Double-target-spot fusion protein capable of enhancing TRAIL antitumor activity

A technology of anti-tumor activity and fusion protein, applied in] The present invention relates to the technical field of genetic engineering drugs, which can solve the problems of increasing the sensitivity of exogenous TRAIL, achieve high recovery and purification efficiency, strong anti-tumor effect, and enhance activity Effect

Inactive Publication Date: 2014-01-22
CHENGDU HUACHUANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the deletion of the XIAP gene did not affect the basal proliferation of the cells, it significantly increased the sensitivity of the cells to exogenous TRAIL

Method used

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  • Double-target-spot fusion protein capable of enhancing TRAIL antitumor activity
  • Double-target-spot fusion protein capable of enhancing TRAIL antitumor activity
  • Double-target-spot fusion protein capable of enhancing TRAIL antitumor activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Splicing and synthesis of full-length fusion gene sequences 1 and 2

[0058] The target of TRAIL protein (114-281aa) in the designed fusion protein is TRAIL receptor DR 4 、DR 5 , SMAC N7 peptide is aimed at the apoptosis antagonist XIAP, in order to have a significant synergistic effect on TRAIL.

[0059] The full-length fusion gene sequence to be synthesized (see sequence 1, 4) is divided into two parts: the fusion front sequence (see sequence 3, 6) and the TRAIL sequence (see sequence 7). The fusion front sequence includes (R8+SMAC N7+AP) (see sequence 3) and (TAT+SMAC N7+AP) (see sequence 6) peptide coding cDNA sequences. The double-strand splicing primers (see sequence 8-18) were used as raw materials, and the synthesized double-strand splicing primers were reacted together. Under the condition of no Taq DNA Polymerase, the spliced ​​fusion front sequence (R8+SMAC N7+AP) and (TAT +SMAC N7+AP) encoding cDNA. At the same time, by using the plasmid containing the T...

Embodiment 2

[0156] Ligation of full-length fusion gene sequences 1 and 2 with pMD19-T vector

[0157] In order to increase the success rate of expression vector construction, the full-length fusion gene sequences 1 and 2 were first ligated and transformed with the pMD19-T vector, and then the successfully transformed clones were subcloned into the expression vectors pTWIN1 and pET-32a to construct the full-length Prokaryotic expression vector of long fusion gene sequences 1 and 2.

[0158] (1) Experimental materials, reagents and equipment

[0159] 1. Materials: Full-length fusion gene sequences 1 and 2 are derived from the results of Example 1.

[0160] 2. See Table 11 for reagents.

[0161] Table 11. Reagents used for linking full-length fusion gene sequences 1 and 2 to pMD19-T vector

[0162] Reagent name Specification batch number Manufacturer pMD19-T Vector 20T CK2401AA TAKARA Top10 Competent Cells 100μl 111108 Tiangen Bio tryptone 500g 8...

Embodiment 3

[0182] Ligation of full-length fusion gene sequences 1 and 2 with prokaryotic expression vector

[0183] The prokaryotic expression vectors pTWIN1 and pET-32a were selected, and the vectors pTWIN1, pET-32a vectors and positive cloning vectors pMD19 / full-length fusion gene sequence 1 and pMD19 / full-length fusion gene sequence 2 were respectively cut with NdeI and PstI. In pTWIN1, the two cohesive ends of the Intern region (about 1549 bp) were removed, and then the same double-digested full-length fusion gene sequences 1 and 2 were inserted into this region; while pET-32a was obtained by removing the two cohesive ends of the expression region of the Trx fusion protein tag. Sticky ends, and then insert the same double-digested full-length fusion gene sequences 1 and 2 in this region. Therefore, NdeI and PstI double enzyme digestion was used, and the TaKaRa ligation kit was used to ligate and transform into the Top10 competent cells of Tiangen Bio.

[0184] (1) Experimental mater...

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Abstract

The invention belongs to the technical field of genetic engineering drugs, and discloses a double-target-spot fusion protein capable of enhancing the TRAIL antitumor activity. The cDNA sequence of the overall-length fusion gene sequence 1 is shown in SEQ ID NO1; the coding amino acid sequence of the overall-length fusion gene sequence 1 is shown in SEQ ID NO2; the coding cDNA sequence of the overall-length fusion gene sequence 2 is shown in SEQ ID NO4; and the coding amino acid sequence of the overall-length fusion gene sequence 2 is shown in SEQ ID NO5. In comparison with the prokaryotic expression carrier of separate TRAIL protein soluble fragment coding cDNA sequence, the fusion protein of the prokaryotic expression carrier has higher soluble expression, and the recycling and purifying efficiency of the fusion protein is higher; and the in-vitro biological activity analysis indicates that the fusion protein can activate death receptors on the cell membrane and inhibit expression of an XIAP gene in the cell plasma, enhance the activity of inducing tumor cell apoptosis through the double-target-spot function in apoptotic pathway and ensure stronger antitumor effect.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering drugs, in particular to the coding cDNA sequence of the wild-type TRAIL protein and the N-terminal 7-peptide coding cDNA of the antagonistic protein SMAC of an endogenous apoptosis inhibitor molecule XIAP through a specific cell endogenous protease The splicing site AP encoding cDNA sequence is connected, and a high-efficiency membrane-penetrating peptide sequence is added in front of the fusion gene to construct R8+SMAC N7+AP+TRAIL (114-281aa) or TAT+SMAC N7+AP+TRAIL (114- 281aa) fusion gene sequence, the above fusion protein was expressed by the Escherichia coli expression vector transformed with pTWIN1, and the above fusion protein was isolated and purified to obtain a dual-target fusion protein capable of enhancing the anti-tumor activity of TRAIL. Background technique [0002] 1. TRAIL protein structure and molecular biology [0003] Tumor necrosis factor-related apoptosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/17A61P35/00
CPCA61K38/00A61P3/00A61P35/00C07K14/70575C07K2319/00C07K2319/10C07K2319/50
Inventor 陈守春潘凤闫娟徐琦胡海洋李昭君朱文彦
Owner CHENGDU HUACHUANG BIOTECH CO LTD
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