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Construction and application of Claudin18.2 reporter gene CHO-K1 stably transfected cell strain

A CHO-K1 and reporter gene technology, applied in the field of biomedicine, can solve problems such as interference, small signal-to-noise ratio window of the method, and poor sensitivity

Pending Publication Date: 2022-05-13
宁波熙宁检测技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to characterize the functional activity of Claudin18.2 antibody drugs, the CDC effect is usually selected by co-incubating human serum and target cells in the presence of Claudin18.2 antibody drugs, by detecting the release of the contents of the target cells such as lactate dehydrogenase , detected by a lactate dehydrogenase detection kit (such as the merchant Promega, product number J2380); the ADCC effect is usually selected by co-incubating human peripheral blood PBMCs and target cells in the presence of Claudin18.2 antibody drugs, by detecting the target The release of the contents of the cells, such as lactate dehydrogenase, is detected by the detection kit of lactate dehydrogenase; the release of the contents of the target cells, such as lactate dehydrogenase, is used to characterize the sensitivity of the ADCC effect or CDC effect of the usual method Relatively poor, the signal-to-noise ratio window of the method is small, and it is susceptible to interference from the release of lactate dehydrogenase from dead PBMC itself.

Method used

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  • Construction and application of Claudin18.2 reporter gene CHO-K1 stably transfected cell strain
  • Construction and application of Claudin18.2 reporter gene CHO-K1 stably transfected cell strain
  • Construction and application of Claudin18.2 reporter gene CHO-K1 stably transfected cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction process of expressing Claudin 18.2 stably transfected cell line

[0025] Claudin 18.2 lentivirus (customized by Jinweizhi) was used to infect 1E4 cells / well of CHO-K1 cells (ATCC, Cat. No. CCL-61) in a 24-well plate at an MOI of 20, with a total volume of 1000 μL; The lentivirus was removed; on the third day, the digested cells were transferred to a 6-well plate and cultured under pressure in F12 medium with 500 μL / mL Hygromycin added. After maintaining 500 μg / ml Hygromycin in the F12 medium for growth, passage and pressurization for two generations, cells were plated in a 96-well plate on the 10th day; on the 30th day, the monoclonal observed in the 96-well plate was transferred to The orifice plate was transferred to a 6-well plate after evaluation and screening, and T25, T75, and T175 were cultured to obtain a single clone, clone number CA03.

Embodiment 2

[0026] Example 2 Evaluation of Transfection Efficiency of Monoclonal Cell Line Expressing Claudin 18.2

[0027] The optimal clone CA03 was selected and cultured in F12 medium supplemented with 500 μL / mL Hygromycin, and untransfected CHO-K1 cells were cultured at the same time. After trypsin digestion and counting, take 1E6 cells to a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, leave about 100ul supernatant to resuspend the cells, add 5μL Anti-18.2-362Antibody, the final concentration is 5μg / ml, and mix well Minimize the generation of air bubbles, incubate at 4°C for 30 minutes, remove and add an appropriate amount of PBS, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, repeat this operation twice, and add 5 μL Goat pAb to Hu IgG (PE) to the remaining 100 μL sample, with a final concentration of 5 μg / mL, incubate at 4°C for 30 min, take out and add appropriate amount of PBS, centrifuge at 1000 rpm for 5 min, discard the supern...

Embodiment 3

[0029] Example 3 Construction process of CMV-Luciferase reporter gene stably transfected cell line

[0030] CMV-Luc2 lentivirus (customized by Heyuanbio) MOI is 20, infects 1E5 cells / well of CHO-K1Claudin18.2 (CA03) cells in a 24-well plate with a total volume of 1000 μL; Virus; on the third day, the digested cells were transferred to a 6-well plate and cultured under pressure in F12 medium with 5 μL / mL Puromycin and 500 μg / mL Hygromycin. After maintaining pressurized 5 μL / mL Puromycin and 500 μg / mL Hygromycin in F12 medium for two passages, cells were plated in a 96-well plate at 1 cell / well on the 10th day; The single clone was transferred to a 24-well plate. After evaluation and screening, it was transferred to a 6-well plate. T25, T75, and T175 were cultured to obtain a single clone, the clone number being CL25.

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Abstract

The invention discloses construction of a Claudin18.2 reporter gene CHO-K1 stably transfected cell strain, which comprises the following steps: transfecting CHO-K1 cells by using a lentivirus containing a Claudin18.2 gene, adding eukaryotic antibiotics for screening and monoclonal selection, and carrying out expression abundance detection and selection to obtain an appropriate high-expression Claudin18.2 stably transfected cell strain monoclonal; a Claudin18.2 stably transfected cell strain monoclonal antibody is infected by lentivirus containing a CMV-luciferase gene, and a proper Claudin18.2 reporter gene stably transfected cell strain is obtained by adding eukaryotic antibiotics for screening and monoclonal selection and performing functional evaluation. The invention also relates to a detection method for CDC killing by using the reporter gene stably transfected cell strain, a detection method for ADCC killing by using the reporter gene stably transfected cell strain, and the like. According to the present invention, the Claudin18.2 reporter gene CHO-K1 stably transfected cell strain is used for the CDC and ADCC cell killing determination method, such that the sensitivity is high, the external interference is not easily generated, and the stability of the used method is good.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the construction and application of a Claudin18.2 reporter gene CHO-K1 stably transfected cell line. Background technique [0002] Claudin18.2 (UniProt ID: P56856-2) is a transmembrane protein located on the cell membrane, which is highly expressed in various tumors, especially digestive system tumors and their metastases. Claudin18 is a tight junction (tight junction, TJs) protein that plays an important role in the important function of normal epithelial cells and cell adhesion. sex. [0003] There are two alleles in the first exon of the human CLDN18 gene, which express Claudin18.2 protein and Claudin18.1 protein respectively. Although Claudin18.1 and claudin18.2 are very similar in structure, they are highly expressed in tumors Not the same, in normal tissues, claudin18.1 is only expressed in the lung, while claudin18.2 is limitedly expressed in the stomach; The expres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12N5/071C12N15/12G01N33/68
CPCC12N15/86C07K14/47C12N5/0682G01N33/68C12N2740/15043
Inventor 黄启宽朱国振余跃云肖敏任亚哲
Owner 宁波熙宁检测技术有限公司
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