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Construction, screening and application of shRNAs expression vector of lung cancer targeted CHRNA5 gene

A technology for expressing vectors and genes, which is applied in the fields of gene therapy drugs, screening, and the construction of shRNAs expression vectors, and can solve problems such as the exact mechanism is not yet clear

Active Publication Date: 2013-10-23
汪运山 +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Nicotine in tobacco is recognized as one of the most important carcinogenic factors causing lung cancer, but its exact mechanism is not yet clear

Method used

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  • Construction, screening and application of shRNAs expression vector of lung cancer targeted CHRNA5 gene
  • Construction, screening and application of shRNAs expression vector of lung cancer targeted CHRNA5 gene
  • Construction, screening and application of shRNAs expression vector of lung cancer targeted CHRNA5 gene

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Embodiment Construction

[0087] 1. Main Reagents

[0088] 1. The pLLU2G vector was purchased from Saiye (Guangzhou) Biotechnology Co., Ltd.

[0089] 2. QIAquick Gel Extraction Kit, QIAGEN, Cat. No. 28704;

[0090] 3. Plasmid mini-extraction kit, TIANGEN, Cat. No. DP103-02;

[0091] 4. dNTP mixture, Fermentas, Cat. No. #R0192;

[0092] 5. DNA marker GeneRuler TM 100bp DNA Ladder, Fermentas, Catalog #SM0241;

[0093] 6. Endonuclease HpaⅠ, TAKARA, product number D1064A;

[0094] 7. Endonuclease XhoⅠ, TAKARA, product number D1094A;

[0095] 8. T4DNA Ligase ligase, TAKARA, Cat. No. D2011A;

[0096] 9. S.O.C medium, Life technologies, product number: 15544034;

[0097] 10.5×Annealing Buffer for DNA Oligos, Beyotime, Cat. No. D0251;

[0098] 11. Taq DNA Polymerase, Fermentas, Cat. No. EP0404;

[0099] 12. Trizol Reagent RNA extraction reagent (Ambion, 15596026);

[0100] 13. DNaseΙ, RNase-free (Thermo, #EN0521);

[0101] 14. RevertAid First Strand cDNA Synthesis Kit Reverse Transcriptase (Thermo, ...

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Abstract

The invention relates to construction, screening and application of a shRNAs expression vector of a lung cancer targeted CHRNA5 gene. A RNA (Ribonucleic Acid) interference technique is utilized, and two expression plasmids CHRNA5-shRNA1 / 2 capable of expressing shRNA in mammalian cells are constructed by aiming at different target sequences of the CHRNA5 gene and taking pLLU2G as a vector. The shRNAs expression vector of the lung cancer targeted CHRNA5 gene is capable of efficiently and specifically inhibiting CHRNA5 gene expression, and is used for preparing gene medicaments for treating lung cancers with high-expression CHRNA5 genes.

Description

technical field [0001] The invention relates to gene therapy drugs for lung cancer expressing CHRNA5 (cholinergic receptor nicotinic alpha5) gene, in particular to the construction, screening and application of shRNAs expression vectors targeting the CHRNA5 gene of lung cancer, belonging to the technical field of biomedicine. Background technique [0002] Lung cancer is a common malignant tumor of the lung, and its mortality rate has accounted for the top of the cancer mortality rate. Nicotine in tobacco is recognized as one of the most important carcinogenic factors causing lung cancer, but its exact mechanism is not yet clear. Nicotinic Acetylcholine Receptor (nAChR) signaling pathway is one of the hotspots in lung cancer targeting research in recent years. In 2008, three independent molecular epidemiological studies on Nature and Nat Genet successively reported that the variation of the nAChR gene cluster CHRNA5 / A3 / B4 located on the long arm of chromosome 15 is closely r...

Claims

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Application Information

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IPC IPC(8): C12N15/85A61K48/00A61P35/00A61P11/00
Inventor 马晓丽郏雁飞肖东杰贾颖祖珊珊赵云韩淑毅汪运山
Owner 汪运山
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