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Construction and application of shRNA recombinant vector specifically inhibiting 3beta-HSD gene expression

A gene expression and recombinant vector technology, applied in the field of genetic engineering, can solve the problems of unstable effect and insignificant effect of 3β-HSD gene

Active Publication Date: 2018-12-18
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on this, it is necessary to provide an interfering shRNA that inhibits the expression of the 3β-HSD gene, a recombinant vector and a construction method thereof for the problem that the effect of RNA interference silencing the expression of the 3β-HSD gene in the prior art is not obvious and the effect is unstable and application

Method used

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  • Construction and application of shRNA recombinant vector specifically inhibiting 3beta-HSD gene expression
  • Construction and application of shRNA recombinant vector specifically inhibiting 3beta-HSD gene expression
  • Construction and application of shRNA recombinant vector specifically inhibiting 3beta-HSD gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Construction of the expression vector for inhibiting the expression of 3β-HSD gene

[0110] (1) Preparation of interfering shRNA

[0111] Prepare four kinds of interfering shRNA, four kinds of interfering RNA are respectively shRNA1, shRNA2, shRNA3 and shRNAc, wherein, shRNAc is a control group, shRNA1 contains the target sequence shown in SEQ ID No.1; shRNA2 is shown in SEQ ID No.2 The target sequence shown; shRNA3 contains the target sequence shown in SEQ ID No.3; shRNAc contains the target sequence shown in SEQ ID No.10.

[0112] Specifically, the sense strand and the antisense strand of the interfering shRNA are synthesized, the synthetic sense strand and the antisense strand are mixed in equal amounts, and annealed to form the interfering shRNA.

[0113] Among them, the annealing program is heating at 95°C for 30s, heating at 72°C for 120s, heating at 37°C for 120s, holding at 25°C for 120s, and holding at 4°C for 20 minutes.

[0114] Wherein, the sense strand of...

Embodiment 2

[0124] Preparation of lentivirus inhibiting 3β-HSD gene expression

[0125] 293FT cells were cultured, and the cells in good growth state were inoculated into six-well plates, 10 per well 6 2 μg of each of the PLVX-shRNA2PshRNA1 recombinant vectors, PLVX-shRNA2PshRNA2 recombinant vectors, PLVX-shRNA2PshRNA3 recombinant vectors, and PLVX-shRNA2PshRNAc recombinant vectors extracted in Example 1 were obtained, and 2 μg of each of the above-mentioned recombinant vectors and 1 μg of pCMV-VSV- G and 2 μg of pCMV-dR8.91 were co-transfected into 293FT cells, cultured at 37°C for 48h and 72h, and the supernatant medium was collected and filtered with a 0.45μm filter membrane to obtain virus liquid containing PLVX-shRNA2PshRNA1 recombinant vector, For the virus liquid containing the PLVX-shRNA2PshRNA2 recombinant vector, the virus liquid containing the PLVX-shRNA2PshRNA3 recombinant vector, and the virus liquid containing the PLVX-shRNA2PshRNAc recombinant vector, the virus titer of eac...

Embodiment 3

[0127] Human breast cancer MCF-7 cells transduced with lentivirus

[0128] MCF-7 cells were cultured, and the cells in good growth state were inoculated into six-well plates, 10 per well 6 The cells reached 50% confluence after 12 hours of culture. 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. Remove the original medium in the six-well plate, and add RPMI-1640 complete medium containing lentivirus. 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 screening time wa...

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Abstract

The invention relates to an interfering shRNA inhibiting 3beta-HSD (3beta-hydroxysteroid dehydrogenase) gene expression, a recombinant vector, a construction method and application thereof. The interfering shRNA inhibiting 3beta-HSD gene expression includes a target sequence, a stem-loop structure, a complementary sequence of the target sequence and a termination site that are connected sequentially, wherein the target sequence is shown 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 sustainably, stably,efficiently and specifically inhibit the expression of 3beta-HSD gene in human cells.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the construction and application of a shRNA recombinant vector that specifically inhibits the expression of 3β-HSD gene. Background technique [0002] 3β-hydroxysteroid dehydrogenase (3β-hydroxysteroid dehydrogenase, 3β-HSD) is widely distributed in the brain, heart, kidney, fat, skin and steroid hormone-producing tissues, and is mainly involved in the synthesis of glucocorticoids, mineralocorticoids and sex hormones. Act as a rate-limiting enzyme. Since 3β-HSD catalyzes the initial step of steroid hormone production, if its expression is abnormal, it may affect the synthesis of all steroid hormones. [0003] Studies have shown that bis(2-ethylhexyl) phthalate (DEHP) can increase the expression level of 3β-HSD mRNA and protein, and DEHP can affect the change of 3β-HSD expression level in the process of steroid hormone synthesis, resulting in reproductive...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N7/01A61K31/713A61P39/02
CPCA61K31/713A61P39/02C12N7/00C12N15/113C12N15/86C12N2310/14C12N2740/15021C12N2740/15043
Inventor 徐新云王利毛吉炎袁建辉黄海燕毛侃琅
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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