA, refer to its instruction manual). Set up pcDNA3.1 (pcDNA) empty plasmid transfection group and untreated group (Untreated) as controls. After 24 hours, the total cellular RNA was extracted with Trizol reagent (Invitrogene, USA, refer to its instruction manual), and the microRNA real-time quantitative detection kit of Guangzhou Funeng Gene Co., Ltd. was used to detect intracellular has -expression level of microRNA-155 mature body, it was found that the expression level of has-microRNA-155 mature body increased by more than 1000 times after transfection of pcDNA3.1-pre-miR-155 plasmid (such as figure 2 shown in A).

Embodiment 3

[0033] Example 3: Detection of expression ability in vitro under the condition of co-transfection of pcDNA3.1-pre-miR-155 plasmid and pVAX1-HBsAg nucleic acid vaccine.

[0034] Using 293T cells as an in vitro model, use Lipofectamine2000 transfection reagent z to transfect the following three groups of plasmids, 1. pVAX1-HBsAg and pcDNA3.1 empty plasmid group (pHBs+pcDNA) transfection; 2. pcDNA3.1-pre-miR- 155 plasmids were transfected with pVAX1-HBsAg (pHBs+pMiR-155); 3. pVAX1-HBsAg was transfected alone (pHBs). After 24 hours, the expression level of HBsAg antigen in the cell culture supernatant was detected by double antibody sandwich ELISA method, and it was found that after co-transfection of pcDNA3.1-pre-miR-155 plasmid and pVAX1-HBsAg, pVAX1-HBsAg could still express HBsAg efficiently Antigen, comparable to the expression level of pVAX1-HBsAg (eg figure 2 shown in B).

[0035] The cells treated in the above three groups were extracted with Trizol reagent from America...

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Abstract

The invention relates to the technical field of medical biology. Micro RNA-155 is micro ribonucleic acid (RNA) for regulating the maturation and natural immunoreaction of T cells and B cells. The invention provides a micro RNA-155-based plasmid for enhancing the immune effect of deoxyribonucleic acid (DNA) vaccine cells. A plasmid pcDNA 3.1-pre-miR-155 is obtained by inserting an expression sequence of a micro RNA-155 precursor molecule into a pcDNA-3.1 plasmid, and the micro RNA-155 precursor molecules can be expressed efficiently without influencing the expression capability of the co-transfected DNA vaccine. In an in-vivo test, the plasmid and the DNA vaccine are injected at the same time and have the capability of effectively introducing cellular immunoreaction, and such capability isrepresented by the increase of antigenically specific spleen IFN-gamma positive cells and enhancement of target killing capability of spleen CD8 positive cells. The plasmid of the invention can be added into the conventional DNA vaccine preparation and injected together so as to obviously promote the cellular immunoreaction induced by the DNA vaccine, and serves as a novel cell immune adjuvant.

Description

technical field [0001] The invention relates to the technical field of medical biology, in particular to the technical field of enhancing the immune activity of nucleic acid vaccines. Background technique [0002] MicroRNAs are abundant endogenous non-coding RNAs that regulate the expression of targeted mRNAs through defective base pairing in the 3′UTR region during transcription, thereby regulating mRNA cleavage and transcriptional repression. (Kato, M. and Slack, F.J. (2008) microRNAs: small molecules with big roles-C.elegans to human cancer. Biol. Cell, 100, 71-81.) MicroRNAs mainly regulate many biological processes, including innate immune response differentiation and activation of many cells in the (Garzon, R. and Croce, C.M. (2008) MicroRNAs in normal and malignant hematopoiesis. Curr. Opin. Hematol., 15, 352-358.) One of the typical MicroRNAs: MicroRNA-155 is a regulatory T cell and Small RNAs for B cell maturation and innate immune responses. (Rodriguez, A., Vigo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/39A61K48/00C12N15/12
Inventor 孙树汉王越唐樱歌郭瀛军章意亮周奇
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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