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Recombinant adeno-associated virus aav-shcdc6 preparation, its preparation and application

A technology of aav-shcdc6 and virus, applied in the direction of recombinant DNA technology, DNA / RNA fragments, drug combination, etc., can solve the problem of lack of effective drug targets for specific killing of tumor cells, and achieve high specificity and low side effects

Active Publication Date: 2016-11-02
汉姆德(宁波)智能医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the targeted therapy of tumors faces great challenges, mainly due to the lack of effective drug targets for specifically killing tumor cells

Method used

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  • Recombinant adeno-associated virus aav-shcdc6 preparation, its preparation and application
  • Recombinant adeno-associated virus aav-shcdc6 preparation, its preparation and application
  • Recombinant adeno-associated virus aav-shcdc6 preparation, its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of recombinant adeno-associated virus AAV-shCdc6

[0027] 1) Design the specific human Cdc6RNAi target sequence and predict by computer software to determine the 1091st to 1110th base of the human Cdc6siRNA target sequence (gagatcaggttctggacaa, as shown in SEQ ID NO.1);

[0028] 2) Chemically synthesize the following primers:

[0029] Primer 1: 5'-GATTC gagatcaggttctggacaaA-3', as shown in SEQ ID NO.2;

[0030] Primer 2: 5'-AGCTTttgtccagaacctgatctcG-3', as shown in SEQ ID NO.3;

[0031] 3) After the above primers were annealed at 55°C, they were ligated into the adenovirus vector pAAV-GFP to obtain pAAV-GFP-shCdc6;

[0032] 4) Co-transfect 293T cells with pAAV-GFP-shCdc6 and helper vector plasmids (pAAV-RC2 and pHelper);

[0033] 5) After 2 days of cell culture, the cell culture supernatant was collected and diluted 1:100 to infect 293T cells again;

[0034] 6) After the cells were cultured for 2 days, the cell culture supernatant was collecte...

Embodiment 2

[0035] Example 2: Infection of recombinant adeno-associated virus AAV-shcdc6 to breast cancer cells

[0036] 1) Breast cancer cells MCF7 and normal diploid breast epithelial cells HBL-100 were cultured in vitro;

[0037] 2) Dilute the virus solution at a ratio of 1:100 (the virus titer is 5×10 per ml) 11 ) adding the above cells and culturing for 3 days;

[0038] 3) Collect the cells, perform protein electrophoresis after lysing, and perform Western Blot with specific rabbit anti-Cdc6 antibody after transfer to confirm the inhibition of Cdc6 protein expression. The results are shown in figure 1 .

Embodiment 3

[0039] Example 3: The specific killing effect of recombinant adeno-associated virus AAV-shCdc6 on breast cancer cells

[0040] 1) Dilute the AAV-shcdc6 virus solution at a ratio of 1:100 (the virus titer is 5×10 per ml) 11 ) to infect breast cancer cells MCF7 and normal diploid mammary epithelial cells HBL-100 respectively; the control group was injected with AAV virus liquid;

[0041] 2) After 3 days, the cells were collected, fixed with 70% ethanol, and flow cytometry was used to detect and analyze the cell growth cycle and apoptosis after PI staining. The results are shown in figure 2 . It can be seen from the figure that AAV-shcdc6 virus liquid has obvious killing effect on breast cancer cell MCF7, but basically has no effect on breast epithelial cell HBL-100.

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Abstract

The invention relates to a recombinant adeno-associated virus AAV-shCdc6 formulation, which contains the recombinant adeno-associated virus AAV-shCdc6 which is obtained by inserting the small interfering RNA (siRNA) of a specific human Cdc6RNAi targeting sequence to the special site of the AAV (Adeno-Associated Virus). The recombinant adeno-associated virus AAV-shCdc6 formulation provided by the invention is capable of efficiently infecting the breast cancer cells and continuously expressing the siRNA of the specific targeting Cdc6, and finally capable of specifically killing the breast cancer cells; on the contrary, the recombinant adeno-associated virus AAV-shCdc6 formulation has no killing effect on normal mammary epithelial cells. Therefore, the recombinant adeno-associated virus AAV-shCdc6 formulation can be used as a novel biological formulation for treating the breast cancer.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant adeno-associated virus preparation, its preparation method and application. Background technique [0002] The global incidence of breast cancer has been on the rise since the late 1970s. 1 in 8 women in the U.S. will be diagnosed with breast cancer in her lifetime. In recent years, the growth rate of the incidence of breast cancer in my country is 1-2 percentage points higher than that of high-incidence countries. According to the 2009 breast cancer incidence data released by the National Cancer Center and the Ministry of Health's Bureau of Disease Control and Prevention in 2012, the incidence of breast cancer in the national tumor registration area ranks first among female malignant tumors, and the incidence (crude rate) of female breast cancer is the highest in the country. The total is 42.55 / 100,000, 51.91 / 100,000 in cities, and 23.12 / 100,000 in rural areas. In rece...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00C12N15/86C12N15/113A61P35/00
Inventor 姜伟朱长军董智雄
Owner 汉姆德(宁波)智能医疗科技有限公司
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