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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 weak antigenicity, insufficient recognition of immune cells, activation and killing, etc., and achieve the effect of simple production and preparation, low cost, and enhanced killing

Active Publication Date: 2014-07-02
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Recombinant polypeptide for treating tumors
  • Recombinant polypeptide for treating tumors
  • Recombinant polypeptide for treating tumors

Examples

Experimental program
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Effect test

Embodiment 1

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

Embodiment 2

[0031] like Figure 3A Induced expression of recombinant protein (pLLO-hEGF) as indicated. First, inoculate the correctly sequenced recombinant monoclonal bacterial solution into 5ml / support LB medium at a ratio of 1:100, add Kana antibiotics at a ratio of 1:1000, and shake at 37°C and 250rpm for culture. OD of the bacterial solution 600 At 0.6 to 0.8, take a 5ml strain of bacteria and use it as a small amount of recombinant protein to induce expression. The inducer IPTG was added to the bacterial solution to make the final concentration 1 mM, and the culture was continued at 37° C. and 250 rpm for 4 h. When the recombinant protein was induced and expressed at 37°C, it mainly existed in the form of inclusion bodies. Take 1ml of pre-induction and post-induction bacteria solution, centrifuge at 10,000 rpm for 1 min, add 100 μl of 1X SDS loading buffer, boil at 100 °C for 10 min, and centrifuge at 12,000 rpm for 5 min after cooling. Take the supernatant as the loading sample f...

Embodiment 3

[0033] exist Figures 4A-4F Among them, a recombinant protein (pLLO-hEGF) can target and bind to 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). The cell slides were washed 3 times with PBS, 2 min each time. 4% paraformaldehyde was fixed at room temperature for 30 min, and the slides were immersed in PBS with small forceps and washed three times for 5 min each time. Blocked with 3% BSA at room temperature for 30 min, washed twice with PBS for 5 min each time. Set up 3 groups: anti-EGFR Rabbit mAb group, namely positive control group; EGF control group; recombinant protein group. EGF and recombinant protein we...

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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 present invention relates to a recombinant polypeptide and its use for the treatment of tumors. The tumor is selected from rectal cancer, lung cancer, and breast cancer. Background technique [0002] At present, one of the most important treatments for cancer patients is still drug therapy. However, traditional anti-tumor 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 the selectivity is poor. Tissue cells also have a large killing effect, produce obvious toxic and side effects, bring great pain to the patient's body and mind, and greatly reduce the anti-tumor therapeutic value of these drugs. In recent years, the rapid development of tumor molecular biology has innovated new models of tumor treatment. Tumor biological therapy and tumor targeted biol...

Claims

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Application Information

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Patent Type & Authority Applications(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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