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Iba1 and LAG-3 dual-gene co-expression recombinant adenovirus vector and preparation method and application thereof

A technology of LAG-3 and recombinant adenovirus, which is applied in the field of preparation of the carrier, can solve the problems of hindering T cell delayed hypersensitivity, many influencing factors, and uneven gene copy of cells, so as to achieve good development and application prospects, inhibit Inflammation response, effect of improving nerve function

Inactive Publication Date: 2015-08-12
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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AI Technical Summary

Problems solved by technology

Furthermore, selective targeted activation of T cells using a LAG-3 toxic antibody blocks T-cell-involved delayed-type hypersensitivity
[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. It makes the experimental results difficult to evaluate and analyze; the above shortcomings limit the further application of this method

Method used

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  • Iba1 and LAG-3 dual-gene co-expression recombinant adenovirus vector and preparation method and application thereof
  • Iba1 and LAG-3 dual-gene co-expression recombinant adenovirus vector and preparation method and application thereof
  • Iba1 and LAG-3 dual-gene co-expression recombinant adenovirus vector and preparation method and application thereof

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Experimental program
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Embodiment 1

[0030] Embodiment 1, the preparation of recombinant adenovirus Ad-LAG-3 / Iba1

[0031] 1. Cloning the full-length cDNA of LAG-3

[0032] 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, and then the total cDNA was used as a template, and F1:5'-gg ctcgagg atgtgggaggctcagttcctg-3' (SEQ ID No.1, the underlined part is the Xho Ⅰ restriction site) and R1: 5'-gg gaattctcagagctgctccggctc-3' (SEQ ID No.2, the underlined part is the EcoR Ⅰ restriction site) was used as the upstream and downstream primers to amplify the full-length cDNA of LAG-3 by PCR. The PCR reaction system was 50 μl, and the cycle condition parameters were: Denaturation for 5 minutes; then denaturation at 94°C for 50 seconds, annealing at 58°C for 50 seconds, and extension at 72°C for 2 minutes, a total of 30 cycles; finally, extension at 72°C for 10 minutes. The PCR products were identified by agarose gel...

Embodiment 2

[0044] Example 2, Preparation of recombinant adenovirus Ad-LAG-3 / Iba1 transfected microglial cells

[0045] 1. Sorting of microglia

[0046] Microglial cells were cultured according to the literature method (Lehnart et al, J Neurosci, 2002; 22(7): 2478-2486, this method is a classic microglial cell culture method, widely used in research). The details are as follows: Take the forebrain tissue of C57BL / 6 mice with a reproductive age of 1-2 days, digest with insulin, grind at 37°C for 20 minutes, transfer the cells to DMEM medium containing calf serum for culture, and shake at 180 rpm for 30 minutes after 1 week. The cell suspension (containing >90% microglial cells) was transferred to an uncoated culture plate, and the non-adherent cell suspension was removed after 15 minutes, washed with PBS for 3 times, and the purity of the cells was identified by immunostaining (90% small glial cells) Glial cells).

[0047] 2. Recombinant adenovirus Ad-LAG-3 / Iba1 transfected microglial ce...

Embodiment 3

[0050] Example 3, Anti-proliferation detection of microglial cells transfected with recombinant adenovirus Ad-LAG-3 / Iba1

[0051] The microglial cells were randomly divided into two groups: the experimental group and the control group. The microglial cells in the experimental group were transfected with Ad-LAG-3 / Iba1 virus, and the control group were cultured normally.

[0052] 1. Detection of the proliferation ability of microglial cells

[0053] Adjust the cell concentration to 1×10 with RPMI 1640 medium containing 10% fetal bovine serum 6 cells / ml, inoculate into 96-well plate, 100 μl per well, set 3 duplicate wells for each group, set the temperature at 37°C, CO 2 Cultivate in an incubator with a gas volume fraction of 5% for 96 hours, add 100 μl of lysed red blood cell suspension, and incubate for 96 hours, then add 0.1ml to each well with a concentration of 1×10 3 μCi / L of 3H-thymidine (3H-TdR) solution, continue to culture for 12 hours, rinse the cells with PBS 3 time...

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Abstract

The invention discloses an Iba1 and LAG-3 dual-gene co-expression recombinant adenovirus vector and a preparation method and application thereof. The recombinant adenovirus vector comprises a dual-gene expression box for expressing Iba1 and LAG-3, wherein the dual-gene expression box sequentially consists of a CMV promoter, LAG-3 genes, an internal ribosome entry site IRES, Iba1 genes and a terminator. A recombinant adenovirus can be prepared by utilizing the recombinant adenovirus vector; after the prepared recombinant adenovirus is transfected to microglial cells, inflammatory reactions after cerebral hemorrhage can be inhibited, the neurological function of a mouse suffering from cerebral hemorrhage is effectively improved,and the lifetime of the mouse suffering from cerebral hemorrhage is prolonged; and the recombinant adenovirus vector can be used for preparing drugs resisting microglial cell activation and has good development and application prospects in the field of treatment of cerebrovascular diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant adenoviral vector for co-expression of Iba1 and LAG-3 double genes, and also relates to a preparation method and application of the vector. Background technique [0002] Intracerebral hemorrhage (ICH) is a common acute cerebrovascular disease with high morbidity, mortality and disability, and its incidence is increasing year by year in my country. Intracranial hematoma formation after ICH is the most important pathology leading to nerve injury. Inflammatory responses induced by the main components of the hematoma and erythrocyte lysates, metabolic abnormalities around the hematoma, and changes in local cerebral blood flow are the main reasons for the aggravation of secondary nerve injury. At present, it is believed that the inflammatory response induced by hematoma components plays an important role in secondary nerve injury and has attracted much attention...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N7/01A61K35/761A61P7/04A61P25/00A61P29/00
CPCY02A50/30
Inventor 杨曌石会李志伟谢鹏
Owner AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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