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A recombinant polypeptide for treating tumors

A technology of recombinant polypeptides and antigenic peptides, applied in the field of tumor treatment, can solve the problems of activated killing, insufficient recognition of immune cells, weak antigenicity, etc., and achieve the effects of enhanced killing, low cost, and simple production and preparation

Active Publication Date: 2016-04-13
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the human body, one of the main reasons why tumor cells are difficult to be completely eliminated by the body is that tumor cells are derived from abnormally mutated normal cells, and their antigenicity is not strong, so immune cells cannot fully recognize, activate and exert their killing function

Method used

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  • A recombinant polypeptide for treating tumors
  • A recombinant polypeptide for treating tumors
  • A recombinant polypeptide for treating tumors

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Experimental program
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Effect test

Embodiment 1

[0029] A 336bp DNA sequence was designed, including three dominant antigenic peptide nucleotide sequences from LLO and hEGF nucleotide sequence, and the connecting peptide nucleotide sequence was between different peptide coding sequences. The designed sequence is artificially synthesized by chemical synthesis and constructed on the template plasmid: A-336_pMA-T (Amp + ). exist Figure 2A In the method, the expression plasmid pET-30a(+) was extracted by alkaline lysis method, and 1 μg was added to 50 μl competent cell DH5a and 50 μl competent cell BL21(DE3). Heat shock at ℃ for 60s, place on ice for 2min, add 800μl of LB medium respectively, place on a shaker at 37℃ at 200rpm, shake for 90min, then centrifuge at 3000rpm for 5min, pour off the supernatant, leave 100μl of medium, and carefully resuspend the bacteria After in vivo, spread on a Kana-resistant flat plate with a glass rod. The plates were placed in a 37°C incubator and incubated upside down for 16 hours. Single ...

Embodiment 2

[0031] Such as Figure 3A Induced expression of the recombinant protein (pLLO-hEGF) is shown. First inoculate the recombinant monoclonal bacteria liquid with correct sequencing into 5ml / tube of LB medium at a ratio of 1:100, add Kana antibiotics at a ratio of 1:1000, and culture on a shaking table at 37°C and 250rpm. OD of the bacteria solution 600 At 0.6-0.8, take one 5ml bacteria for small-scale induced expression of recombinant protein. The inducer IPTG was added to the bacterial solution to make the final concentration 1 mM, and the shaking culture was continued at 37° C. and 250 rpm for 4 h. When the recombinant protein was induced to express at 37°C, it mainly existed in the form of inclusion bodies. Take 1ml of the bacterial solution before and after induction, centrifuge at 10,000rpm for 1min, add 100μl of 1X SDS loading buffer, boil at 100°C for 10min, cool and centrifuge at 12,000rpm for 5min, take the supernatant as the loading sample for 12% SDS-PAGE, gel The g...

Embodiment 3

[0033] exist Figures 4A-4F Among them, the recombinant protein (pLLO-hEGF) can target a variety of tumor cells. Cell immunochemical staining was used to analyze whether the recombinant protein could bind to tumor cells with high expression of EGFR. Preparation of cell slides of different tumor cells, including colorectal cancer cells HCT116 and HT29 ( Figure 4A and 4B ), lung cancer cells A549 and NCI-H157 ( Figure 4C and 4D ), breast cancer cells SK-BR-3 and MDA-MB-231 ( Figure 4E and 4F). Wash the cell slides with PBS 3 times, 2 min each time. Fix with 4% paraformaldehyde at room temperature for 30 minutes, clamp the slides with tweezers and immerse them in PBS to wash 3 times, 5 minutes each time. Block with 3% BSA at room temperature for 30 min, wash with PBS twice, 5 min each time. Three groups were established: anti-EGFR Rabbit mAb group, namely positive control group; EGF control group; recombinant protein group. The EGF group and the recombinant protein g...

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Abstract

The invention relates to a recombinant polypeptide for treating tumors. Especially, the recombinant polypeptide comprises three immunogenic antigen peptides and a human epidermal growth factor (hEGF) mature peptide, which are connected through a connecting peptide which is 2-10 amino acids long.

Description

technical field [0001] The invention relates to a recombinant polypeptide and its use for treating tumors. The tumor is selected from rectal cancer, lung cancer, breast cancer. Background technique [0002] Currently, drug therapy is still one of the most important clinical treatments for cancer patients. However, traditional antineoplastic drugs exert their curative effect by distributing the whole body to reach a certain blood concentration, and the mechanism of action of these drugs is generally to exert a toxic effect on cells with vigorous metabolism and rapid proliferation, and their selectivity is poor, so they have poor selectivity for normal cells. Tissue cells also have a large killing effect, produce obvious toxic and side effects, bring great pain to patients physically and mentally, and greatly reduce the anti-tumor therapeutic value of these drugs. In recent years, the rapid development of tumor molecular biology has created a new model of tumor treatment. Tu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/18A61K47/48A61P35/00
Inventor 刘玉琴孙蕊朱琰
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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