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Dual expression vector of immunostimulatory RNA and liver cancer target gene Pim-3 silent RNA and application thereof

An immune stimulation and gene-targeting technology, which is applied in the fields of genetic engineering and biotherapy, can solve problems such as no Pim-3 proto-oncogene reported, and achieve good inhibitory effect, good application prospects, and enhanced killing effect

Inactive Publication Date: 2012-01-18
SHANDONG UNIV
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Problems solved by technology

In recent years, scientists have discovered some proto-oncogenes that are closely related to the occurrence and development of liver cancer by using technologies such as bioinformatics, but there is no report on a proto-oncogene such as Pim-3

Method used

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  • Dual expression vector of immunostimulatory RNA and liver cancer target gene Pim-3 silent RNA and application thereof
  • Dual expression vector of immunostimulatory RNA and liver cancer target gene Pim-3 silent RNA and application thereof
  • Dual expression vector of immunostimulatory RNA and liver cancer target gene Pim-3 silent RNA and application thereof

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Experimental program
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Embodiment

[0029] 1. Construction of a double expression vector:

[0030] (1) For the sequence of the Pim-3 gene, design the specific silencing RNA and the corresponding DNA sequence for the Pim-3 gene, and add the double restriction site sequences Sal I and Xbal I at both ends of the sequence, and anneal to form Double-stranded structure, the sequence is shown in seq.1-8;

[0031] (2) The pTZU6+1 vector is subjected to double enzyme digestion of Sal I and Xbal I, and the vector skeleton is recovered;

[0032] (3) Ligate the sequence obtained in step (1) with the pTZU6+1 vector obtained in step (2) to construct pTZU-shRNA; respectively denoted as pTZU-shRNA1, pTZU-shRNA2, pTZU-shRNA3, pTZU-shRNA4.

[0033] (4) Use cationic liposome Lipofectamine2000 (purchased from invitrogen) to transfect the pTZU-shRNA constructed in step (3) into the mouse liver cancer cell line Hepa1-6, and detect the expression level of Pim-3 gene, the results show that pTZU-shRNA1 has a more obvious silencing abi...

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Abstract

The invention discloses a dual expression vector of immunostimulatory RNA and liver cancer target gene Pim-3 silent RNA, which is a recombinant vector comprising a DNA sequence transcribing shRNA, a DNA sequence transcribing ssRNA, and a pTZU6+1 vector, and has a sequence as shown in seq.12. The dual expression vector of immunostimulatory RNA and liver cancer target gene Pim-3 silent RNA of the invention is used to transfect mouse liver cancer Hepa1-6 cells; after 24 hours, the cells are detected by Sem-qPCR and Western-blot, and it is found that the expression of Pim-3 in Hepa1-6 cells is down-regulated; after the transfection of Hepa1-6 cells for 24 hours, detection finds that the apoptosis of Hepa1-6 cells is obviously increased; the transfection of Hepa1-6 cells can significantly up-regulate the secretion of I-type interferon (IFN-alpha and IFN-beta) in supernatant of Hepa1-6 cells; therefore, the dual expression vector of immunostimulatory RNA and liver cancer target gene Pim-3 silent RNA of the invention is likely to become a new biological means for liver cancer treatment, and has good application prospects.

Description

technical field [0001] The invention relates to a double expression vector of immunostimulatory RNA and liver cancer target gene Pim-3 silencing RNA, a construction method and application thereof, and belongs to the technical field of genetic engineering and biotherapy. Background technique [0002] Liver cancer is one of the most common malignant tumors in my country. Since the 1990s, the annual mortality rate of liver cancer has risen to the second and the first place in the mortality rate of malignant tumors in urban and rural areas. As for the treatment of liver cancer, radiotherapy, chemotherapy and surgery are still the main treatments at present, among which surgical treatment plays a decisive role, especially surgical resection is still dominant. However, due to the high degree of malignancy of liver cancer, early spread is very easy For reasons such as dissemination and transfer, the above-mentioned therapies all have certain limitations. In recent years, with the ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/113C12N15/117C12N15/66A61K48/00A61P35/00A61P1/16A61P31/20
Inventor 张彩郭切兰培祥田志刚
Owner SHANDONG UNIV
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