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Double-gene recombinant complex macrophage vaccine and preparation method and application thereof

A macrophage and complex technology, which is applied in the field of vaccines prepared by the same and its preparation, and dual-gene recombination complexes, can solve the problems of many influencing factors, unfavorable target gene expression and subsequent treatment, difficult evaluation and analysis of experimental results, and the like, Achieve the effect of inhibiting inflammatory response and brain edema, good development and application prospects, and improving neurological function scores

Pending Publication Date: 2019-06-28
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0005] There are two main methods of gene combination therapy: one is to simultaneously transfect target cells with a variety of recombinant expression vectors carrying different genes. The construction of recombinant expression vectors is cumbersome, time-consuming, and has many influencing factors. In addition, due to the randomness of the transfection process, the transfected cells have the problem of uneven gene copies, which is not conducive to the expression of the target gene and subsequent treatment. The experimental results are difficult to evaluate and analyze, so the above shortcomings limit the further application of this method

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  • Double-gene recombinant complex macrophage vaccine and preparation method and application thereof
  • Double-gene recombinant complex macrophage vaccine and preparation method and application thereof
  • Double-gene recombinant complex macrophage vaccine and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] The preparation of embodiment 1 double gene recombination compound

[0044] 1.1 Cloning of IRAK2RNAi

[0045] According to the instructions of the Trizol kit, the total RNA of human liver tissue was extracted, and the total cDNA was obtained by reverse transcription. Then, using the total cDNA as a template, F1:5'-aat ctgcag acttttcgcccgcacagagcgg-3' (SEQ ID No.3, the underlined part is the restriction site of Bgl1) and R1: 5'-tac gaattc aagcgcgtgcacgaggaggtcacg-3' (SEQ ID No.4, the underlined part is the restriction site of Apo1) is the upstream and downstream primers to amplify the full-length RNAi of IRAK2 by PCR. ; Then denature at 94°C for 45 seconds, anneal at 58°C for 60 seconds, and extend at 72°C for 1 minute, a total of 30 cycles; finally extend at 72°C for 5 minutes. The PCR product was identified by agarose gel electrophoresis, recovered and purified by gel extraction kit, and ligated with pT-Easy vector. The ligated product was transformed into Escheric...

Embodiment 2

[0060] Example 2 Preparation of double-gene recombinant complex transfection macrophage vaccine

[0061] 2.1. Sorting of macrophages

[0062] C57BL / 6 normal mice were taken, killed by neck dislocation, and routinely disinfected with 75% alcohol. Inject 5 mL of serum-free RPMI 1640 medium into the abdominal cavity of mice, rub the abdomen repeatedly for 5 minutes, let it stand for 3 minutes, open the abdominal cavity, extract the peritoneal fluid, centrifuge, detect the cell density, and plant it in a 6-well plate for 3 hours to remove unattached cells , add medium and cytokines: IFN-γ 10ng / mL, LPS 100ng / mL; M2 macrophages: IL-4 10ng / mL) to stimulate culture for 24h.

[0063] 2.2. Double-gene recombination complex transfected macrophages

[0064] Inoculate macrophages in a 6-well plate at a cell concentration of 1×106 / ml, add the fourth-generation double-gene recombination complex at a multiplicity of infection (MOI) of 100, collect cells 48 hours after infection, wash with P...

Embodiment 3

[0068] Example 3 Detection of Cerebral Edema in Mice with Cerebral Hemorrhage

[0069] 25 μl of autologous whole blood was injected into the basal ganglia of mice, and 10 minutes later, 1 mg of double-gene recombinant complex macrophage vaccine or control (PBS) was injected at the same site. After 3 days, the mice were sacrificed, the brain tissues were obtained, and the cerebral edema of the mice was detected by dry and wet weight methods. Research results such as Figure 4 As shown, compared with the control (PBS), the double-gene recombinant complex macrophage vaccine can significantly reduce brain edema in mice with cerebral hemorrhage.

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Abstract

The invention belongs to the field of biological medicines, and particularly relates to a double-gene recombinant complex, a vaccine prepared from the same, and a preparation method and application ofthe complex and the vaccine. The recombinant complex is composed of a polypeptide complex and a double-gene expression cassette, and the portion from the upstream to the downstream of the double-geneexpression cassette is sequentially provided with a GFAP promoter, an IRAK2 RNAi gene, a terminator, an internal ribosome entry site IRES, an SMURF1 gene and a terminator. The polypeptide complex isformed by binding cell-penetrating peptide and a positive charge polypeptide. The double-gene recombinant complex macrophage vaccine can be used for transfecting hematoma surrounding tissue of cerebral hemorrhage in vitro, the activity of macrophages can be inhibited, inflammatory reaction and brain edema of cerebral hemorrhage are effectively inhibited, the neurological score is improved, and anideal strategy is provided for treatment of cerebral hemorrhage. The preparation method is simple and low in cost and has good development and application prospects in the field of cerebral hemorrhagetreatment.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a double-gene recombination compound, a vaccine prepared by it, a preparation method and application thereof. Background technique [0002] Intracerebral hemorrhage (ICH) is a serious injury of the central nervous system, which has a high rate of disability and mortality. Brain injury after cerebral hemorrhage includes primary injury and secondary injury caused by hematoma expansion and increased intracranial pressure. Inflammation plays an important role in secondary injury after cerebral hemorrhage, and its specific mechanism involves activity of macrophages infiltration of inflammatory cells and release of cytokines and inflammatory chemokines, ultimately leading to neuronal death . Macrophages are important innate immune cells and antigen-presenting cells. According to the phenotype of macrophages, macrophages are usually divided into M1 macrophages of the classical...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/85C12N5/10A61K39/00A61P9/10A61P7/04A61P7/10
Inventor 赵旺
Owner AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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