Plasmid for detecting and screening circulating tumor cells and detecting and screening method using same
A tumor cell and screening method technology, applied in the field of plasmid detection and screening of circulating tumor cells, can solve the problems of low sensitivity, inability to detect cancer patients, and high difficulty, and achieve the effects of high sensitivity, low cost and strong specificity
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Embodiment 1
[0110] A plasmid for detecting and screening circulating tumor cells, the scFvHer2-Rluc-6*his-pcDNA3 plasmid contains the single-chain variable fragment scFvHer2 of the antibody, the bioluminescent protein sequence Rluc extracted from Renilla, and has a his tag;
[0111] 具体的,从海肾中提取的生物体发光蛋白序列Rluc的氨基酸序列为MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ;
[0112] On the basis of the constructed scFvHer2-Rluc-pcDNA3 plasmid, primers were designed on the upstream of the single-chain variable fragment scFvHer2 of the antibody and the downstream of the bioluminescent protein sequence Rluc extracted from Renilla, in which
[0113] The upstream primer is GCGCAAGCTTaccatggatatccagatgacccag;
[0114] The downstream primers have Hi...
Embodiment 2
[0117] The method for detecting and screening circulating tumor cells using the plasmid of Example 1 comprises the following steps:
[0118] ① The single-chain variable fragment scFvHer2 of the antibody was connected with the bioluminescent protein sequence Rluc extracted from Renilla renilla, and the his tag was used to prepare the scFvHer2-Rluc-6*his-pcDNA3 plasmid; scFvHer2-Rluc-6*his -The construction process of the pcDNA3 plasmid is as follows: construct the scFvHer2-Rluc-pcDNA3 plasmid, then use the scFvHer2-Rluc-pcDNA3 plasmid as a template to amplify the PCR product, digest it with HindIII and XbaI, and connect it to pcDNA3 to obtain scFvHer2-Rluc- 6*his-pcDNA3 plasmid.
[0119] ②Transfect 293ft cells with the scFvHer2-Rluc-6*his-pcDNA3 plasmid obtained in step ① to obtain the scFvHer2-Rluc-6*his fusion protein; specifically,
[0120] ⑴Cultivate 293ft cells until the confluence is 90-95% for transfection;
[0121] (2) In the first centrifuge tube, add 1ml Opti-MEM re...
Embodiment 3
[0128] Using the scFvHer2-Rluc-6*his fusion protein in Example 2 to target circulating tumor cells, including the following steps:
[0129] Add scFvHer2-Rluc-6*his fusion protein to different numbers (0, 10, 100, 1000, 10000) of positive cells and negative cells, incubate on a shaker for 3-5 hours, add 10ml of passive lysate, and place on a shaker Lyse at room temperature for half an hour, centrifuge at 14,000 rpm for 10 minutes, pipette the supernatant into a new tube, discard the cell debris, and measure the photometric value on the machine, the unit is LU / microliter, and each group performs three parallel experiments, the results are shown in the table 1 and figure 2 shown.
[0130] Table 1 Analysis results of fusion proteins targeting circulating tumor cells with different numbers of positive cells
[0131]
[0132] From the data in Table 1 and figure 2 It can be seen from the rising curve that when the number of positive cells is 0, there is little difference in t...
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