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Malignant melanoma T cell nano antibody, coding sequence and application thereof

A malignant melanoma, nanobody technology, applied in the field of nanobody, can solve the problem that the effect cannot be finalized

Inactive Publication Date: 2011-08-24
SHANDONG SHENGBAILING MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the vaccine of malignant melanoma T cell antigen epitope polypeptide (gp100, Mart1) has been clinically tested in foreign countries, and its effect is not yet certain.

Method used

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  • Malignant melanoma T cell nano antibody, coding sequence and application thereof
  • Malignant melanoma T cell nano antibody, coding sequence and application thereof
  • Malignant melanoma T cell nano antibody, coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Screening process of Nanobody against malignant melanoma-specific T cell epitope polypeptide Mart1 polypeptide-GAAGIGILIV:

[0028] (1) 20 micrograms of biotinylated Mart1 polypeptide-GAAGIGILIV were combined with 100 micrograms of avidin magnetic beads (Product by Invitrogen company) for 2 hours at room temperature. At the same time, use 100 microliters of phage (5x1011tfu non-immune camel nanobody phage display gene library) + 100 micrograms of avidin magnetic beads + 500 microliters of 2% skimmed milk 0.01M phosphate buffer saline (PBS), pH7.0, in 2 hours at room temperature. (2) Remove the supernatant of the biotin-avidin magnetic bead conjugate in (1), add the phage adsorbed by the avidin magnetic bead, and combine at room temperature for 2 hours. (3) Wash 5 times each with 0.05% Tween 20 (T) PBS (PBST) and PBS to wash off unbound phages. (4) Use TEA (triethylamine (7.18M)) to dissociate the phage that specifically binds to the polypeptide, and infect Escherichia...

Embodiment 2

[0030] Use phage enzyme-linked immunosorbent assay (ELISA) to screen for a single positive clone specific to the polypeptide:

[0031] (1) Select a single colony from the above-mentioned bacterial culture dish containing phage after 3 to 4 rounds of screening and inoculate it in a 96-well bacterial culture plate, and cultivate it overnight; (2) transfer the supernatant containing phage to the In the ELISA plate coated with avidin and combined with biotinylated Mart1 polypeptide-GAAGIGILIV, place it at room temperature at 37 degrees for 1-2 hours, (3) wash off unbound phage with PBST, (4) add pepper The anti-phage antibody labeled with root peroxidase acts for 1 hour, (5) TMB develops color, and reads the absorbance value (OD) at a wavelength of 450 nm on an ELISA instrument. (5) When the OD value of the sample well is more than 3 times greater than the OD value of the control well, it is judged as a positive clone well. (6) PCR amplifies or purifies the plasmids of positive w...

Embodiment 3

[0034] Construction of specific nanobody expression plasmid:

[0035] The obtained specific Nanobody gene was amplified by PCR to obtain a PCR product with restriction endonuclease BbsI and ApaI sites, and the PCR product and the vector (pSCT1 plasmid-source In the modified pSJF2 plasmid), through ligation and recombination, the plasmids MT-#1~MT-#5 capable of high expression in Escherichia coli were obtained.

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PUM

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Abstract

The invention relates to a nano antibody aiming at malignant melanoma T cell epitope polypeptide Mart1, and simultaneously discloses a coding sequence which provides the nano antibody as well as a host cell which expresses or can express the nano antibody. The nano antibody can be expressed efficiently in colon bacillus and used for preparing pharmaceutical composition for detecting and treating the malignant melanoma.

Description

1. Technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and in particular relates to a nanobody directed against malignant melanoma T cell antigen epitope polypeptide Mart1 polypeptide. 2. Background technology [0002] The incidence of malignant melanoma is not high, but it is a malignant tumor of melanocytes in the epidermis or mucosa, with a high degree of malignancy, and is prone to hematogenous and lymphatic metastasis, with a poor prognosis. According to the data of Shanghai Cancer Hospital, this disease accounts for 10% of skin malignant tumors and 1-2% of all tumors, and it has an increasing trend in recent years. Treatment measures: surgical resection, radiotherapy, chemotherapy (for those with metastases) and immunotherapy (such as BCG, interleukin II, α-interferon, etc. as adjuvant therapy) or a combination of the above four methods "http: / / www.biox.cn / content / 20050910 / 37264.htm”. In recent years, the vaccine...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N1/21G01N33/577G01N33/574A61K39/395A61P35/00C12R1/19
Inventor 王大升
Owner SHANDONG SHENGBAILING MEDICAL TECH CO LTD