CN102526705B - Application of human ribosomal protein L23 label peptide in preparation of anti-cancer medicines - Google Patents

Abstract

The invention discloses the application of the human ribosomal protein L23 tag polypeptide in the preparation of anticancer drugs. The amino acid sequence of the human ribosomal protein L23 tag polypeptide is defined by SEQ ID NO:1. Human ribosomal protein L23-tagged polypeptide has a time- and dose-dependent growth inhibitory rate on human breast cancer Bcap-37. At 0.2, 0.5 and 0.8 μg/ml concentrations, it has a good effect on the growth inhibition of human breast cancer Bcap-37, and at a concentration of 0.5 μg/ml, the human ribosomal protein L23 tag peptide has a good effect on the growth of human breast cancer Bcap-37 cells The inhibition rate was the best, reaching 72.29%.

Description

Application of human ribosomal protein L23 tag polypeptide in the preparation of anticancer drugs

technical field

The invention relates to the application of human ribosomal protein L23 tag polypeptide in the preparation of anticancer drugs, and belongs to the technical field of biomedicine.

Background technique

Ribosomes are organelles that catalyze protein synthesis. Mammalian ribosomes are composed of 4 RNAs and about 80 different proteins. The names of ribosomal proteins are related to the size of the protein in the ribosome. The small subunit ribosomal proteins are named S1 to S31, and the large subunit The base ribosomal proteins are named L1 to L44. The RPL23a protein is a component of the 60S ribosomal protein. The ribosomal protein encoded by the RPL23A gene is a component of the 60S subunit. This protein belongs to the L23P family of ribosomal proteins. It exists in the cytoplasm. In recent years, some research results have shown that the RPL23A gene is involved in cell growth and regulation. The cDNA and structural gene of the human ribosomal protein RPL23A have been cloned, sequenced and analyzed. The structural gene is about 4.0kb in length, contains 5 exons and 4 introns, and encodes a protein subunit of 156 amino acids. RPL23A protein subunit may be one of the substances involved in mediating growth-inhibiting interferon (Jiang et al., 1997). It has also been shown that the RPL23A gene is involved in the joint gene transcription of the U42A, U42B, U101A and U101B small nucleolar RNA genes (Rogan et al., 2001). At present, many mammalian RPL23A genes and their encoded proteins have been studied, and it is found that the amino acid sequence of the RPL23A protein subunit is highly conserved, and there is a 16-amino acid functional site, which is called ribosomal protein L23 tag (Ribosomal protein L23 signature). When we studied the giant panda RPL23A gene, we found that the giant panda RPL23A gene also encodes a 156-amino acid protein subunit, and there is also a 16-amino acid ribosomal protein L23 tag, but there is a mutation site in the giant panda RPL23A protein subunit , that is, the fifth position of the giant panda RPL23A protein subunit is proline (P), while the fifth position of the human and other mammal RPL23A protein subunits is alanine (A).

In order to further study the function of the giant panda RPL23A protein subunit, we constructed the expression vector of the giant panda RPL23AcDNA, transformed it into Escherichia coli for expression, purified the expression product, and obtained the recombinant protein of the giant panda RPL23A gene. The molecular weight of the protein is 21.265KDa; the isoelectric point is 10.57. During the research, we unexpectedly found that the recombinant protein of the giant panda RPL23A gene has a strong inhibitory effect on human laryngeal cancer (Hep-2) (the result has been applied for a Chinese patent , patent application number: 201110369377.0).

So far, there has not been any report at home and abroad on the strong growth inhibitory effect of human ribosomal protein L23-tagged polypeptide on human breast cancer cells.

