CYP3A5 protein, encoding gene and recombinant vector and application thereof in preparing drugs for resisting tumor recurrence or metastasis

A technology of encoding genes and recombinant vectors, applied in the field of biomedicine, can solve the problems of few research results of CYP3A5, achieve the effect of inhibiting migration and invasion ability, liver cancer recurrence and metastasis prevention

Inactive Publication Date: 2016-09-28
王红阳 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

CYP3A5 is the most deeply studied low-abundance member of the CYP3A subfamily, but existing research is still mainly focused on the relationship between CYP3A5 and tumorigenesis and drug metabolism, and the existing research results are mainly the results of the epidemiological field , there are few or no research results on CYP3A5 in anti-tumor, especially anti-hepatic cancer

Method used

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  • CYP3A5 protein, encoding gene and recombinant vector and application thereof in preparing drugs for resisting tumor recurrence or metastasis
  • CYP3A5 protein, encoding gene and recombinant vector and application thereof in preparing drugs for resisting tumor recurrence or metastasis
  • CYP3A5 protein, encoding gene and recombinant vector and application thereof in preparing drugs for resisting tumor recurrence or metastasis

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Experimental program
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Embodiment 1

[0035] This embodiment provides CYP3A5 protein, the coding gene of CYP3A5 protein, the expression cassette containing the coding gene, the recombinant vector and the recombinant bacteria.

[0036] 1. Construction of a vector carrying the gene encoding the CYP3A5 protein

[0037] The eukaryotic expression plasmid pcDNA3.1-CYP3A5 carrying the gene encoding CYP3A5 protein and the lentiviral expression plasmid lenti-CYP3A5-GFP carrying the gene encoding CYP3A5 protein were both constructed by Shanghai Jikai Gene Chemical Technology Co., Ltd. Wherein, the amino acid sequence of the CYP3A5 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the CYP3A5 protein is shown in SEQ ID NO.2.

Embodiment 2

[0039] This embodiment provides viral vectors, such as lentiviral vectors, and recombinant cells containing genes encoding CYP3A5 protein.

[0040] 1. Vector transformation to construct SMMC-7721 and HCC-LM3 two stable cell lines overexpressing CYP3A5 protein

[0041]The SMMC-7721 and HCC-LM3 cells in good growth state were transferred to 6-well plates respectively, and when the cell confluence rate was 60%-70%, the serum-free DMEM medium containing the above-mentioned lenti-CYP3A5-GFP was added (MOI=20 ), continue culturing for 8-9 hours, and then replace with fresh serum-containing DMEM medium. After culturing for another 6-10 days, the cells with green fluorescence were sorted by flow cytometry, which were stable cell lines with CYP3A5 overexpression. Two stable cell lines overexpressing CYP3A5 protein, SMMC-7721 and HCC-LM3, were obtained respectively.

Embodiment 3

[0043] This embodiment provides the application of the above CYP3A5 protein in inhibiting the recurrence or metastasis of tumor cells such as liver cancer cells.

[0044] 1. In vitro experiments to detect the effect of overexpression of CYP3A5 protein on the migration ability and invasion ability of liver cancer cells

[0045] The SMMC-7721 and HCC-LM3 cells transformed with the above-mentioned lentiviral vectors expressing CYP3A5 protein were used as the treatment group; the SMMC-7721 and HCC-LM3 cells transformed with the lentiviral vectors expressing the GFP protein were used as the control group. The effect of overexpression of CYP3A5 protein on the metastasis and invasion ability of liver cancer cells SMMC-7721 and HCC-LM3 was detected.

[0046] (1) Transwell method was used to detect the migration ability of liver cancer cells in the treatment group and the control group. The specific steps are as follows.

[0047] Rehydrate the inner chamber of Matrigel precoated trans...

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Abstract

The invention discloses a CYP3A5 protein, encoding gene and recombinant vector and application thereof in preparing drugs for resisting tumor recurrence or metastasis. The protein has an amino acid sequence as shown in SEQ ID NO.1, and the encoding gene has a nucleotide sequence as shown in SEQ ID NO.2. The protein can inhibit metastasis and invasion of hepatoma cells, and a very wide application prospect in preparation of the drugs for resisting tumor recurrence or metastasis is achieved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to CYP3A5 protein, coding gene, recombinant vector and the application thereof in the preparation of anti-tumor recurrence or metastasis medicine. Background technique [0002] At present, hepatocellular carcinoma is one of the deadliest malignant tumors, accounting for the third cause of tumor-related mortality worldwide, and it is the leading cause of death in patients with liver cirrhosis. According to reports, there were more than 700,000 newly diagnosed patients with HCC in 2008. According to age stratification, there are 16 newly diagnosed patients with HCC in every 100,000 people. Patients with early-stage liver tumors who have been carefully screened can be effectively treated through liver resection, liver transplantation and radiofrequency ablation. However, patients with advanced tumors, despite active comprehensive treatment, have poor prognosis due to postoperative recurrenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53A61K38/44A61P35/00A61P35/04
CPCC12N9/0071A61K38/00
Inventor 王红阳陈磊
Owner 王红阳
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