Applications of TRAIL truncated mutant in activating NF-kappaB and in inflammatory responses

A truncated and variant technology, applied to medical preparations containing active ingredients, anti-inflammatory agents, non-central analgesics, etc., can solve the problem of reduced ability of BX439 to activate NF-κB

Inactive Publication Date: 2013-02-06
SINOGENOMAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Among the TRAIL variants, the only difference between BX439 and BX424 is that BX439 lacks exon 2 (138 nucleotides), but the ability of BX439 to activate NF-κB is greatly reduced when overexpressed, indicating that the exon 2 encoded product is essential for activation NF-κB is critical

Method used

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  • Applications of TRAIL truncated mutant in activating NF-kappaB and in inflammatory responses
  • Applications of TRAIL truncated mutant in activating NF-kappaB and in inflammatory responses
  • Applications of TRAIL truncated mutant in activating NF-kappaB and in inflammatory responses

Examples

Experimental program
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Embodiment 1

[0027] Structural analysis and protein sequence alignment of embodiment 1, 7 truncated TRAIL variants

[0028] Using EST database analysis, combined with UCSC genome browser viewing, we mapped the gene structure of 7 truncated TRAIL variants. The protein sequences of the seven truncated TRAIL variants were compared with those of RefSeq using bioinformatics software such as DNAstar and DNAMAN.

[0029] The results are shown in Figure 1, in which 1A shows that the intron boundaries spanned by all these variant sequences conform to the GT / AG rule, where AK (encoding 65 amino acids) and DA (encoding 98 amino acids) are in the first and DA lacks exon 2, and contains a new exon between exons 4 and 5. E2 (coding 88 amino acids), E3 (coding 123 amino acids) and E4 (coding 145 amino acids) were extended on the original exons 2, 3, and 4, respectively. BX424 (encoding 101 amino acids) lacks exons 3 and 4. BX439 lacks exons 2, 3, and 4. Black squares represent known exons, while gray...

Embodiment 2

[0030] Example 2, Expression of 7 truncated TRAIL variants in different cell lines

[0031] Eight kinds of cells (Raji, Jurkat, THP-1, HCT116, BT-325, HepG2, PC12, 293T, all purchased from ATCC, USA) were routinely cultured in a six-well plate (NEST Biotecl, 703001) at 300,000 / well 24 hours. RNA was extracted according to the instructions of the Invitrogen reverse transcription kit, reverse-transcribed into cDNA, PCR amplification conditions were 94°C for 5 minutes, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, and 30 cycles.

[0032] The result is as figure 2 As shown, the mRNA levels of 7 truncated TRAIL variants in different cell lines were detected by RT-PCR. Except that DA was only detected in THP-1, other variants were expressed in various cell lines.

Embodiment 3

[0033] Example 3, the expression of 7 truncated TRAIL variants on the cell membrane

[0034] HEK293T cells (purchased from ATCC, USA) were routinely cultured in a six-well plate (NEST Biotecl, 703001) at 300,000 / well for 18 hours, and 3 μg pCMV-sport6 ​​(negative control), 7 truncated TRAIL variants Plasmid (pCMV vector), RefSeq (pCMV vector, positive control) According to the instructions, the plasmid was transfected into HEK293T cells using VigoFect (Vigorous Company) cationic transfection reagent. After continuing to culture for 24 hours, collect the cell culture supernatant in a centrifuge tube, trypsinize the cells, transfer them to a centrifuge tube, centrifuge at 1,500rpm for 5 minutes, discard the supernatant, wash the cells twice with PBS, add 2% FBS FACS blocking solution (PBS containing 2% FBS), the cells to be tested were made into a single-cell suspension with a density of approximately 5×10 5 / ml. Subsequently, the anti-Myc antibody (purchased from MBL Company) ...

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Abstract

The invention belongs to the field of tumor necrosis factor-(TNF-)-related apoptosis-inducing ligand (TRAIL). Especially, the invention relates to 7 truncated mutants (AK, E2, E3, E4, DA, BX424 and BX439) of TRAIL. A protein sequence coded by DA is completely same with that of TRAILbeta, and a protein sequence coded by BX424 is completely same with that of TRAILdelta. As a result of experiments, among the truncated mutants, except BX439, the mutants have different degrees of capabilities for activating NF-kappaB. As a result of further studies, over-expressions of DA, BX424 and E4 assist in substantially activating macrophage inflammatory protein-1beta(MIP-1beta/CCL4), macrophage inflammatory protein-3alpha(MIP-3alpha/CCL20), and interleukin8(IL8/CXCL8) promoter reporter gene. The TRAIL mutant provided by the invention has wide application prospect in treating inflammatory responses.

Description

technical field [0001] The invention relates to the application of seven truncated TRAIL variants in the preparation of medicaments for treating inflammatory response, in particular to the effect of activating NF-κB in cells and treating inflammation. Background technique [0002] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, and its coding gene is located on chromosome 3 (3q26), consisting of 5 exons , encoding 281 amino acids, of which the 18th to 38th amino acids are transmembrane regions, and the 39th to 281st are extracellular regions, belonging to type II transmembrane proteins. TRAIL can be expressed in many normal tissues, has no obvious toxicity to most normal cells, but can selectively kill tumor cells, so it has great potential in tumor therapy (for example, see Stegehuis JH.TRAIL receptor targeting therapies for non-small cell Lung cancer: current status and perspectives. Drug Resist Update ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/19A61P29/00A61P35/00
Inventor 王平章卢艳李长清李娜石太平于鹏马大龙
Owner SINOGENOMAX
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