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Construction method of cell model for detecting pyrogens, cell model and pyrogen detection kit

A cell model, construction method technology, applied in microorganism-based methods, receptors/cell surface antigens/cell surface determinants, cells modified by introducing foreign genetic material, etc., can solve the problem of inability to transmit LPS signals, lack of cytoplasm Segmentation and other problems, to achieve high detection sensitivity, good reactivity, and overcome the effects of the new human monocyte activation test

Active Publication Date: 2016-11-23
牛刚
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] CD14 is a protein expressed on the surface of cytoplasm and leukocytes as glucose-phosphoinositide-linked protein, which has a high affinity with LPS, but the CD14 molecule lacks a cytoplasmic segment and cannot directly transmit LPS signals into cells

Method used

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  • Construction method of cell model for detecting pyrogens, cell model and pyrogen detection kit
  • Construction method of cell model for detecting pyrogens, cell model and pyrogen detection kit
  • Construction method of cell model for detecting pyrogens, cell model and pyrogen detection kit

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Experimental program
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Embodiment 1

[0098] The construction of embodiment 1 cell model

[0099] 1. Preparation of cDNA from human peripheral blood mononuclear cells

[0100] 1.1 Isolation and culture of human peripheral blood mononuclear cells

[0101] Take 5mL fresh anticoagulated whole blood from normal people, operate according to the manual of the human mononuclear cell separation medium kit, extract human peripheral blood mononuclear cells, add RPMI1640 medium to wash and centrifuge once, centrifuge at 1500r / min for 10min, discard supernatant. Add 3 to 4 mL of RPMI1640 culture medium containing 10% fetal bovine serum per mL of blood sample to resuspend the cells, count the cells, and adjust the cell density to 2×10 7 / mL cultured in 10cm 2 Place in a Petri dish at 37°C, 5% CO 2 After cultured in a cell culture box for 2-3 hours, the supernatant was removed to obtain adherent human mononuclear cells. Then draw 10 μL of the cell suspension diluted according to the required dilution ratio and mix it with ...

Embodiment 2

[0187] Example 2 Detection of pyrogen marker LPS using cell model

[0188] 1ELISA to identify the responsiveness of cells to LPS stimulation, and to detect the expression of IL-6 and TNFα

[0189] 293T, 293T / TLR4, 293T / CD14 / MD2, 293T / TLR4 / CD14 / MD2 four cell lines were stimulated with LPS, and the release of IL-6 and TNFα cytokines in the supernatant was detected by ELISA, 293T, 293T / TLR4 No response to LPS stimulation, 293T / CD14 / MD2, 293T / TLR4 / CD14 / MD2 release IL-6 or TNFα after LPS stimulation, but the release amount of 293T / CD14 / MD2 is not as much as 293T / TLR4 / CD14 / MD2, such as Figure 13A and Figure 13B shown.

[0190] Continue to use 293T / TLR4 / CD14 / MD2 cell line for follow-up experiments. The release of IL-6 at different time points after LPS stimulation was detected. It was found that the release of IL-6 reached a peak around 5-6 hours, such as Figure 14 shown. Detect the 6h IL-6 release after 293T / TLR4 / CD14 / MD2 with different cell numbers stimulated with 5EU / mL L...

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Abstract

The invention provides a construction method of a cell model for detecting pyrogens, the cell model and a pyrogen detection kit. The cell model utilizes specific locations of CRISPR / CAS9 induced genomes to form double-bond fission, TLR4 and CD14-MD2 are knocked into two chromosomes of a cell line respectively by the aid of the homologous recombination repair principle, green fluorescence GFP and red fluorescence RFP are respectively used for tracing finally successfully constructed TLR4 / CD14 / MD2 fixed-point knocked-in fluorescent tracer cell models, and the LPS stimulating cell model can detect release of IL-6 and TNF-a cytokines by means of ELISA, Western Blot, mass spectrum and immunomagnetic beads. The cell model is good in stability and high in sensitivity, and the lowest detectable limit can reach 0.005EU / mL and is far lower than 0.025EU / mL of the Tachypleus Amebocyte Lysate method.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the detection of pyrogens in medicines and biological products (including genetic engineering products), specifically a method for constructing a cell model for detecting pyrogens, a cell model, a method for detecting pyrogens, and a kit . Background technique [0002] 1.1 Overview of pyrogens [0003] As we all know, the use of injection drugs or medical equipment in clinical treatment often leads to adverse reactions such as chills, chills, nausea, fever, vomiting, headache, leukopenia, increased vascular permeability, coma, shock and other adverse reactions that aggravate the condition. It not only increases the pain of the patient, but also affects the effect of rescue and treatment, and may even endanger the life of the patient. In 1875, Burdon-Sanderson (Li Zenghong, Wang Ruidong. Journal of Foshan Institute of Science and Technology, 2007, 25 (3): 43-45) showed that substances...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85G01N33/68C12R1/91
CPCC07K14/47C07K14/705C07K14/70596C12N15/85G01N33/6866G01N33/6869G01N2333/5412G01N2333/56C12N2800/80C12N2800/107C12N15/907G01N33/5047C12N5/0686C12N15/64C12N15/86G01N33/5044G01N33/6863G01N2333/525C12N2523/00G01N33/96
Inventor 牛刚谭焕然
Owner 牛刚
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