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Construction and application of shrna recombinant vector for inhibiting star gene expression

A technology of gene expression and recombinant vectors, applied in the direction of DNA / RNA fragments, retroRNA viruses, applications, etc., can solve the problems of insufficient interference effect, interference, and inability to achieve long-term stability

Active Publication Date: 2020-09-18
SHENZHEN CENTER FOR DISEASE CONTROL AND PREVENTION (SHENZHEN HEALTH INSPECTION CENTER SHENZHEN INSTITUTE OF PREVENTIVE MEDICINE)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the siRNA expression vector can silence gene expression in a short period of time, its interference effect is not obvious enough to achieve long-term and stable interference

Method used

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  • Construction and application of shrna recombinant vector for inhibiting star gene expression
  • Construction and application of shrna recombinant vector for inhibiting star gene expression
  • Construction and application of shrna recombinant vector for inhibiting star gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Construction of an expression vector for inhibiting the expression of StAR gene

[0134] (1) Preparation of interfering shRNA

[0135] Four interfering shRNAs were prepared, and the four interfering RNAs were shRNA1, shRNA2, shRNA3 and shRNAc, respectively, and shRNAc was used as a control group. Among them, the target sequence contained in shRNA1 is siRNA1, and siRNA1 is shown in SEQ ID No.1; the target sequence contained in shRNA2 is siRNA2, and siRNA2 is shown in SEQ ID No.2; the target sequence contained in shRNA3 is siRNA3, and siRNA3 is shown in SEQ ID No. Shown in No.3; the target sequence contained in shRNAc is siRNAc, and siRNAc is shown in SEQ ID No.10.

[0136] Specifically, synthesize the sense strand and antisense strand of the interfering shRNA, mix the synthesized sense strand and antisense strand in equal amounts, and anneal to form the interfering shRNA. 120s, 25°C for 120s, 4°C for 20min. Wherein, the sense strand of shRNA1 is shown in SEQ ID No.4, ...

Embodiment 2

[0141] Preparation of lentivirus inhibiting StAR gene expression

[0142] 293FT cells were cultured, and the cells in good growth state were inoculated into six-well plates, 10 per well 6 Take 2 μg each of the PLVX-shRNA2PshRNA1 recombinant vector, PLVX-shRNA2PshRNA2 recombinant vector, PLVX-shRNA2PshRNA3 recombinant vector, and PLVX-shRNA2PshRNAc recombinant vector extracted in Example 1, mix 6 μL of Lipofectamine2000, 2 μg of the above recombinant vector, and 1 μg of pCMV- VSV-G plasmid and 2 μg of pCMV-dR8.91 plasmid were co-transfected into 293FT cells, cultured at 37°C for 48h and 72h, the supernatant medium was collected and filtered with a 0.45μm filter to obtain LVX-shRNA2PshRNA1 recombinant vector Virus liquid, virus liquid containing PLVX-shRNA2PshRNA2 recombinant vector, virus liquid containing PLVX-shRNA2PshRNA3 recombinant vector, virus liquid containing PLVX-shRNA2PshRNAc recombinant vector, use the Lenti-X GoStix kit to detect the virus titer of each virus liqui...

Embodiment 3

[0144] Human breast cancer MCF-7 cells transduced with lentivirus

[0145] Each virus solution obtained in Example 2 was taken and diluted 10:1 with RPMI-1640 complete medium, and then polybrene (polybrene) was added to a final concentration of 5 μg / mL for use. MCF-7 cells were cultured, and the cells in good growth state were inoculated into six-well plates, 10 per well 6 After 12 hours of culture, the confluence of the cells reached 50%, the original medium in the six-well plate was removed, and RPMI-1640 complete medium containing lentivirus was added. After 24 hours of transfection, the RPMI-1640 complete medium containing lentivirus was removed, and the normal RPMI-1640 complete medium was added to culture for another 24 hours, and then the cells were screened with 0.5 μg / mL puromycin. The medium was changed every 3 days, and the concentration of puromycin was increased every time the medium was changed until the concentration of puromycin was increased to 1.0 μg / mL. The...

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Abstract

The invention relates to an interfering shRNA inhibiting the expression of a StAR gene, a recombinant vector, a preparation method and application. The interfering shRNA inhibiting the expression of the StAR gene comprises a target sequence, a stem loop structure, a complementary sequence of the target sequence and a termination site which are sequentially connected, wherein the target sequence isshown as SEQ.ID.NO.1, or the target sequence is shown as SEQ.ID.NO.2, or the target sequence is shown as SEQ.ID.NO.3.The interfering shRNA can continuously, stably, efficiently and specifically inhibit the expression of the StAR gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the construction and application of a shRNA recombinant vector for inhibiting StAR gene expression. Background technique [0002] Steroidogenic acute regulatory protein (StAR, Steroidogenic Acute Regulatoryprotein), also known as steroid hormone sensitive regulatory protein, can promote the transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane. StAR plays a key rate-limiting role in the metabolism of cholesterol and the synthesis of steroid hormones. 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. [0003] In the traditional StAR gene expression regulation technology, the main indirect means to regulate the expression of the StAR gene, for example, by adding ractopamine or clenbuterol hydrochloride to regulate ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/713A61P39/02
CPCA61K31/713A61P39/02C12N15/113C12N15/86C12N2310/14C12N2740/15043
Inventor 徐新云毛吉炎毛侃琅袁建辉黄海燕彭鹏
Owner SHENZHEN CENTER FOR DISEASE CONTROL AND PREVENTION (SHENZHEN HEALTH INSPECTION CENTER SHENZHEN INSTITUTE OF PREVENTIVE MEDICINE)
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