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A recombinant fusion protein that specifically recognizes tumor cells and its application

A fusion protein and tumor cell technology, applied in the field of recombinant fusion protein, can solve the problems of high cost, high operation threshold of colorectal cancer early diagnosis technology, lack of genetic engineering means for specific identification of intestinal tumors, etc. Easy to use effect

Active Publication Date: 2021-08-31
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the existing technology for early diagnosis of colorectal cancer has high operating threshold and high cost, and lacks genetic engineering means that can specifically identify intestinal tumors.

Method used

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  • A recombinant fusion protein that specifically recognizes tumor cells and its application
  • A recombinant fusion protein that specifically recognizes tumor cells and its application
  • A recombinant fusion protein that specifically recognizes tumor cells and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Construction of plasmid bacterial surface display pCDFDuet1-INP-tPep of

[0040]Plasmid pET-InaZN-EGFP (S.Bao, S.Yu, X.Guo, et al.Journal of AppliedMicrobiology, 2015 (119):. 236-244) as a template and primers designed and SacI-INPF HindIII-tPep- INPR PCR amplification, to obtain a DNA fragment of INP-tPep. Wherein an upstream primer SacI-INPF as: CGAGCTCCTGTACCATGGATCTCGACAA (SEQ ID NO: 1); downstream primer HindIII-tPep-INPR as: CCCAAGCTTGGGTTAAATACGAGAAGCAGGAGACCAAGCATACCAACGATGCCAAACAATCGTGGATCCGTAAGTGG (SEQID NO: 2). Further by the SacI and HindIII double enzyme digestion, DNA fragments having cohesive ends. Which was constructed between the enzyme-linked by the process of digestion of plasmid pCDFDuet1 (available from Novagen, 71340-3 Num) of SacI and Hindlll cloning site, forming plasmid pCDFDuet1-INP-tPep with INP-tPep of transform E. coli, using streptomycin screened to obtain transformed strains. After plasmid extraction and purification, DNA sequencing, the plasm...

Embodiment 2

[0042] Construction of recombinant bacteria INP-tPep-EGFP

[0043] Construction of pGEX-4T-1-EGFP plasmid: by PCR from the plasmid pEGFP-N1 (preferably available from Takara Bio) as a template, amplified EGFP-FP with the following primers: GCGGATCCATGGTGAGCAAGGGCGAGGAG (SEQ ID NO: 6); EGFP- RP: GCGAATTCTTACTTGTACAGCTCGTCCATGCCG (SEQ ID NO: 7) product recovery after double digestion with BamHI and EcoRI, connected to the same double digestion of pGEX-4T-1 plasmid (preferably available from Takara bio), transformation, positive clones were obtained by screening ampicillin . After by cotransformation The plasmid pCDFDuet1-INP-tPep plasmid pGEX-4T-1-EGFP was transformed into E. coli BL21 (genotype F-ompThsdS (rB-mB-) galdcm (DE3)), the expression allowed. Specific steps are as follows:

[0044] 1) Transformation: each 5μl pCDFDuet1-INP-tPep with pGEX-4T-1-EGFP plasmid was added 50μl of E. coli competent cells, placed on ice for 30 minutes, 42 ℃ heat shock 90s, then returned to ice for...

Embodiment 3

[0048] Cells climbing film identified recombinant bacteria INP-tPep-EGFP and human HT29 colorectal cancer cell specifically recognizing placed 2cm diameter climbing film cells in 24-well cell culture plate, each well 0.5ml of 5% of the density of HT29 cells; cell proliferation when the rear cover 70% to 80% of the slide, the medium was changed to normal saline (0.5ml / well) was added 100μlINP-tPep-EGFP EGFP recombinant bacterium or bacteria (i.e., only into the bacterial pGEX-4T-1-EGFP plasmid) was (OD600≈1.0), placed in a 37 [deg.] C shaker were incubated 0.5 hours after incubation, cells were removed climbing film, with a 4% paraformaldehyde fixed cells, and treated with nuclei were stained DAPI staining solution; DAPI fluorescence microscope at 488 and climbing film passage cells were photographed, and the combined image of two channels. Such as figure 1 Indicated (shown) figure 1 A is INP-tPep-EGFP recombinant bacterial binding to cells; figure 1 B is EGFP recombinant bacteri...

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Abstract

The invention discloses a recombinant fusion protein, which relates to the technical field of genetic engineering, and comprises an ice nucleoprotein N-terminal domain and a T antigen-binding short peptide. Among them, the N-terminal domain of the ice nucleoprotein provides the surface anchoring function of the bacteria, enabling the fusion protein to be anchored and displayed on the cell surface; the T-antigen-binding short peptide is an oligopeptide that can specifically bind to the T-antigen on the surface of intestinal tumor cells . In the present invention, the T antigen-binding short peptide is displayed on the surface of bacteria in the form of fusion protein. This strategy can realize the specific recognition of bacteria on intestinal tumors, solve the limitation of the existing intestinal exploration and detection technology that must invade the body, and lay the foundation for the further development of targeted drugs for early diagnosis and treatment of intestinal tumors .

Description

Technical field [0001] The present invention relates to the field of genetic engineering, particularly to a bacterial surface display system based on a recombinant fusion proteins and their application. Background technique [0002] Currently, cancer is greatly endanger the lives and health of severe disease in humans. In recent years, with the advancement of basic research in tumor biology and immunology, cancer surgery, chemotherapy, radiation therapy and biological efficacy in the treatment of certain types of tumors have been significantly improved, but the key to improve the survival rate of cancer patients still depends early detection and early diagnosis of cancer. [0003] Of colorectal cancer (Colorectal cancer, referred to as CRC) is the third most common cancer in humans, accounting for 9.7% of all cancers, with more than half of the cases occur in developed areas of the world because of an increase in the elderly population, unhealthy eating habits and environmental r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N15/65C12N1/21A61K47/68A61K47/42A61K49/00A61P35/00C12R1/19
CPCA61K47/42A61K49/0047A61K49/0058A61K47/6863A61P35/00C07K7/08C07K2319/035C12N15/65C12N15/70
Inventor 马钢王毓舒洪艺瑞梁智涵孙诗语郑书钰方清伟冉运聪刘致祥萧家宜钟博子韬何沛祥张凯然夏若瑜吴蔚文姚宽贺林
Owner SHANGHAI JIAO TONG UNIV
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