Lung cancer cell-specific adhesion short peptide and its application
A lung cancer cell and specific technology, applied in the field of protein peptides, can solve the problem of few reports on screening lung cancer targeting ligands, and achieve the effects of small molecular weight, high specificity and broad application prospects.
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Embodiment 1
[0022] Example 1 Screening of Lung Cancer Specific Binding Polypeptides
[0023] In this example, a phage display random 12-peptide library is used for subtractive screening of polypeptides that specifically bind to lung cancer cells, and the specific steps are as follows:
[0024] After digesting human lung cancer cells (A549) and human lung normal cells (MRC-5) with trypsin, adjust the cell density, inoculate in a culture dish pre-coated with poly-lysine, and wait until the cells grow to 85%-90% Screening experiments were performed for fusion.
[0025] Take the above-mentioned A549 cells, culture them with serum-free DMEM, add bovine serum albumin BSA to block, then add 10 μl phage peptide library, after incubation for 1.5 h, pour to remove unbound phage, and shake upside down to remove the residual solution. Rinse 3 times with washing solution 0.1% (v / v) TBST buffer, add non-specific buffer 0.2M glycine-hydrochloric acid (pH2.1) 1ml, suck out eluate, add 150μl 1MTris-HCl (...
Embodiment 2
[0029] Example 2 The amount of phage after screening and amplification
[0030] In Example 1, the phage obtained in each round of screening was diluted 100 times with LB medium, and 10 μl of the diluted phage was mixed with 200 μl of E. After the LB top layer agar, quickly pour it on the LB solid plate containing IPTG / Xgal, overnight, count the number of spots on the plate where the phage can grow, and then multiply this number by the dilution factor to get the plaque forming unit (pfu) drops per 10 μl phage Spend.
[0031] Enrichment of phage polypeptides specifically bound to lung cancer cells: As shown in Table 1, the recovery rate of phage clones positive for A549 was increased by 100 times after three rounds of selection.
[0032] Table 1 The recovery rate of positive phage after three rounds of screening
[0033]
Embodiment 3
[0034] Embodiment 3ELISA identification phage polypeptide
[0035] Identification of positive clones of phage polypeptides specifically binding to lung cancer: In Example 1, after three consecutive rounds of subtractive screening of the phage peptide library, 12 phage clones were randomly selected, and the phage clones were preliminarily identified for A549, A549, Affinity for H1299.
[0036] Press A549, H1299 by 1×10 5 The density per well was seeded in a 96-well plate and placed in CO 2 After culturing in the incubator for 24 hours, the cells were treated with serum-free for 1 hour, washed, fixed with paraformaldehyde, washed with PBS, treated with TritonX-100, blocked with PBS-BSA, added phage monoclonal, incubated for 2 hours; added HRP-antiM13 Antibody, incubate for 1 h at 37°C; develop color with TMB (50 μl TMB per well, develop color for 10-15 minutes), add an equal volume of 1NHCl or 2NH 2 SO 4 To terminate the reaction, the reaction solution in the microplate well...
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