Lentiviral expression vector for specifically promoting high expression of CYP3A4 (cytochrome P450 3A4) gene in hepatocytes, and construction method and application thereof

An expression vector and liver cell technology, applied in the field of genetic engineering, to achieve high transfection efficiency, reduced sequence synthesis costs, and low dosage

Inactive Publication Date: 2013-01-09
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no vector that can continuously, stably and efficiently express CYP3A4 gene in human cells

Method used

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  • Lentiviral expression vector for specifically promoting high expression of CYP3A4 (cytochrome P450 3A4) gene in hepatocytes, and construction method and application thereof
  • Lentiviral expression vector for specifically promoting high expression of CYP3A4 (cytochrome P450 3A4) gene in hepatocytes, and construction method and application thereof
  • Lentiviral expression vector for specifically promoting high expression of CYP3A4 (cytochrome P450 3A4) gene in hepatocytes, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Design of CYP3A4 gene primers.

[0026] According to the CYP3A4 gene coding sequence (GenBank NM_001202855.2), 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 Xma 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 CYP3A4 gene in hepatocytes.

[0029]After diluting the synthesized primers, use Premix PrimeSTAR HS enzyme to amplify the coding sequence of CYP3A4 gene, recover it by electrophoresis and perform A-tailing reaction, then use T4 DNA ligase to connect to the pGM-T vector to obtain the ligation product (3A4-T vector), the ligation product was transformed into competent Escherichia coli DH5α, spread evenly on the LB medium plate containing ampicillin, and cultured at 37°C for 12 h, and set 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-CYP3A4 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 high expression of CYP3A4 (cytochrome P450 3A4) gene in hepatocytes. The lentiviral expression vector for specifically promoting high expression of CYP3A4 (cytochrome P450 3A4) gene in hepatocytes comprises fundamental sequences, resistance gene sequences, multiple-cloning-site sequences, promoter subsequences, and CYP3A4 gene cDNA (complementary DNA) sequences in a pLVX-AcGFP-C1 expression vector. Multiple cloning sites include an XhoI restriction enzyme cutting site and an XmaI restriction enzyme cutting site. The CYP3A4 gene cDNA sequences include XhoI restriction enzyme cutting sites, CYP3A4 gene coded sequences and XmaI restriction enzyme cutting sites. The CYP3A4 gene cDNA sequences are forwardly inserted into the multiple-cloning-site sequences. The lentiviral expression vector is capable of expressing the CYP3A4 gene specifically, continuously, efficiently and stably with high transfection efficiency and low consumption, and can be used as a powerful tool applied to pharmaceutical research and development related to CYP3A4.

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 CYP3A4 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. CYP3A4 belongs to the CYP3A subfamily of the CYP450 family, encoded by the CYP3A4 gene, and mainly exists in the liver, accounting for about 30-40% of the total liver CYP enzymes. It is the most abundant CYP in the liver and the most important human drug metabolism enzyme. CYP3A4 is involved in the metabolism of about 38 types of more than 150 clinical therapeutic drugs, including antibiotics, antifungal dr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66A61K48/00
Inventor 徐新云毛侃琅何晓阳毛吉炎应明
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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