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A gene expression element for inhibiting tumor cell growth and its application

A technology of gene expression and components, applied in application, gene therapy, anti-tumor drugs, etc., can solve the problems of inability to kill tumor cells, block tumor cells, and the effect of blocking DNA replication is not ideal, and achieve complete gene therapy. Effect

Active Publication Date: 2017-08-08
东莞兰卫医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] DNA polymerases responsible for DNA synthesis in eukaryotic cells mainly include α, δ, and ε. In the existing tumor-targeted gene expression elements used to inhibit the expression of DNA polymerases, the microRNAs carried by them all target a single DNA Polymerases, which are less effective at blocking DNA replication
Moreover, only blocking the proliferation of tumor cells can not kill tumor cells, it is difficult to cure cancer

Method used

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  • A gene expression element for inhibiting tumor cell growth and its application
  • A gene expression element for inhibiting tumor cell growth and its application
  • A gene expression element for inhibiting tumor cell growth and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The construction of the expression test element of the targeting DNA polymerase α, δ, ε artificial microRNA of embodiment 1 single and tandem connection

[0044] 1. PCR amplification

[0045] The coding sequences of artificial microRNA targeting DNA polymerase α, δ, ε are all based on the natural structure of human miR-30 (miRBaseaccession number: MI0000088), and the core sequence is obtained from RNAi Codex (http: / / cancan.cshl.edu / cgi-bin / Codex / Codex.cgi), named and miR-polε. The primer sequences of the coding sequences of the three artificial miRNAs were designed with software and sent to Invitrogen Shanghai Branch for synthesis. Two rounds of PCR were used to amplify the DNA sequences encoding the three artificial miRNA precursors, in which the amplification primers of the first round of PCR were corresponding Forward and Reverse, and the primers served as templates for each other; the second round of PCR amplification was used to recover and purify The amplif...

Embodiment 2

[0067] Embodiment 2 The inhibitory effect of each expression test vector on the mRNA level of DNA polymerase gene expression in liver cancer cell Hep3B (fluorescence real-time quantitative PCR method)

[0068] Human liver cancer cells Hep3B in logarithmic growth phase 0.9×10 6 , and uniformly inoculated in 24-well plates in 3 wells, and took the five expression test vectors constructed in Example 1, and transiently transfected liver cancer cells Hep3B according to the ratio of 1.5 g vectors and 3 ul Lipofectamine LTX. After 72 hours, the cells were collected, and the mRNA levels of DNA polymerase α, δ, ε and the internal reference protein β-Actin were detected with a fluorescent real-time quantitative PCR kit QuantiFast SYBRGreen PCR (Qiagen, Germany). Three replicate wells were set up for each sample, and the experiment was repeated at least once. Test results such as Figure 1A , Figure 1B , Figure 1C shown.

[0069] Depend on Figure 1A , Figure 1B , Figure 1C It ...

Embodiment 3

[0071] Example 3 Construction of Gene Expression Elements for Inhibiting the Growth of Hepatocellular Carcinoma Cells

[0072] 1 Construction of the first expression cassette

[0073] (1) Recombinant AFP promoter

[0074] The base sequence of the recombinant AFP promoter is shown in SEQ ID No.1, wherein the 1st to 6th are restriction endonuclease BglII enzyme cutting sites; the 1013th to 1018th are restriction endonucleases Restriction site of enzyme BamHI.

[0075] The recombinant AFP promoter contains the enhancer region from -4120 to -3300 and the basic promoter region from -180 to +1 of the human AFP gene. For the cloning method, please refer to the literature: Ye Jingjia, Chen Ping, Jia Zhenyu, Li Chunchun, Chen Lihong , Cao Jiang. The study of enhancing the efficiency of human alpha-fetoprotein promoter by cytomegalovirus enhancer. Chinese Journal of Cell Biology, 2013, 35(4): 486-493; obtained pMD19-T / AFP.

[0076] (2) Coding sequence of artificial microRNA targeti...

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Abstract

The invention discloses a gene expression component for inhibiting tumor cell growth and application thereof. The gene expression element consists of a first expression frame and a second expression frame, the first expression frame consists of a tumor-specific promoter, coding sequences of three mircoRNAs targeting DNA polymerase α, δ, and ε respectively, and a transcription termination signal The sequences are connected sequentially; the second expression box is formed by sequentially connecting a tumor-specific promoter, an apoptosis factor gene and a transcription termination signal sequence. On the one hand, the present invention targets the three DNA polymerase genes necessary for chromosome replication, and effectively inhibits their expression at the same time, so that tumor cells cannot perform chromosome replication and thus cannot proliferate due to the lack of these three DNA polymerases; On the other hand, apoptotic factors are used to promote the apoptosis of tumor cells that have stopped proliferating, and finally achieve the goal of more thorough gene therapy.

Description

technical field [0001] The invention belongs to the technical field of tumor gene therapy, and in particular relates to a gene expression element for inhibiting tumor cell growth and its application. Background technique [0002] Malignant tumor is one of the diseases that seriously endanger human health. Its treatment consumes a lot of medical resources. The curative effect and prognosis of conventional methods such as surgery, chemotherapy and radiotherapy are not ideal. Therefore, it is of great significance to study and explore new cancer treatment methods. [0003] Emerging tumor biotherapeutic methods have already been applied clinically. The treatment of malignant tumors that are ineffective to radiotherapy and chemotherapy has shown its unique advantages and good application prospects. Among them, tumor gene therapy has always been one of the main directions of tumor biological therapy. [0004] Targeting is a very important aspect of tumor gene therapy, that is, it...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/57C12N15/861A61K48/00A61K38/48A61K31/7088A61P35/00
Inventor 贾振宇曹江刘昊魏群
Owner 东莞兰卫医学检验实验室有限公司