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Protein plasmid for expressing mouse p53a or p53b as well as construction method and application thereof

A construction method and protein technology, which is applied in the expression of mouse p53a or p53b protein particles and its construction, can solve the problems of different C-terminal sequences, easy formation of spontaneous tumors, and uncertain biological properties of p53 protein, etc., and achieve excellent amplification efficiency effect

Pending Publication Date: 2021-11-02
THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mice deficient in the p53 gene develop normally but are prone to spontaneous tumor formation
Current studies have found that the mouse p53 gene contains only one promoter, which is different from the selective promoter of the human p53 gene, and because of the different splicing methods, it encodes different types of p53 proteins (two types of p53a and p53b are currently found), among which p53a Encodes 390 amino acids, p53b encodes 381 amino acids, the main difference is that the C-terminal sequence is different, and the biological properties of different types of mouse p53 proteins have not yet been determined

Method used

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  • Protein plasmid for expressing mouse p53a or p53b as well as construction method and application thereof
  • Protein plasmid for expressing mouse p53a or p53b as well as construction method and application thereof
  • Protein plasmid for expressing mouse p53a or p53b as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This embodiment is a construction method for expressing mouse p53a or p53b protein particles, which construction method comprises the following steps:

[0039] (a) extract the RNA in the mouse liver tissue, and reverse transcribe it into cDNA;

[0040] (b) Using cDNA as a template to perform PCR amplification on the genes expressing p53a and p53b proteins respectively, wherein the upstream primer sequence expressing the p53a protein gene is shown in SEQ ID NO: 1, and the specific sequence is 5′-CGGGATCCCGGCAGGGTGTCACGCTTCT-3′ The downstream primer sequence is shown in SEQ ID NO:2, and the specific sequence is 5'-CGGAATTCCG AGGGACCGGGAGGATTGT-3'; the upstream primer sequence expressing the P53b protein gene is shown in SEQ ID NO:3, and the specific sequence is 5'-CGGGATCCGGCAGGGTGTCACGCTTCT- 3', the downstream primer sequence is shown in SEQ ID NO: 4, and the specific sequence is 5'-GCGAATTCGGAGGGATGAAGTGATGGGA-3';

[0041] The amplification conditions for expressing th...

Embodiment 2

[0051] This embodiment is a host cell, which is prepared by the following method:

[0052] (a) Inject 8-10 IU PMSG in 3-week-old Kunming female mice of SPF grade, kill the mice 48 hours later, take ovaries, and then separate to obtain preovulatory granulosa cells;

[0053] (b) Inoculate mouse preovulatory granulosa cells in a 6-well plate containing DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody, and replace it with no double antibody when the cells grow to 80% confluence DMEM / F12 culture fluid, after 12h, according to the transfection method in lipofectamine LTX, adopt the plasmid transfection cell that the embodiment 1 prepares to obtain host cell, wherein, pCMV-HA-p53a, pCMV-HA-p53b plasmid The transfection amount was 2 μg; the transfection time was 24h.

Embodiment 3

[0055] This example is the expression of p53 expression plasmid in mouse granulosa cells

[0056] Transfect pCMV-N-HA-p53a, pCMV-N-HA-p53b respectively according to the method in embodiment 2, transfect empty expression vector pCMV-N-HA simultaneously as negative control and the blank control of non-transfection; After transfecting pre-GCs with 2 μg for 24 hours, the cells were collected, and 20 μg of total protein of granulosa cells were extracted respectively. After the total protein was separated by SDS-PAGE gel, it was transferred to PVDF membrane by wet transfer; the hybridization membrane was sequentially passed through blocking solution (containing 5% TBST of skimmed milk powder) at 37°C for blocking for 1 hour, 1:750 dilution of mouse HA monoclonal primary antibody and 1:1000 dilution of mouse β-actin primary monoclonal antibody were incubated overnight at 4°C, 1:3000 dilution of HRP-labeled sheep After the anti-mouse secondary antibody was incubated at room temperatur...

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Abstract

The invention discloses a protein plasmid for expressing mouse p53a or p53b protein as well as a construction method and application thereof, and the construction method comprises the following steps: extracting RNA (Ribonucleic Acid) in mouse tissues, and carrying out reverse transcription to obtain cDNA (Complementary Deoxyribonucleic Acid); respectively carrying out PCR (Polymerase Chain Reaction) amplification on genes for expressing p53a and p53b proteins by taking the cDNA as a template; carrying out BamH I and EcoR I double enzyme digestion on a gene segment expressing p53a protein, a gene segment expressing p53b protein and pCMV-HA, recovering enzyme digestion products, connecting and transforming escherichia coli, and extracting recombinant plasmids; carrying out transfection amplification on recombinant plasmids which are subjected to enzyme digestion identification and correct sequencing, and extracting plasmids; the construction method has the characteristics of rapidness, accuracy and high efficiency.

Description

technical field [0001] The invention relates to the technical field of plasmid construction, in particular to a plasmid expressing mouse p53a or p53b protein and its construction method and application. Background technique [0002] The tumor suppressor gene p53 plays a very important role in the regulation of cell genome integrity and cell growth and development. Deletion of the p53 gene can lead to abnormal cell growth, therefore, the expression and activity of p53 are tightly regulated. The half-life of p53 protein is very short, and the content of p53 protein in normal cells is very small. When cells face external stress, DNA damage stimulus and chronic mitotic stimulus, p53 protein is transiently stabilized and activated. Depending on the cell type, cell environment, and oncogenic factors, activated p53 inhibits the cell cycle and induces senescence, differentiation, or apoptosis. Mice deficient in the p53 gene develop normally but are prone to spontaneous tumor form...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/12C12N15/10C12N5/10C12N5/071
CPCC12N15/85C07K14/4746C12N15/1096C12N5/0682C12N2800/107C12N2510/00C12Q2531/113
Inventor 刘晓明陈放霍立军程静张帆朱春芳赵军招
Owner THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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