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Galpha gene overexpression lentiviral vector and lentivirus and construction method thereof

A technology of lentiviral vector and construction method, applied in the field of Gα gene overexpression lentiviral vector, can solve the problems of low success rate and low transfection efficiency, and achieve the effect of promoting stable expression

Inactive Publication Date: 2016-12-07
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 the Gα 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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  • Galpha gene overexpression lentiviral vector and lentivirus and construction method thereof
  • Galpha gene overexpression lentiviral vector and lentivirus and construction method thereof
  • Galpha gene overexpression lentiviral vector and lentivirus and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of Gα gene overexpression lentiviral vector.

[0044] 1. Artificially synthesized Gα gene, the DNA of which is shown in SEQ ID NO.1.

[0045] 2. Using PrimeSTAR high-fidelity enzyme, using the artificially synthesized Gα gene as a template, perform PCR amplification with the primers shown in Table 1:

[0046] Table 1

[0047]

[0048] The PCR reaction system and conditions are shown in Table 2:

[0049] Table 2

[0050]

[0051] The PCR program is shown in Table 3:

[0052] table 3

[0053]

[0054] 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 electrophoresis gel, and recover and purify it with a DNA recovery kit;

[0055] 4. Use EcoRI and BamHI restriction endonucleases to perform double enzyme digestion on the PCR product of the Gα gene and the lentiviral expression vector pLVX-IRES-mCherry (digestion a...

Embodiment 2

[0068] Example 2: Packaging of Gα lentiviruses

[0069] 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 / LCO 2 Culture in the incubator;

[0070] 2. Prepare four plasmid DNA solutions in the lentiviral packaging system:

[0071] pRSV-Rev 10 μg,

[0072] pMDLg-pRRE 15 μg,

[0073] pMD2G 7.5 μg,

[0074] pLVX-IRES-mCherry-Gα 20 μg;

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

[0076] 3. Transfect when the cell density reaches 60%-70%. At this time, 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, d...

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 attached to the wall and reach a confluence of 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 to incubate for 1 h.

[0085] 2. Add 5 μL of the Gα 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 Gα lentivirus solution The only difference is that it does not contain the Gα gene, and the rest of the preparation methods are the same), the other well is used as a blank cell control, shake well and put it in 37°C, 5% (v / v) CO 2 , 95% relative humidity incubator culture

[0086] 3. After 6...

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Abstract

The invention relates to a Galpha gene overexpression lentiviral vector and lentivirus and a construction method thereof, and belongs to the technical field of molecular biology. Construction is conducted by taking a pLVX-IRES-mCherry lentiviral expression vector as a basis through a gene recombination technology, a Galpha gene is integrated on the vector, and the Galpha gene overexpression lentiviral vector pLVX-IRES-mCherry-Galpha is obtained. The Galpha gene overexpression lentiviral vector pLVX-IRES-mCherry-Galpha is packaged through lentiviral packaging plasmids including pRSV-Rev, pMDLg-pRRE and pMD2.G, HEK 293 cell transfection is conducted, and the Galpha lentivirus is obtained. The Galpha gene overexpression lentiviral vector constructed through the method is high in host cell transfection efficiency and can specifically, continuously and efficiently promote stable expression of the Galpha gene in host cells.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a Gα gene overexpression lentiviral vector, a Gα 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, and they function in the form of T1R2+T1R3 heterodimers to 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 be...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66
CPCC07K14/4722C12N15/66C12N15/86C12N2740/15043
Inventor 夭建华朱洲海李雪梅缪明明米其利管莹高茜唐萍
Owner CHINA TOBACCO YUNNAN IND
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