Contents of the invention

In order to explore the anti-cancer mechanism and mechanism of the RPL23A gene recombinant protein, and to develop solid scientific data for the accumulation system of anti-cancer protein drugs, we cut the giant panda RPL23A gene recombinant protein into several small polypeptides, and screened the peptide pairs one by one. The effect of different types of human cancer cell growth, found that a group of polypeptides near the ribosomal protein L23 tag have different effects on the growth of different types of human cancer cells. Because the ribosomal protein L23 tag of giant pandas and other mammals is exactly the same as that of humans, we artificially synthesized the human ribosomal protein L23 tag polypeptide, and used this tag polypeptide to act on different types of cancer cells and normal cells, and found that The tag polypeptide has almost no effect on the growth of normal cells, and shows different effects on the growth of different types of cancer cells, among which it has a very strong growth inhibitory effect on human breast cancer cells, and its growth inhibition rate is as high as 72.29%; Human laryngeal carcinoma cells have a growth-promoting effect.

The application of the human ribosomal protein L23 tag polypeptide in the preparation of anticancer drugs, the amino acid sequence of the human ribosomal protein L23 tag polypeptide is defined by SEQ ID NO:1.

Significance of the present invention is as follows:

1. The human ribosomal protein L23-tagged polypeptide is a small molecular polypeptide, which is easy to prepare.

2. For the first time, it was found that the tag polypeptide had almost no effect on the growth of normal cells, and showed different effects on the growth of different types of cancer cells. Among them, it had a very strong growth inhibitory effect on human breast cancer cells, and its growth inhibition rate was as high as 72.29% %; and has a growth-promoting effect on human laryngeal cancer cells. It shows that human ribosomal protein L23 tag polypeptide has obvious targeting effect on cells.

3. The human ribosomal protein L23-tagged polypeptide has a very strong growth inhibitory effect on human breast cancer cells. This discovery provides a scientific basis and direction for the research and development of anticancer protein drugs. It also provides a valuable resource for studying the mechanism of anticancer.

Description of drawings

Figure 1 shows the effect of human ribosomal protein L23 tag polypeptide on the growth of normal human liver cells Chang Liver, human laryngeal cancer Hep-2 cells, and human breast cancer Bcap-37 cells (1-8 in the figure are: 0.05, 0.1, 0.2, 0.5, 0.8, 1.5, 3.0, and 6.0 μg/ml. human ribosomal protein L23 tag peptide concentration);

Figure 2 is the effect of human ribosomal protein L23 tag polypeptide on the morphology of normal human liver cells Chang Liver, human laryngeal cancer Hep-2 cells, and human breast cancer Bcap-37 cells (1-9 in the figure are: 0, 0.05, 0.1, 0.2, 0.5, 0.8, 1.5, 3.0, and 6.0μg/ml. Human ribosomal protein L23-tagged peptide concentration; C: Chang liver; B: Human breast cancer Bcap-37; H: Human laryngeal carcinoma Hep-2)

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments.

1.1 Materials and methods

1.1.1 Materials

1.1.1.1 Cell lines: normal human liver cells Chang Liver, human laryngeal cancer Hep-2 cells, human breast cancer Bcap-37 cells, the above cell lines are all commercialized biomaterials.

1.1.1.2 Reagents: RPMI1640 powder (purchased from Gibco, USA), fetal bovine serum (purchased from Sijiqing Biological Products Company, China), double antibodies (penicillin, streptomycin, purchased from Gibco).

1.1.1.3 Human ribosomal protein L23 signature polypeptide (Ribosomal protein L23 signature), synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. The amino acid sequence of the polypeptide is: KKAYVRLAPD YDALDV, the molecular weight is 1.8368KDa, and the isoelectric point is 6.50.

1.1.2 Method

1.1.2.1 Cell culture

Human normal liver cells Chang Liver, human laryngeal cancer Hep-2 cells, and human breast cancer Bcap-37 cell lines were treated with 10% inactivated fetal bovine serum (fetalbovine serum, FBS) 100 U/ml penicillin, 100 μg/ml chain Mycin and RPMI-1640 complete culture solution with pH 7.4 were cultivated in a 37°C, 5% CO ₂ incubator.

