Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Mir-124 and HER2-shRNA double-gene expression cassette virus vector, construction method,virus and application

A mir-124, 1.mir-124 technology, applied in the field of molecular biology, can solve the problems of breast cancer patients with recurrence and metastasis, the effect of inhibiting proliferation cannot meet the actual needs, and the overall survival rate is less than 25%.

Active Publication Date: 2018-10-26
云笛生物科技有限公司
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemotherapy is the main therapy for invasive breast cancer, but breast cancer patients still have the risk of recurrence and metastasis after treatment
So far, recurrent and metastatic breast cancer is still an incurable disease, and its 5-year overall survival rate is still less than 25%
However, the currently developed HER2-shRNA virus vectors can not meet the actual needs after transfection of breast cancer, ovarian cancer and other cells with high HER2 expression. Inhibitory proliferation of target cells

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Mir-124 and HER2-shRNA double-gene expression cassette virus vector, construction method,virus and application
  • Mir-124 and HER2-shRNA double-gene expression cassette virus vector, construction method,virus and application
  • Mir-124 and HER2-shRNA double-gene expression cassette virus vector, construction method,virus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] To construct the mir-124 and HER2-shRNA double gene expression cassette lentiviral vector, the specific steps are as follows:

[0053] 1) Artificial synthesis of the mir-124 sequence shown in SEQ ID NO.1 and the HER2-shRNA sequence shown in SEQ ID NO.2 and SEQ ID NO.3;

[0054] 2) Using KOD high-fidelity enzyme, using step 1) artificially synthesized mir-124 sequence and HER2-shRNA sequence as templates, respectively, PCR amplification was performed with the primers shown in Table 1:

[0055] Table 1

[0056] Primer name

[0057] The specific reagent dosage for PCR amplification is shown in Table 2 below:

[0058] Table 2

[0059]

[0060]

[0061] The program conditions for PCR amplification are shown in Table 3 below:

[0062] table 3

[0063]

[0064] 3) Agarose gel electrophoresis to check whether the size of the PCR amplified product is consistent with expectations, cut the target fragment band in the agarose gel electrophoresis gel, recover and purify it with a DNA recovery...

Embodiment 2

[0084] To construct a mir-124 and HER2-shRNA double gene expression cassette lentivirus, the specific steps are as follows:

[0085] 1. Digest the HEK 293 cells in the logarithmic growth phase with membrane protease, with a cell density of 5×106, and re-inoculate them in a cell culture dish containing 25 mL of antibiotic-free DMEM medium containing 10% (v / v) FBS with a diameter of 15 cm , 37℃, 5% CO2 incubator;

[0086] 2. Prepare four plasmid DNA solutions in the lentivirus packaging system, and the granulation map is as follows Figure 1~4 Shown:

[0087] Table 9

[0088] Plasmid name

quality

pRSV-Rev

1Oμg

pMDLg-pRRE

15μg

pMD2G

7.5μg

pCDH-CMV-mir124-HER2shRNA-EF1-copGFP-T2A-Puro

20μg

[0089] Add the above plasmid to 1800μl of sterile water, then add 200μl of CaCl2 (2.5mol / L) solution and mix well, add 2000μl of 2×PBS buffered salt solution, and leave it at room temperature for 20-30min;

[0090] 3. Transfection is performed when the cell density in step 1 reaches ab...

Embodiment 3

[0098] Cell transfection and screening of the mir124 and HER2-shRNA double gene expression cassette lentivirus constructed in Example 2: The specific steps are:

[0099] Ⅰ: Culture MCF-7 cells in a 6-well cell culture plate, and cell transfection can be carried out after the cells are fully adherent and confluent at 30%-40%: Before transfection, cells in each well are replaced with fresh DMEM for complete culture Add 1μL of polybrene to each well of the 6-well plate and place it in the incubator to incubate for 1h;

[0100] Ⅱ: Add 0.1 μL of the mir124 and HER2-shRNA double gene expression cassette lentiviral solution obtained in Example 2 to the 5 wells of the above 6-well cell culture plate, shake well and put it in 37°C, 5% ( v / v) Incubate in an incubator with CO2 and 95% relative humidity for 1 to 5 days, add 20μL / well of CCK8 reagent every day, and continue to incubate for 3 hours, then measure the absorbance OD value of each well at 450nm;

[0101] Ⅲ: Follow the same method as ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the technical field of molecular biology, in particular to a mir-124 and HER2-shRNA double-gene expression cassette virus vector, a construction method, a virus and application. The double-gene expression cassette virus vector is constructed by integratively connecting a mir-124 sequence and a HER2-shRNA sequence in a virus expression vector, wherein the mir-124 sequence is shown as SEQ ID NO.1; the HER2-shRNA sequence is shown as SEQ ID NO.2 and SEQ ID NO.3. The transfection rate of the mir-124 and HER2-shRNA double-gene expression cassette virus vector for breast cancer cells is as high as 80 to 90 percent. The mir-124 and HER2-shRNA double-gene expression cassette virus vector can express mir-124 and HER2-shRNA efficiently and stably in the breast cancer cells,plays a synergistic role, can remarkably inhibit the proliferation of the breast cancer cells and promote apoptosis,and can be applied to gene therapy of HER2-positive tumors such as breast cancer andovarian cancer.

Description

Technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a mir1-24 and HER2-shRNA double gene expression cassette viral vector, a construction method, a virus, and an application. Background technique [0002] Breast cancer is the most common tumor in women all over the world, and its incidence in my country accounts for 7% to 10% of systemic malignant tumors, and the age of onset tends to be younger. Although the mortality rate of breast cancer is declining year by year through early screening and diagnosis, the incidence rate still ranks first among female tumors, which seriously threatens women's health. At present, the main methods of treating breast cancer include surgery, chemotherapy, radiotherapy, and endocrine therapy. Chemotherapy is the main therapy for invasive breast cancer, but breast cancer patients are still at risk of recurrence and metastasis after treatment. So far, recurrent and metastatic breast canc...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/867C12N15/66C12N7/01A61K48/00A61P35/00
CPCA61K48/005A61P35/00C12N7/00C12N15/66C12N15/86C12N2740/15021C12N2740/15043
Inventor 李志强孙向东付建喜王新震孙华辉
Owner 云笛生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com