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Nuclear transcription factor mutant rFoxO3a-MTSS as well as recombinant vector and application thereof

A nuclear transcription factor, peasy-rfoxo3a-mt32 technology, applied in the nuclear transcription factor mutant rFoxO3a-MTSS and its recombinant vector and application fields, can solve the problems of different activation sites and the like

Inactive Publication Date: 2014-08-06
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain differences in the FoxO3a gene sequences of human and rat origin, and the sites activated by Akt in vivo are different

Method used

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  • Nuclear transcription factor mutant rFoxO3a-MTSS as well as recombinant vector and application thereof
  • Nuclear transcription factor mutant rFoxO3a-MTSS as well as recombinant vector and application thereof
  • Nuclear transcription factor mutant rFoxO3a-MTSS as well as recombinant vector and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] CDS fragment amplification and cloning of embodiment 1rFoxO3a gene

[0042] (1) Primer design

[0043] Query the mRNA information of rat FoxO3a gene through GeneBank database (http: / / www.ncbi.nlm.nih.gov / genbank), and use Primer Primer5.0 software to design primers CDS-F and CDS-R for amplifying large Murine FoxO3a gene CDS coding region. Wherein, the primer sequence is:

[0044] CDS-F: 5'-GAGAGGAGAGAGCAAGAGCCCA-3';

[0045] CDS-R: 5'-CTCAGTGATCCTTCAGCCTGGC-3';

[0046] (2) PCR amplification of CDS of rat FoxO3a gene coding region

[0047] Total RNA was extracted from heart tissue of SD rats (purchased from Guangdong Provincial Medical Experimental Animal Center) with TRIzol kit (Invitrogen), and cDNA was synthesized with reverse transcription kit (TOYOBO) and diluted to 200 ng / μL. Using cDNA as a template, KOD-Plus-Neo high-fidelity enzyme (TOYOBO) was used for PCR amplification. The specific reaction system is as follows:

[0048] Table 1 PCR amplification react...

Embodiment 2

[0052] Example 2 Preparation of recombinant vector pEASY-rFoxO3a-MT32

[0053] (1) Design of mutation primers MT32-F and MT32-R: According to the primer design principles and mutation requirements, use Primer Primer5.0 software to design mutation primers. The sequence of the mutant primers is as follows:

[0054] MT32-F: 5'-TGT TGGCCCCTGCAGAGGCCGGAGCT-3';

[0055] MT32-R: 5'-GGAGCGTGGCCGACTCTGTGGCTCGAACT-3';

[0056]Wherein, the lowercase base g in the MT32-F primer is a mutated base, and the codon ACG in the wild-type gene can be mutated into gCG, so that the encoded threonine can be mutated into alanine.

[0057] (2) Dilute the above mutation primers to 10 pmol / μL, dilute the template plasmid DNA (pEASY-rFoxO3a-CDS) to 50 ng / μL, and use the KOD-Plus-Mutagenesis Kit (TOYOBO) to carry out the mutation reaction. The specific reaction system is as follows Show:

[0058] Table 2 Plasmid pEASY-rFoxO3a-CDS mutation reaction system

[0059] Sterilized DDW

[0060] M...

Embodiment 3

[0061] Example 3 Preparation of recombinant vector pEASY-rFoxO3a-MT32S252

[0062] (1) Design of mutation primers MS252-F and MS252-R: According to the primer design principles and mutation requirements, use Primer Primer5.0 software to design mutation primers. The sequence of the mutant primers is as follows:

[0063] MS252-F: 5'-GTC ATGGACAACAGCAACAAGTACAC-3';

[0064] M S252-R: 5'-GGCCCGCCGCCGGGGAGCCTTCCCACTCTTT-3';

[0065] Wherein, the lowercase base g in the MS252-F primer is a mutated base, and the codon TCC in the wild-type gene can be mutated into gCC, so that the encoded serine can be mutated into alanine.

[0066] (2) Dilute the above mutation primers to 10pmol / μL, use the mutant plasmid pEASY-rFoxO3a-MT32 as the DNA template, and dilute the template plasmid DNA to 50ng / μL, and use the KOD-Plus-Mutagenesis mutation kit (TOYOBO) to carry out Mutation reaction, the specific reaction system is as follows:

[0067] Table 3 Plasmid pEASY-rFoxO3a-MT32 mutation react...

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Abstract

The invention discloses a nuclear transcription factor mutant rFoxO3a-MTSS as well as a recombinant vector and application thereof. The nuclear transcription factor mutant rFoxO3a-MTSS is obtained by replacing the 32nd threonine, the 252nd serine and the 314th serine in a wild type rat FoxO3a peptide chain into alanine simultaneously, wherein the amino acid sequence of the mutant rFoxO3a-MTSS is shown in the SEQ ID NO.1; the sequence of nucleotide coding the mutant is shown in the SEQ ID NO.2. Compared with a wild type rFoxO3a, the mutant rFoxO3a-MTSS discloses by the invention has the characteristic of capable of avoiding phosphorylation by an upstream regulatory factor Akt, can keep in a continuous active state in vivo, and has important scientific significance and potential application value on research on functions of FoxO3a genes and targeted therapy of diseases related to the FoxO3a genes.

Description

technical field [0001] The invention relates to a transcription factor mutant, in particular to a nuclear transcription factor mutant rFoxO3a-MTSS and its recombinant vector and application. Background technique [0002] The nuclear transcription factor FoxO3a can regulate the transcription of multiple downstream target genes such as Bim, Fas ligand (FasL), TNFR2, p27, p53, and antioxidant enzymes to play different roles in promoting apoptosis, cell cycle arrest, and antioxidation. biological functions. Previous studies have shown that FoxO3a is widely distributed in various tissues and cells of mammals, and participates in physiological and pathological processes such as glucose metabolism, skeletal muscle differentiation and development, angiogenesis, inhibition of cell proliferation, and inhibition of cardiac hypertrophy. Therefore, the study of the function of FoxO3a transcription factor under different pathological and physiological conditions has attracted more and mo...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/85C12N15/66C12Q1/68
CPCC07K14/4702
Inventor 齐绪峰李昀键陈卓莹夏景波蔡冬青龙颖妍赵天琪吴彩红王健欢
Owner JINAN UNIVERSITY
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