Lentiviral expression vector for specifically promoting hepatic cell CYP2E1 gene high expression, construction method of vector and application of vector

A CYP2E1 and expression vector technology, applied in the field of genetic engineering, to achieve high transfection efficiency, good operation effect, and less dosage

Inactive Publication Date: 2013-01-16
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the prior art, there is no vector that can continuously

Method used

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  • Lentiviral expression vector for specifically promoting hepatic cell CYP2E1 gene high expression, construction method of vector and application of vector
  • Lentiviral expression vector for specifically promoting hepatic cell CYP2E1 gene high expression, construction method of vector and application of vector
  • Lentiviral expression vector for specifically promoting hepatic cell CYP2E1 gene high expression, construction method of vector and application of vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Design of CYP2E1 gene primers.

[0026] According to the CYP2E1 gene coding sequence (GenBank NM_000773.3), use Oligo7 to analyze it, find the upstream primer and downstream primer (requires no primer dimer as much as possible and the annealing temperature difference is small), and then in the upstream primer and downstream primer Protective bases and enzyme cleavage sites Xho I and EcoR I were added to the 5' end respectively, and the designed primer sequences are shown in Table 1. The designed PCR primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0027]

Embodiment 2

[0028] Example 2 Construction of a lentiviral vector that specifically promotes the high expression of CYP2E1 gene in hepatocytes.

[0029]After diluting the synthesized primers, use Premix PrimeSTAR HS enzyme to amplify the coding sequence of CYP2E1 gene, recover it by electrophoresis, then perform A-tailing reaction, and use T4 DNA ligase to connect to the pGM-T vector to obtain the ligation product (2E1-T vector), the ligation product was transformed into competent Escherichia coli DH5α, evenly spread on the LB medium plate containing ampicillin, and incubated at 37°C for 12 h, and a negative control group 1 (competent The cells were evenly spread on the plate without ampicillin), the negative control group 2 (the competent cells were evenly spread on the plate containing 100 μg / ml ampicillin), the positive control group 1 (the double enzyme cut empty vector The ligation products of the samples were evenly spread on the plate containing 100 μg / ml ampicillin), positive con...

Embodiment 3

[0035] Example 3 Lentiviral packaging.

[0036] 293FT cells were cultured, and the cells in good growth state were inoculated into six wells, 10 per well 6 Use the lentivirus packaging auxiliary kit to transfect 2 μg of the extracted recombinant plasmid pLVX-CYP2E1 into 293FT cells, collect the supernatant medium containing the virus after 48 hours, filter the virus liquid with a 0.45 μm sieve, and use it for infection L-02 hepatocytes, the titer of the virus detected by the Lenti-X GoStix kit was 5×10 6 ~5×10 7 IFU.

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Abstract

The invention provides a lentiviral expression vector for specifically promoting hepatic cell CYP2E1 gene high expression. The lentiviral expression vector comprises a fundamental sequence, a resistant gene sequence, a multiple cloning site sequence, a promoter sequence and a CYP2E1 gene cDNA sequence of a pLVX-AcGFP-C1 expression vector, multiple cloning sites include XhoI enzyme cutting sites and EcoRI enzyme cutting sites, and the CYP2E1 gene cDNA sequence comprises XhoI enzyme cutting sites, a CYP2E1 gene coding sequence and EcoRI enzyme cutting sites and is forwardly inserted into the multiple cloning site sequence. The lentiviral expression vector has the advantages of high transfection efficiency, low consumption and capability of specifically, continuously, efficiently and stably expressing CYP2E1 genes, and can serve as a powerful tool applied to drug research and development related to CYP2E1.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a lentiviral expression vector for specifically promoting the high expression of CYP2E1 gene in liver cells, a construction method and application thereof. Background technique [0002] Cytochrome P450 enzymes (Cytochrome P450, CYP450) are an important mixed-function oxidase system, mainly distributed in the endoplasmic reticulum and mitochondria of organisms, and participate in the biotransformation process of many endogenous and exogenous substances. CYP2E1 belongs to the CYP2E subfamily of the CYP450 family, encoded by the CYP2E1 gene, mainly exists in the liver, accounts for about 7% of the total liver CYP enzymes, and mainly metabolizes small molecular substances. At present, there are more than 70 substrates identified as CYP2E1, which are involved in the metabolism of nitrosamines, isoniazid, carbon tetrachloride, clovaxazone, acetaminophen, theophy...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66A61K48/00A61P43/00
Inventor 徐新云毛侃琅程锦泉彭朝琼
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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