Novel polypeptide targeting multiple tumor cells and application of novel polypeptide
A tumor cell, targeting technology, applied in the field of biomedicine, can solve the problems of strong toxic and side effects, large trauma, lack of efficient treatment methods, etc., and achieve the effects of weak immunogenicity, high efficiency, and simple synthesis and purification.
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Embodiment 1
[0038] Example 1 Three rounds of subtractive screening of positive polypeptides specifically binding to liver cancer cells using phage display technology.
[0039] 1.1 Recovery and cultivation of host bacteria E.coli ER2738.
[0040]To prepare an E. coli plate, take the LB-TET culture plate and preheat it in a 37°C incubator for 1 hour. After the E.coli ER2738 bacterial liquid melts, use an inoculation loop to dip a small amount of bacterial liquid evenly on the culture plate, and then place it upside down at 37°C. overnight in a constant temperature incubator. Prepare the host bacterial solution, pick a single colony from a well-grown culture plate, place it in LB bacterial culture solution containing tetracycline, and cultivate overnight at 37°C with shaking at 180rpm, so that the bacteria are in the logarithmic growth phase. The prepared LB-Tet plate containing Escherichia coli was stored in a 4°C refrigerator for later use, and the host bacterial solution was stored in a ...
Embodiment 2
[0062] Example 2 Enzyme-linked immunosorbent assay detection of target affinity between positive phage clones and liver cancer cells
[0063] 2.1 Purification of positive phage clones.
[0064] 2.1.1 Amplification of positive phages: Add 20ml LB / Tet liquid medium to the Erlenmeyer flask, then add Escherichia coli liquid and phage to be amplified at a ratio of 1:100, place at 37ºC, shake vigorously in a constant temperature shaker for 4.5 h, the amplification solution of phage was obtained.
[0065] 2.1.2 Purification of positive phage: centrifuge the phage amplification solution obtained through the above steps at 4ºC and 12000r / min for 10min, take the supernatant, add 1 / 6 volume of PEG-NaCl to precipitate overnight, centrifuge at 12000r / min for 15min, discard Remove the supernatant, dissolve the precipitate with TBS buffer, give 1 / 6 volume PEG-NaCl again, and incubate on ice for 1 h. 4ºC, 14000r / min, centrifuge for 15min, discard the supernatant, dissolve the obtained preci...
Embodiment 3
[0078] Example 3 Determination and analysis of DNA sequences of positive phage clones.
[0079] 3.1. Selection of positive monoclonal phages.
[0080] The phage liquid obtained after the third round of screening was titrated and spread on LB plates. On the plates with less than 100 growing spots, 20 well-growing blue spots at an interval of 5 mm were randomly selected. Add 20 randomly picked blue spots to 1ml pre-logarithmic host bacterial solution (same as phage amplification), and amplify at 37°C and 200rpm with rapid shaking for 4.5 hours.
[0081] 3.2 Positive monoclonal phage single-stranded DNA extraction.
[0082] Centrifuge the amplified monoclonal phage liquid at 4°C and 14,000 rpm for 30 seconds, transfer the supernatant to a new tube, centrifuge at 4°C and 1,000 rpm for 30 seconds, take 80% of the supernatant and transfer to a new nuclease-free centrifuge In the tube, take 300ul of bacterial liquid and add 300ul of glycerol at a ratio of 1:1, and store it in a -20 d...
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