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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

Pending Publication Date: 2017-11-07
杭州京北生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Circulating tumor cell (CTC) detection and screening are becoming more and more important in daily operations. The screened CTC cells can be used for primary culture, gene or protein detection, but the current screening methods are low in sensitivity and cannot be detected in the early stages of cancer patients. Carry out CTC detection, this is due to the difficulty of CTC detection and screening in the current technology, this is because every 10 6 ~10 7 One mononuclear cell contains one CTC cell, because human blood contains a large number of red blood cells (5~9×10 9 / mL), white blood cells (5~10×10 6 / mL) and platelets (2.5~4×10 8 / mL), the detection and screening of CTC cells has a high background, and ultra-high sensitivity methods and technologies are required in the detection and screening process.

Method used

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  • Plasmid for detecting and screening circulating tumor cells and detecting and screening method using same
  • Plasmid for detecting and screening circulating tumor cells and detecting and screening method using same
  • Plasmid for detecting and screening circulating tumor cells and detecting and screening method using same

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a plasmid for detecting and screening circulating tumor cells and a detecting and screening method using the same, and relates to the technical field of detecting and screening of cells. By the detecting and screening method, the plasmid for detecting and screening the circulating tumor cells can screen surface antigens of the circulating tumor cells in a targeted manner, can screen nickel magnetic beads in combination with a his tag, and is good in specificity and high in sensitivity. By the circulating tumor cell detecting and screening method, a single-chain variable fragment scFvHer2 of an antibody is connected with a biological light-emitting protein sequence Rluc extracted from sea pansy, the plasmid with the his tag transfects 293ft cells to obtain fusion protein, and the fusion protein can target the surface antigens of the circulating tumor cells and is transformed into an amplified signal for detecting and screening the circulating tumor cells; the detecting and screening method is safe in operation, low in cost, high in specificity and high in sensitivity; the detecting and screening method can be applied to detection and screening of the CTCs (circulating tumor cells) in peripheral blood of a patient suffering from breast cancer, and the screened CTCs are applied to primary culture and gene or protein detection.

Description

technical field [0001] The invention relates to the technical field of cell detection and screening, in particular to a plasmid for detection and screening of circulating tumor cells and a detection and screening method using the plasmid. Background technique [0002] Circulating tumor cell (CTC) detection and screening are becoming more and more important in daily operations. The screened CTC cells can be used for primary culture, gene or protein detection, but the current screening methods are low in sensitivity and cannot be detected in the early stage of cancer patients. Carry out CTC detection, this is due to the difficulty of CTC detection and screening in the current technology, this is because every 10 6 ~10 7 One mononuclear cell contains one CTC cell, because human blood contains a large number of red blood cells (5~9×10 9 / mL), white blood cells (5~10×10 6 / mL) and platelets (2.5~4×10 8 / mL), the detection and screening of CTC cells has a high background, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/09G01N33/569G01N33/574G01N21/64
CPCG01N21/6486G01N33/56966G01N33/57415C12N5/0693C12N15/85C12N2509/00C12N2800/107
Inventor 唐东起赵小刚王文娟李世武吴琦
Owner 杭州京北生物科技有限公司
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