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Carrier PCD-VEGF able to stable express VEGF shRNA

A technology for stable expression and vector, applied in the field of the vector pCD-VEGF that can stably express VEGF shRNA, can solve the problems of application limitation and high synthesis cost, achieve the best effect, avoid non-specific toxicity, and avoid the effect of cumbersome operation

Inactive Publication Date: 2006-09-20
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the method of in vitro synthesis of siRNA is simple and fast, but the high cost of synthesis limits its application.

Method used

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  • Carrier PCD-VEGF able to stable express VEGF shRNA
  • Carrier PCD-VEGF able to stable express VEGF shRNA
  • Carrier PCD-VEGF able to stable express VEGF shRNA

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: the design of VEGF shRNA target sequence and its DNA template

[0037] The selection of shRNA target sequences mainly follows the Tushl principle (Methods 2002, 26: 199-213), such as avoiding as much as possible within 50 nucleotides downstream of the start codon and within 100 nucleotides upstream of the stop codon. Sequence; since there are many regulatory protein binding sites in the 5' or 3' untranslated region and near the start codon, these sites are generally not selected; the sequence should avoid excessive GC content, and should avoid continuity in the sequence 3 or more G's appear, etc.

[0038] After the selection, a Blast search is required to ensure that the homology between the target sequence and other genes is very low, and as far as possible to ensure that the designed siRNA has the greatest specific gene silencing effect and the smallest non-specific toxicity and off-target (off-target) effect .

[0039] After the design of the target s...

Embodiment 2

[0057] Embodiment 2: Construction of pCD-VEGF plasmid

[0058] In order to enable the long-term sustained expression of the plasmid in mammalian cells, the plasmid vector needs to have a eukaryotic replication signal and a promoter. For this reason, the present invention has selected the main sequence of the commonly used eukaryotic expression vector pcDNA3.0 as the basic sequence ( figure 1 ). In order to prevent the original CMV promoter from affecting the expression of the shRNA sequence, the CMV promoter was removed by cutting with a restriction endonuclease, and then connected into the H1 promoter for shRNA expression. The specific steps are as follows:

[0059] 1) Use BamHI and BglII endonucleases to double-digest pcDNA3.0 to remove the CMV promoter. Since there is only one BamHI and one BglII restriction site on pcDNA3.0, only two bands appear in the electrophoresis after digestion, the lengths are about 0.9 kb and 4.5 kb respectively, and a large fragment of about 4....

Embodiment 3

[0065] Example 3: Transfection of Interfering Sequence Expression Plasmids and Screening of Mammalian Cell Lines Continuously Expressing shRNA Sequences

[0066] The present invention adopts LipofectAMINE TM 2000 liposomes (Life Technologies, Cat. No. 11668-027) were transfected into human fibrosarcoma cell line HT1080, and the ratio of transfected plasmid mass (μg) to liposome volume (μl) was 1:2.5. The plasmid concentration used was 2 μg / ml. All operations were carried out according to the instructions of liposomes. Twenty-four hours after the transfection, the cells were replaced with a culture solution containing 400 μg / ml G418. After that, the medium was changed every 2-3 days for the cells. After all the cells in the control group (that is, the group not transfected with any plasmid) died, the cells of the transfected pCD-VEGF plasmid group and the cells transfected with the pCD-MOCK plasmid group were digested, and after counting, the selective culture medium ( MEM-...

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Abstract

This invention relates to a carrier for stable expression of VEGF shRNA in mammal cells, and its application as an inhibitor to tumor cell invasion and metastasis. Research results show that after the reansfection to tumor cells by the pCD-VEGF carrier designed for VEGF gene, the invasion ability of tumor cells to the matrix membrane is weakened, the mobility of tumor cells is limited, and the growth of tumor cells in nude mouse is obviously suppressed. The pCD-VEGF carrier has a potential application in effective antitumor drugs.

Description

Technical field: [0001] The invention relates to a carrier capable of stably expressing VEGF shRNA in mammalian cells; the invention also relates to the application of the carrier in tumor treatment. Background technique: [0002] Blood vessels play an important role in the occurrence and development of tumors. When the tumor grows to 1-2mm 3 , requires the formation of new blood vessels. New blood vessels connect the tumor with the surrounding circulatory system, providing the necessary material exchange for tumor growth. In addition, primary tumor cells also enter the blood circulation through blood vessels, causing tumor metastasis. Therefore, angiogenesis is the only way for tumor growth and development, and is closely related to the occurrence and metastasis of tumors. This is why many tumors appear clinical symptoms only after new angiogenesis. Anti-tumor therapy targeting angiogenesis has advantages that cannot be replaced by conventional chemotherapy drugs: for e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12A61K48/00A61P35/00
Inventor 邵荣光张敏刘铁刚何红伟欧阳志钢张胜华
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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