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Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof

A BEAS-2B, CYP2A13 technology, applied in the field of human recombinant gene lung epithelial cells, can solve the problems of unstable expression of the target protein, time-consuming and laborious, low success rate, etc., and achieve stable expression, high transfection efficiency, and responsive effects.

Inactive Publication Date: 2013-05-22
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mainly due to transiently expressed cells, the expression of the target protein is unstable, and the difference between different experiments is sometimes very large
In addition, the efficiency of constructing stable expression cells with adenovirus is low, the success rate is small, and it is often time-consuming and laborious

Method used

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  • Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof
  • Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof
  • Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Construction and verification of pLJM1-GFP-CYP2A13 plasmid

[0036] The lentiviral plasmid pLJM1-GFP-CYPs containing the target CYP gene was constructed by endonuclease double digestion and enzyme ligation, which is the pFastBac existing in our laboratory. TMThe 1-CYP2A13 plasmid and the lentivirus-specific empty plasmid pLJM1-GFP were digested at the same time, and the digested products were recovered and enzyme-ligated respectively, and then transformed into DH5α competent cells, screened by ampicillin, and positive clones were picked to extract recombinant plasmids. Recombinant plasmids were verified by PCR using specific primers for each gene.

[0037] 2. Lentiviral vector packaging and infection of BEAS-2B cells

[0038] Transfer 293T cells to a 60mm culture dish, and transfect when the cell density reaches 80-90% confluence. Prepare the liposome transfection complex: DMEM (serum-free, double-antibody-free), the complex for transfection per dish contains 6 μg ...

Embodiment 2

[0048] 1. Dose-dependent application research on the effect of cells on AFB1

[0049] Using the BEAS-2B / CYP2A13 cells obtained in Example 1, and using the empty cell BEAS-2B / Vector as a control, the cells were given 0, 10, 100, 1000 and 10,000 nM of AFB1, treated for 24 hours, and then detected by the MTT method Cell viability under different doses of treatment, evaluate the toxic effect of cells under different doses of AFB1 treatment, and construct the dose-effect relationship.

[0050] 2. Time-dependent application research on the effect of cells on AFB1

[0051] Using the BEAS-2B / CYP2A13 cells obtained in Example 1, and using the empty cell BEAS-2B / Vector as a control, the cells were treated with 100 nM AFB1 for 24, 48 and 72 hours, and then the MTT method was used to detect the CYP2A13 cells treated at each time point. Cell viability, evaluate the toxic effect of cells under different time of AFB1 treatment, and construct the time-effect relationship.

[0052] Results:...

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Abstract

The invention relates to a human recombination gene lung epithelial cell, in particular to a human lung cell BEAS-2B / CYP2A13 and application thereof. The human lung cell BEAS-2B / CYP2A13 is characterized in that: the cell strain is collected in China Center for Type Culture Collection, and the collection number is CCTCC No: C2010127. In the method, the BEAS-2B cell for stably expressing CYP2A13 is constructed by utilizing a slow virus system for the first time at home and abroad, which is the key technology in the invention. Compared with other virus vector systems, the technology has the advantages of high transfection efficiency, stable expression, easiness in screening and the like, and has the unique advantage on transfection of immortalized cells.

Description

technical field [0001] The present invention relates to human recombinant lung epithelial cells, in particular to a human lung cell BEAS-2B / CYP2A13 and its application. Background technique [0002] Choosing an ideal model is a prerequisite for doing a good job in scientific research. Cells are undoubtedly an ideal and commonly used research model in scientific research. On the one hand, a single cell can reduce the error between experiments and ensure the controllability and repeatability of the research results; on the other hand, the cell test cycle is short, the cost is low, and the experimental conditions are not demanding. Therefore, it is favored by the majority of scientific researchers. With the continuous deepening of research, human beings have entered the era of post-genome research, the core content of which is to study the function of genes. Due to the low or no expression of specific proteins in most cell lines, it is urgent to construct cell models that hig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12Q1/02
Inventor 王守林陆慧媛李孜音李建民杨雪娇
Owner NANJING MEDICAL UNIV
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