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Lentiviral vector efficiently mediating overexpression of T1R2 gene and lentivirus, and construction methods thereof

A technology of gene overexpression and lentiviral vector, applied in the field of molecular biology, can solve the problems of low success rate of cell lines, low transfection efficiency of liposome transfection plasmid vector, etc., 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 T1R2 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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  • Lentiviral vector efficiently mediating overexpression of T1R2 gene and lentivirus, and construction methods thereof
  • Lentiviral vector efficiently mediating overexpression of T1R2 gene and lentivirus, and construction methods thereof
  • Lentiviral vector efficiently mediating overexpression of T1R2 gene and lentivirus, and construction methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Construction of T1R2 gene overexpression lentiviral vector.

[0045] 1. Artificially synthesized T1R2 gene, the DNA sequence of which is shown in SEQ ID NO.1.

[0046] 2. Using PrimeSTAR high-fidelity enzyme, using the artificially synthesized T1R2 gene as a template, 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 (10μM)

1μL

Downstream primer (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 electrophore...

Embodiment 2

[0072] Example 2: Packaging of T1R2 lentivirus

[0073] 1. Replace the HEK293 cells with a confluence of more than 80% with antibiotic-free medium DMEM+10% (v / v) FBS, 37°C, 5% (v / v) CO 2 Incubator for 2 hours.

[0074] 2. Mix the packaging plasmid mixture (pLP / VSVG+pLP1+pLP2, 3 μg each) and 4 μg T1R2 gene overexpression lentiviral vector in 400 μL 0.9% (w / v) saline, and add 50 μL transfection reagent Fitran, room temperature After standing and incubating for 20 minutes, slowly and evenly drop it into the culture dish containing HEK293 cells in step 1, and shake it properly; record it as the start time of transfection.

[0075] 3. After 4-6 hours of transfection, prepare to change the medium. After discarding the old medium, absorb an appropriate amount of PBS to gently rinse the cells for 1-2 times, and replace with DMEM medium containing 2% (v / v) FBS.

[0076] 4. Collect the supernatant 72 hours after transfection, centrifuge the collected supernatant at 3000rpm at 4°C for ...

Embodiment 3

[0079] Example 3: Cell Transfection

[0080] 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.

[0081] 2. Add 5 μL of the T1R2 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 T1R2 lentivirus solution The only difference is that it does not contain the T1R2 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.

[0082] 3. After 6 hours of transfection, suck away the medium in the well, and wash it twice with PBS; r...

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Abstract

The invention relates to a lentiviral vector efficiently mediating overexpression of a T1R2 gene and a lentivirus, and construction methods thereof, belonging to the technical field of molecular biology. According to the invention, construction is carried out by using gene recombination technology on the basis of a pCDH-CMV-MCS-Sv40-Neo lentiviral vector and the T1R2 gene is integrated, so the lentiviral vector pCDH-CMV-MCS-Sv40-Neo-T1R2 for overexpression of the T1R2 gene is obtained. Lentivirus packaging plasmids pLP / VSVG, pLP1 and pLP2 are used for packaging of the lentiviral vector for overexpression of the T1R2 gene T1R2, and the T1R2 lentivirus is obtained after transfection of HEK293 cells with the plasmids. The lentiviral vector constructed in the invention has high host cell transfection efficiency and can specifically, continuously and efficiently promote stable expression of the T1R2 gene in host cells.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a lentiviral vector for efficiently mediating T1R2 gene overexpression, a 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N7/01
CPCC12N15/86C07K14/705C12N7/00C12N15/66C12N2740/15021C12N2740/15043C12N2740/15051
Inventor 朱洲海夭建华李雪梅缪明明高茜米其利管莹唐萍
Owner CHINA TOBACCO YUNNAN IND
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