1.1.2.2 MTT method to detect cell viability

Human normal liver cells Chang Liver in logarithmic growth phase, human laryngeal cancer Hep-2 cells and human breast cancer Bcap-37 cells were digested with trypsin and the number of cells was adjusted to 1× ¹⁰⁵ cells/mL, and seeded in 96 wells Plate, the volume of cell fluid in each well is 200 μL, put it in a CO ₂ incubator, and incubate at 37°C, 5% CO ₂ and saturated humidity until the cell monolayer covers the bottom of the well (96-well flat-bottom plate), and then add Human ribosomal protein L23-tagged peptides were made to have final concentrations of 0, 0.05, 0.1, 0.2, 0.5, 0.8, 1.5, 3.0, and 6.0 μg/ml. Set up 6 replicate wells, and the blank group was cultured in RPMI1640 without cells After 24, 36 and 48 hours of culture in a CO ₂ incubator, take out the 96-well plate, add 10 μL of MTT (5 mg/mL) solution, continue to cultivate for 4 hours, and then add 150 μL of dimethylsulfoxide (DMSO) to shake for 10 minutes , after the blue-purple crystals are completely dissolved, measure the absorbance (OD) value of each well with a microplate reader at a wavelength of 490nm, and calculate the human ribosomal protein L23-tagged polypeptide at different concentrations (this embodiment adopts direct addition to culture and grows well The observation results in cancer cells, clinically, protein or polypeptide biopharmaceuticals are mostly in the form of injections, intramuscular injection or intravenous drip) the inhibition rate (%) of various types of cells. The formula for cell growth inhibition rate is: cell growth inhibition rate=(1-A experimental group/A control group)×100%.

On the basis of the above experiments, human breast cancer cell line Bcap-37, human laryngeal cancer cell line Hep-2, and human normal liver cell line Chang Liver, which had the best growth inhibitory effect on human ribosomal protein L23 tag polypeptide, were selected for comparative experiments. The time is 36h, and the specific method and steps are the same as above.

1.1.2.3 Morphological observation

The 96-well plate was observed under an inverted microscope, and the morphological changes of cells at different concentrations were recorded by taking pictures.

1.2 Results

1.2.1 The effect of human ribosomal protein L23-tagged polypeptide on the growth of normal human liver cells Chang Liver, human laryngeal cancer Hep-2 cells, and human breast cancer Bcap-37 cells

In this example, the MTT method was used to detect that different concentrations of human ribosomal protein L23 tagged polypeptides had almost no effect on the growth inhibition of normal human liver cells Chang Liver at 36 hours, and had a promoting effect on the growth of human laryngeal cancer Hep-2 cells. Human breast cancer Bcap-37 cell growth has a strong inhibitory effect. It can be seen from Figure 1 that the growth inhibition rate of human ribosomal protein L23 tag polypeptide on human breast cancer Bcap-37 is time- and dose-dependent. It was found that at the concentration of 0.2, 0.5 and 0.8 μg/ml, it has a good effect on the growth inhibition of human breast cancer Bcap-37. When the concentration is 0.5 μg/ml, the human ribosomal protein L23 tag polypeptide has a good effect on the growth of human breast cancer Bcap-37 cells. The growth inhibition rate was the best, reaching 72.29% (see Figure 1).

1.2.2 Effects of human ribosomal protein L23-tagged peptides on the morphology of normal human liver cells Chang Liver, human laryngeal cancer Hep-2 cells, and human breast cancer Bcap-37 cells

Use an inverted microscope to observe the morphological changes of human breast cancer Bcap-37 cells treated with different concentrations of human ribosomal protein L23 tag polypeptide (0.05, 0.1, 0.2, 0.5, 0.8, 1.5, 3.0, and 6.0 μg/ml) for 36 hours (Figure 2) . As can be seen from Figure 2, under the treatment time of 36h, compared with the control group, as the concentration of the human ribosomal protein L23 tagged polypeptide changes, the cells appear rounded, agglomerated, shed or even cracked into fragments (see figure 2).

It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should belong to the protection scope of the appended claims of the present invention.

Claims (1)

1. The application of the human ribosomal protein L23 tag polypeptide in the preparation of anti-human breast cancer medicine, characterized in that the amino acid sequence of the human ribosomal protein L23 tag polypeptide is defined by SEQ ID NO: 1, and the human ribosomal protein L23 tag polypeptide has growth inhibitory effect on human breast cancer cells.

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