Antitumor peptide with pH selectivity and application of antitumor peptide
An anti-tumor and selective technology, applied in the field of anti-tumor drug development and application, can solve problems such as toxic and side effects, and achieve the effect of enhancing selectivity
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Embodiment 1
[0068] Example 1. Preparation of polypeptides
[0069] (1) The ribosome display vector (pRDV, Genbank No. AY327136) was used for screening, the empty vector and the library were cut with restriction endonucleases BamHI and HindIII respectively, and the library was inserted into the restriction sites of the vector BamHI and HindIII (Such as figure 1 ). DNA libraries containing 19, 22 and 25 random polypeptide sequences consist of a GNN codon and 18, or 21 or 24 NNS codons, N is adenine (A), guanine (G), A mixture of cytosine (C) and thymine (T), S is a mixture of C and G. NNS can encode all 20 essential amino acids, but only one of the three terminators. The library can encode polypeptides of 19 amino acids, 22 amino acids and 25 amino acids in length. Then, PCR amplifies the above vector inserted into the library, and introduces the T7 RNA polymerase promoter sequence, ribosome binding position (sequence) and 5' stem-loop of stable structure before the sequence of the l...
Embodiment 2
[0082] Example 2. Anticancer activity test of polypeptide
[0083] The anticancer activity of the polypeptide was detected in human lung cancer cell line A549, human breast cancer cell line MCF-7, human lung fibroblast cell line CCD-13Lu and human breast epithelial cell line MCF-10A. Press 5×10 3 The cells were seeded into a 96-well plate at a density of 1 cell / well and incubated at 37°C for 24 hours. Dilute the polypeptide to different concentrations with culture solution (pH to be tested) to replace the original culture solution. Incubate at 37°C for 2 hours. Then, according to the standard experimental method of MTT, the activity of the polypeptide at different pH values was measured.
[0084] Table 2. Activity of peptides at pH 7.5 and 5.5.
[0085]
[0086] *: IC 20 (μM): Peptide concentration that inhibits 20% cell viability.
[0087] pH selectivity = (IC at pH 7.5 50 ) / (IC at pH 5.5 50 )
[0088] The results showed that all the peptide...
Embodiment 3
[0089] Example 3. In vivo experiments
[0090] Feeding and maintenance of Nu / Nu nude mice. Mice were housed in pathogen-free cages on a 12-hour light / 12-hour dark rhythm. Subcutaneously inject 100 mL containing 1.7′10 6 A549 cell suspension. Every other day, the tumor size was measured with a vernier caliper, and the volume was calculated according to the following formula: volume = 0.5 ′ W ′ L ′ H, where W, L, and H represent width, length, and height, respectively. When the tumor grows to about 100 mm 3 start intravenous injection. Mice were divided into four groups: (1) saline (control); (2) ZTU0 in saline (pH 7.5); (3) ZTU17 in saline (pH 7.5); (4) ZTU17 in saline (pH 5.5). Each group consisted of 6 to 7 mice. Peptide injection (20 nmol peptide / mouse, dissolved in 100 ml saline) was injected every three days. Tumor volumes were measured every four days after the start of polypeptide injection. 24 days after the start of injections, all mice were sacri...
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