Polypeptide specifically binding to human CD47 molecule and application thereof
A peptide and composition technology, applied in the field of immunology, can solve the problems of immunogenicity, cytotoxicity and high price
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Embodiment 1
[0049] Example 1 Verifying the expression of CD47 in lung cancer tissues, primary lung cancer cells and cell lines
[0050] Experimental Materials
[0051] Lung cancer tissue with bone metastases and surrounding normal tissues were obtained from bone surgery resection samples of Zhongshan Hospital affiliated to Fudan University; human lung cancer cell line H1229 was purchased from ATCC; primary lung cancer cells were obtained from bone metastases lung cancer specimens and digested After treatment, they were cultivated in the center of Zhongshan Hospital Affiliated to Fudan University; C57 / B6 mice were provided by the Department of Experimental Animal Science, Fudan University School of Medicine.
[0052] 1.1 The lung cancer tissue with bone metastases and surrounding normal tissue were taken, fixed and stained with H&E to detect the infiltration of inflammatory cells and histological structure of the cancer tissue; the expression of CD47, SIRP-a, and CD68 was detected by immun...
Embodiment 2
[0057] Example 2 Screening of phage polypeptide library
[0058] 1.1 Construction of phage polypeptide library
[0059] The human recombinant rCD47 protein was coated onto the MaxiSorp 96-well plate, and then the phage containing the polypeptide library (Ph.D.Phage display libraries, New England BioLabs, Inc, Ipswich, MA, USA) was added to the coated well to make the containing The phage of the specific polypeptide fragment binds to rCD47, and after the unbound phage is eluted, the phage that binds to rCD47 for the first time is used for the second binding, elution, concentration and amplification process, after repeated 3-5 times of screening , selected 20 individual clones, and carried out DNA sequencing, which was entrusted to Bosun Biotechnology (Shanghai) Co., Ltd. for DNA sequencing. The DNA sequences of these 20 clones were analyzed and compared, and the DNA sequence with the highest occurrence rate among the 20 clones was selected as the CD47 targeting-specific sequen...
Embodiment 3
[0071] Example 3 investigates the growth of polypeptide CD47KLA on lung cancer cells in a mouse lung cancer model
[0072] In order to investigate the inhibition of CD47KLA on the growth of lung cancer cells in a mouse lung cancer model, mouse lung cancer cells were treated with 30 μM CD47KLA, and 1×10 6 A cell dose was subcutaneously injected into C57 / B6 adult mice through the groin, and the tumor growth was observed. The tumor size was observed at different time points, and the growth curve was drawn. As a control, the same dose (1×10 6 LLC lung cancer cells) were subcutaneously injected into C57 / B6 adult mice through the groin, and the tumor growth was observed; after the mouse lung cancer cells were treated with 30 μM control peptide Scramble peptide (SCKLA), 1×10 6 A cell dose was subcutaneously injected into C57 / B6 adult mice through the groin, and the tumor growth was observed. The tumor growth curve is shown in image 3 Middle A. The results showed that CD47KLA si...
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