Tetrahymena cell line containing luciferase gene, construction method and applications thereof

A technology of luciferase gene and Tetrahymena, which is applied in the field of genetically engineered cell lines, can solve the problems that Tetrahymena cell lines have not been reported, and achieve the effect of simple and fast detection process, rapid response and detection operation

Inactive Publication Date: 2013-02-13
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there have been reports at home and abroad that the whole-cell biosensor constructed by bacteria, yeast and Tetrahymena has been used in the biological monitoring of water environment, but the Tetrahymena cell line containing luciferase constructed by the present invention has not been reported yet.

Method used

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  • Tetrahymena cell line containing luciferase gene, construction method and applications thereof
  • Tetrahymena cell line containing luciferase gene, construction method and applications thereof
  • Tetrahymena cell line containing luciferase gene, construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Amplification of Luciferase LUC Gene and Construction and Identification of Intermediate Plasmid pBX-LUC

[0029] Design primers, use PCR method to add BamH I and Asc I restriction sites on both ends of the firefly luciferase gene, and optimize the N-terminal and C-terminal sequences of the LUC gene to be able to The codons expressed efficiently in Tetrahymena, the PCR product is recovered and mixed with the vector pEASY-T1 in an appropriate ratio, ligated at 25°C for 20 minutes, and the ligated product is transformed into E. coli DH5а for large-scale amplification, and the plasmid pEASY-T1-LUC is extracted and combined Carry out restriction digestion identification.

[0030] PEASY-T1-LUC and the intermediate plasmid pBX were digested with BamH I and Asc I, and the digested product was recovered. The digested LUC and the vector were mixed in proportion, and ligated with T4 ligase overnight at 16°C. The product was transformed into Escherichia coli DH5а, coated wit...

Embodiment 2

[0031] Example 2 Construction and identification of Tetrahymena cell strain containing luciferase gene

[0032] The constructed intermediate vector was linearized by Xho Ⅰ restriction enzyme digestion, purified and concentrated with ethanol, then wrapped on gold particles, and transformed into Tetrahymena B2086 starved for 18-24 hours by gene gun particle bombardment, and then added with Paromonas 4 hours later (Final concentration is 100ug / ml) and CdCl 2 (The final concentration is 0.5ug / ml). Dispense them into 96-well plates. After incubating at 30°C for 3 days, gradually increase the concentration of paromomycin to screen recombinant Tetrahymena B2086-LUC. The principle is the process of gene recombination ( figure 2 ), and finally expand the recombinant Tetrahymena B2086-LUC, extract genomic DNA for PCR identification ( image 3 ). M: DNA standard molecular weight; 1: PCR products of LUC gene and MTT1 gene were detected using recombinant Tetrahymena B2086-LUC genome as templa...

Embodiment 3

[0033] Example 3 Expansion culture and preservation of Tetrahymena cell line B2086-LUC containing luciferase gene

[0034] Under the microscope, single cells of recombinant Tetrahymena were gradually expanded and cultured to obtain a large number of homotypic recombinant cell lines B2086-LUC, which were frozen in liquid nitrogen.

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Abstract

The invention relates to a tetrahymena cell line containing luciferase gene, a construction method and applications thereof. According to the present invention, a PCR technology is adopted to optimize a N-terminal sequence and a C-terminal sequence of firefly luciferase gene into tetrahymena preference codons, the optimized firefly luciferase gene LUC is linked to a carrier pBX to obtain firefly luciferase gene-containing recombinant plasmid pBX-LUC, a gene gun particle bombardment method is adopted to transform the firefly luciferase gene-containing recombinant plasmid pBX-LUC into tetrahymena thermophila B2086 cells, intracellular homologous recombination and paromomycin resistance screening are performed, and MTT1 gene in the cells are replaced by the LUC gene to obtain the engineered cell line B2086-LUC, wherein the engineered cell line B2086-LUC expresses luciferase under specific heavy metal induction, coloration is performed after a fluorescent substrate D-Luciferin is added, and the enlarged fluorescence intensity is detected on so as to reflect conditions of heavy metal pollutants in a reaction environment through quantitative data, such that the detection method is rapid and sensitive, and easy to operate. The tetrahymena cell line containing luciferase gene has a wide application prospect in biological monitoring of water body environment heavy metal pollution.

Description

Technical field [0001] The present invention relates to a genetic engineering cell line, specifically a Tetrahymena cell line containing a luciferase gene LUC and a construction method and application thereof, and more specifically a Tetrahymena cell line containing LUC preference codons Insect cell line and the cell line's monitoring of heavy metal pollution in water environment. Background technique [0002] Environmental pollution is currently one of the most serious problems in the world. At present, the most commonly used monitoring method in practical applications is to use instruments to perform physical and chemical monitoring of heavy metals in the environment. However, the monitoring process needs to clarify the pollutant composition, and the monitoring results cannot reflect some key points. Parameters such as bioavailability, toxicity and genotoxicity make the detection targets limited and the detection results one-sided. With the in-depth study of model organisms by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N1/11C12N15/79C12Q1/02C12R1/90
Inventor 王伟张鹏幸许静梁爱华
Owner SHANXI UNIV
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