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Expression vector for promoting star gene expression and its construction method and application

A technology for gene expression and expression vector, which is applied in the field of expression vector and its construction to promote StAR gene expression, and can solve the problem of lack of human cells and so on.

Active Publication Date: 2020-10-27
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above studies all use indirect means to regulate the expression of the StAR gene. At present, there is a lack of expression vectors that can be transfected into human cells and express the StAR gene continuously, stably and efficiently.

Method used

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  • Expression vector for promoting star gene expression and its construction method and application
  • Expression vector for promoting star gene expression and its construction method and application
  • Expression vector for promoting star gene expression and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Construction of expression vectors to promote the expression of StAR gene

[0086] (1) Design of primers for StAR target gene expression fragment.

[0087] The StAR gene coding sequence was designed according to the NCBI database GenBank NM_000349, and the StAR gene coding sequence (cDNA sequence of StAR) whose nucleotide sequence was shown in SEQ ID No.1 was obtained. 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.

[0088] Table 1: Primers for PCR amplification of StAR gene coding s...

Embodiment 2

[0095] Preparation of lentivirus containing StAR gene

[0096] 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-StAR 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. The virus supernatant was collected, and the virus titer was determined using the Lenti-X GoStix Gold Label Kit, and then stored at -80°C.

Embodiment 3

[0098] Human breast cancer MCF-7 cells transduced with lentivirus

[0099] 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, and 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-StAR cell line) with high expression of StAR gene was screened.

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Abstract

The invention discloses an expression vector for promoting StAR gene expression and a construction method and application thereof. The expression vector comprises a basic sequence of lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro, a resistant gene sequence, a polyclonal site sequence, a promoter sequence, and a target gene expression fragment. The target gene expression fragment is forwardly inserted between EcoRI restriction enzyme cutting site and NotI restriction enzyme cutting site. The expression vector can be transfected to human cells and express StAR gene in the human cells in continuous, stable and efficient manner, and the expression vector is also capable of enhancing the effect of a plasticizer to damage tumor cells and is expectedly applicable to antitumor drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an expression vector for promoting the expression of StAR gene and its construction method and application. Background technique [0002] Steroidogenic acute regulatory protein (StAR), also known as steroid hormone sensitive regulatory protein, is a transport protein that promotes the transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane. Plays a key rate-limiting role in synthesis. Studies have shown that StAR plays an important role in regulating the synthesis of testosterone and is an important protein necessary to maintain normal physiological functions. In the traditional StAR gene expression technology, for example, Ren Xin et al. used ractopamine to regulate the expression of StAR gene in rat testis, and Ma Jinkui et al. studied the effect of clenbuterol hydrochloride on the expression of StAR in rat testis. The above studies all u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/66A61K48/00A61P35/00
CPCA61K48/005C12N15/66C12N15/86C12N2740/15043C12N2800/107
Inventor 徐新云彭鹏袁建辉毛侃琅王利
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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