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T1R3 gene overexpressed lentiviral vector and lentivirus, and construction methods thereof

A lentiviral vector and gene overexpression technology, applied in the field of molecular biology, can solve the problems of low success rate of cell lines and low transfection efficiency, and achieve the effect of promoting stable expression

Inactive Publication Date: 2016-12-21
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing method uses the plasmid vector integrated with T1R3 gene to transfect host cells through liposomes, but the transfection efficiency of liposome-transfected plasmid vectors is low, and it is necessary to construct a stable co-expression of T1R2 and T1R3 and Gα genes have a lower success rate in cell lines

Method used

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  • T1R3 gene overexpressed lentiviral vector and lentivirus, and construction methods thereof
  • T1R3 gene overexpressed lentiviral vector and lentivirus, and construction methods thereof
  • T1R3 gene overexpressed lentiviral vector and lentivirus, and construction methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: T1R3 gene overexpression lentiviral vector construction.

[0045] 1. Artificially synthesize the T1R3 gene, the DNA of which is shown in SEQ ID NO.1.

[0046] 2. Use PrimeSTAR high-fidelity enzymes, use the artificially synthesized T1R3 gene as a template, and perform PCR amplification with the primers shown in Table 1:

[0047] Table 1

[0048]

[0049]

[0050] The PCR reaction system and conditions are shown in Table 2:

[0051] Table 2

[0052] Reagent

volume

template

1μL

Upstream primer T1R3-F (10μM)

1μL

Downstream primer T1R3-R (10μM)

1μL

Prime STAR Max (2x)

10 μL

wxya 2 o

7μL

total capacity

20 μL

[0053] The PCR program is shown in Table 3:

[0054] table 3

[0055]

[0056] 3. Agarose gel electrophoresis to detect whether the size of the PCR amplification product is consistent with the expectation, cut the target fragment band in the agarose gel electr...

Embodiment 2

[0071] Example 2: Packaging of T1R3 lentivirus

[0072] 1. Digest HEK293 cells in the logarithmic growth phase with trypsin, and the cell density is 5×10 6 re-inoculated in a cell culture dish with a diameter of 15 cm containing 25 mL of antibiotic-free DMEM medium containing 10% (v / v) FBS, at 37 °C, 50 mL / L CO 2 Culture in the incubator;

[0073] 2. Prepare four plasmid DNA solutions in the lentiviral packaging system:

[0074]

[0075] Add the above plasmid to sterile water to make up to 1800μL, then add CaCl 2 (2.5mol / L) solution 200μL, mix well, add 2000μL of 2×PBS buffered saline solution, and place at room temperature for 20-30min;

[0076] 3. Transfect when the cell density reaches 60%-70%. At this point, transfer the DNA solution and calcium phosphate mixture prepared in step 2 to the cell culture dish containing monolayer cells obtained in step 1, mix well, discard the culture solution after culturing for 12 h, and wash the cells with 15 mL of PBS solution for ...

Embodiment 3

[0083] Example 3: Cell Transfection and Screening

[0084] 1. Cultivate HEK293 cells in a 6-well cell culture plate. After the cells are completely adherent and confluent to 40%-50%, the cells can be transfected; before transfection, replace the cells in each well with 500 μL of fresh DMEM for complete culture Base, and 1 μL of polybrene (polybrene) was added to each well, and placed in an incubator for 1 h.

[0085] 2. Add 5 μL of the T1R3 lentivirus solution obtained in Example 2 to the two wells of the above-mentioned 6-well cell culture plate, and add 10 μL of the empty lentivirus solution to the cells in one well (the empty lentivirus solution and the T1R3 lentivirus solution The only difference is that it does not contain the T1R3 gene, and the rest of the preparation methods are the same), and the other well is used as a blank cell control. 2 , 95% relative humidity incubator culture

[0086] 3. After 6 hours of transfection, suck away the medium in the well, and wash...

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Abstract

The invention relates to a T1R3 gene overexpressed lentiviral vector and a T1R3 lentivirus, and construction methods thereof, and belongs to the technical field of molecular biology. By a gene recombination technology, construction is performed by taking a pLVX-Puro lentiviral expression vector as the basis, a T1R3 gene is integrated, and the T1R3 gene overexpressed lentiviral vector pLVX-Puro-T1R3 is obtained. Lentiviral packaging plasmids pRSV-Rev, pMDlg-pRRE and pMD2.G are used for packaging the T1R3 gene overexpressed lentiviral vector pLVX-Puro-T1R3, and HEK293 cells are transfected to obtain the T1R3 lentivirus. The constructed T1R3 gene overexpressed lentiviral vector has high transfection efficiency to host cells, and can specifically, continuously and efficiently promote stable expression of the T1R3 gene in the host cells.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a T1R3 gene overexpression lentiviral vector, a T1R3 lentivirus and a construction method thereof. Background technique [0002] Taste function studies have shown that sweetness is mainly mediated by the G protein-coupled receptors (GPCRs) family of taste cells, that is, taste receptor family 1 members (T1Rs). Among them, T1R2 and T1R3 are sweet taste receptors, they function in the form of T1R2+T1R3 heterodimer, and jointly recognize sweet taste. After the sweet taste receptor binds to the ligand, the downstream G protein is activated, its α subunit is separated from the β and γ subunits, and the Gα subunit enters the cytoplasm to activate downstream effectors, eventually leading to an increase in the intracellular calcium ion concentration. Therefore, by constructing a cell line co-expressing T1R2, T1R3 and Gα genes, the calcium flux detection method can ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66C12N7/01
CPCC12N15/86C07K14/705C12N7/00C12N15/66C12N2740/15021C12N2740/15043C12N2740/15051
Inventor 夭建华朱洲海李雪梅缪明明管莹高茜米其利唐萍
Owner CHINA TOBACCO YUNNAN IND
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