A novel polypeptide targeting various tumor cells and its application
A tumor cell and tumor technology, applied in the field of biomedicine, can solve the problems of large trauma, strong side effects, and lack of efficient treatment methods, and achieve the effects of weak immunogenicity, good effect, and simple synthesis and purification
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Embodiment 1
[0039] Example 1 Three rounds of subtractive screening for specific binding of positive polypeptides to liver cancer cells using phage display technology.
[0040] 1.1 Recovery and culture of host strain E.coli ER2738.
[0041] Prepare Escherichia coli plates, take LB-TET culture plates and preheat them in a 37°C incubator for 1 hour. After the E.coli ER2738 bacterial solution is thawed, dip a small amount of bacterial solution with an inoculating loop and spread it evenly on the culture plates, then place it upside down at 37°C. Incubate overnight in a constant temperature incubator. The host bacterial solution was prepared, and a single colony was picked from a well-grown culture plate, placed in LB bacterial culture solution containing tetracycline, and cultured overnight at 37 °C and 180 rpm shaking to keep the bacteria 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 bacteria...
Embodiment 2
[0063] Example 2 Enzyme-linked immunosorbent assay detects the targeted affinity of positive phage clones with liver cancer cells
[0064] 2.1 Purification of positive phage clones.
[0065] 2.1.1 Amplification of positive phage: Add 20ml of LB / Tet liquid medium to the conical flask, then add E. coli bacteria liquid and phage to be amplified at 1:100, place at 37ºC, and shake vigorously in a constant temperature shaker for 4.5 h, the amplified solution of phage was obtained.
[0066] 2.1.2 Purification of positive phage: Centrifuge the phage amplification solution obtained by the above steps at 4ºC, 12000r / min for 10min, take the supernatant, add 1 / 6 volume of PEG-NaCl to precipitate overnight, centrifuge at 12000r / min for 15min, discard the Remove the supernatant, dissolve the precipitate with TBS buffer, give 1 / 6 volume of PEG-NaCl again, and incubate on ice for 1 h. 4ºC, 14000r / min, centrifuge for 15min, discard the supernatant, dissolve the obtained precipitate with TBS-...
Embodiment 3
[0079]Example 3 Determination and analysis of the DNA sequences of positive phage clones.
[0080] 3.1. Positive monoclonal phage selection.
[0081] The phage solution obtained after the third round of screening was used for titer determination and LB plates were prepared. On the plate with less than 100 growing spots, 20 well-grown locus coeruleus were randomly selected at intervals of 5 mm. 20 randomly picked locus coeruleus were added to 1 ml of logarithmic pre-host bacterial solution (same as phage amplification), and were amplified by rapid shaking at 37 °C and 200 rpm for 4.5 hours.
[0082] 3.2 Extraction of positive monoclonal phage single-stranded DNA.
[0083] Centrifuge the amplified monoclonal phage solution at 4°C and 14000rpm for 30 seconds, transfer the supernatant to a new tube, centrifuge at 4°C and 1000rpm for 30 seconds, and transfer 80% of the supernatant to a new nuclease-free centrifuge. In the tube, take 300ul bacterial solution and add 300ul glycerol...
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