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Expression vector for enhancing gene expression of 3 beta-HSD, construction method and application thereof

A technology of gene expression and expression vector, which is applied in the field of expression vector and its construction to promote the expression of 3β-HSD gene, and can solve the problem of lack of human cells

Active Publication Date: 2017-10-24
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The above studies all use indirect methods to regulate the expression of 3β-HSD gene. At present, there is a lack of expression vectors that can be transfected into human cells and express 3β-HSD genes continuously, stably and efficiently in human cells.

Method used

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  • Expression vector for enhancing gene expression of 3 beta-HSD, construction method and application thereof
  • Expression vector for enhancing gene expression of 3 beta-HSD, construction method and application thereof
  • Expression vector for enhancing gene expression of 3 beta-HSD, construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0087] Construction of an expression vector promoting the expression of 3β-HSD gene

[0088] (1) Design of primers for 3β-HSD target gene expression fragment.

[0089] Design the 3β-HSD gene coding sequence according to the NCBI database GenBank NM_001328615, and obtain the 3β-HSD gene coding sequence (cDNA sequence of 3β-HSD) whose nucleotide sequence is shown in SEQ ID No.1. Use Oligo7 to analyze it, search for upstream and downstream primers (requires no primer dimers as much as possible and the annealing temperature difference is small), and then add protective bases and EcoR I restriction site sequences at the 5' end of the upstream primer, downstream The 5' end of the primer was added with a protective base and a Not I restriction site sequence, and the designed primer sequence is shown in Table 1. The designed PCR primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0090] Table 1: Primers for PCR amplification of the 3β-HSD gene c...

Embodiment 2

[0097] Preparation of lentivirus containing 3β-HSD gene

[0098] 293FT cells were cultured, and the cells in good growth state were inoculated into six wells, 10 per well 6 Using the lentiviral packaging auxiliary kit, transfect 2 μg of the pLVX-3β-HSD recombinant vector extracted in Example 1 into 293FT cells, culture at 37°C for 48 hours, and filter the supernatant medium with a 0.45 μM filter membrane Filter, collect the virus supernatant, use the Lenti-X GoStix Gold Label Kit to measure the virus titer, and then store it at -80°C.

Embodiment 3

[0100] Human breast cancer MCF-7 cells transduced with lentivirus

[0101] Take the virus supernatant obtained in Example 2, dilute it 1:1 with RPMI-1640 complete medium, and then add Polybrene to a final concentration of 6 μg / mL-10 μg / mL for use. will be 3×10 5 MCF-7 cells were inoculated in T25 cell culture flasks, and the confluence of the cells reached 50% after 18 hours of culture. The original complete medium in the culture flasks was removed, washed twice with PBS, and then added to the above-mentioned RPMI-1640 containing lentivirus for complete culture. base. After transfection for 24 hours, remove the RPMI-1640 complete medium containing lentivirus, add normal RPMI-1640 complete medium and culture for another 24 hours, then replace the cells with 1 μg / mL puromycin for selection, and change the medium every 2 days 1 The second time, the screening time was 7 days, and the MCF-7 cell line (pLVX-3β-HSD cell line) with high expression of 3β-HSD gene was screened.

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Abstract

The invention discloses an expression vector for enhancing gene expression of 3 beta-HSD, a construction method and an application thereof. The expression vector comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence, a starter sequence and a target gene expression segment of a lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro. The target gene expression segments are forwards inserted between an EcoRI restriction enzyme cutting site and a NotI restriction enzyme cutting site. The expression vector disclosed by the invention can be transfected into human cells, and expresses 3 beta-HSD genes in the human cells in a continuous-stable-efficient manner, also can improve the damage action of a plasticizing agent on tumor cells and is expected to application in anti-tumor drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an expression vector for promoting the expression of 3β-HSD gene and its construction method and application. Background technique [0002] Steroid hormones play an important role in the differentiation, development, growth and physiological functions of most vertebrate cells. The production of steroid hormones depends on the supply of raw material cholesterol and the catalysis of a series of steroidogenic enzymes. Among them, 3β-supersteroid deaminase (3β-HSD) plays an important role in the biosynthesis of glucocorticoids, mineralocorticoids and sex hormones. [0003] In the traditional 3β-HSD gene expression technology, for example, Liu Haiyan et al. used gonadotropin-releasing hormone agonists to realize the regulation of 3β-HSD gene expression in rat Leydig cells through the ERKMAPK pathway; Zhu Hui et al. studied the effect of hCG on adult Regulation of 3β-HSD expression in mo...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66C12N7/01A61K48/00A61P35/00
CPCA61K48/005C12N7/00C12N15/66C12N15/86C12N2740/15021C12N2740/15043C12N2800/107
Inventor 徐新云王利毛侃琅彭鹏
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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