Human pifl gene, its coding protein and use thereof

A protein and protein activity technology, applied in the fields of molecular biology and genetic engineering, can solve problems such as limiting the growth ability and shortening of transformed cells

Inactive Publication Date: 2008-04-16
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Thus closely linking this enzyme to immortalization and tumor formation! In normal human cells, programmed shortenin

Method used

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  • Human pifl gene, its coding protein and use thereof
  • Human pifl gene, its coding protein and use thereof
  • Human pifl gene, its coding protein and use thereof

Examples

Experimental program
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Embodiment 1

[0092] Cloning and identification of embodiment 1 human PIF1 gene

[0093]The inventors previously reported that human PIF1 protein has 32% homology with yeast PIF1 protein in a region of 438 amino acids (Zhou, etc Science 2000). No full-length clone of human PIF1 gene was found from the database. But the inventors found the cDNA clone gene of Drosophila. Then, the cloned genes of Drosophila were compared in the NCBI database, and the inventors found two human gene clones AK026045 and BC033254. These two sequences were compared with yeast PIF1, and the N-terminus had 9% identity and the C-terminus had 43% identity. In addition, these two cloned fragments are both distributed in the range of 11Kb on human chromosome 15, implying that they are two parts of human PIF1 gene. Therefore, the inventors designed two sets of primers and performed PCR amplification from the Hek293 cDNA library to obtain a 2.1 Kb fragment. The sequencing results of these two independent clones showed...

Embodiment 2

[0094] Example 2 Preparation of four kinds of polyclonal antibodies against human Pif1 protein

[0095] In order to detect the expression of hPIF1 at the protein level, the inventors made some polyclonal antibodies. N-terminal (76aa-230aa) respectively Ag0 , middle (309aa-435aa) Ag1 , near the C-terminal (438aa-549aa) Hp43 , C-terminal (550aa-641aa) Ag2 . Four polyclonal antibodies were identified with strong specificity and high titer. These antibodies were affinity purified.

[0096] The genes corresponding to the four peptides were cloned into the pGEX (AmerPharmarcial Co) prokaryotic expression vector, transfected into Bl21 strain for expression, affinity purified with GST gel beads, and immunized into rabbits after dialysis: Take 300 μg of purified GST- hPif1 (0, 1, 2, 43) fusion protein, dissolved in 0.5mL PBS, added an equal volume of Freund's complete adjuvant (Sigma Co), mixed thoroughly, intradermally injected into 2.0kg female New Zealand white rabbits ba...

Embodiment 3

[0098] Example 3 Human PIF1 protein encodes 5'-3' DNA helicase

[0099] Since human PIF1 protein has obvious sequence homology with yeast PIF1 protein and contains 7 helicase domains[ image 3 ], the inventor wants to prove that it is a DNA helicase. The coding sequence of hPIF1 gene was cloned into bacterial, yeast and baculovirus expression vectors simultaneously. In all three expression vectors, the inventors detected the overexpression of recombinant PIF1 protein, however, the inventors only purified hPIF1 protein in the yeast expression system. The hPIF1 cDNA clone yeast expression vector pEG(KT), [this system expresses the recombinant Rrm3p and Pfhlp (Zhou et, al 2003, Teng et, al2003)], make pEG(KT)-hPIF1, transform this plasmid To the yeast strain BCY123, as shown in the figure, the hPif1-GST fusion protein was expressed under the induction of galactose. The fusion protein was affinity purified with GST gel beads. 【 Figure 5 a]

[0100] Because the helicase uses ...

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Abstract

Human Pifl gene, its coding protein and use are disclosed. It also relates antibody of Pifl protein, agonist, detecting reagent box and composition containing the substance above.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a human Pif1 gene capable of inhibiting telomeric DNA lengthening, its encoded protein and its application. Background technique [0002] Telomeres are protein-DNA structures at the end of chromosomes in eukaryotic cells, which play an important role in protecting the integrity of chromosomes and maintaining the replication ability of cells. Its function is to complete the replication of chromosome ends and prevent chromosome fusion, recombination and degradation. [0003] The inventors found that yeast Pif1 protein (5' to 3' DNA helicase) can inhibit the elongation of telomeric DNA, and it works by inhibiting telomerase. Mutations in the Pif1 protein have the same phenotype as the absence of the Pif1 protein, both elongating telomeres. If the enzymatically active Pif1 protein is overexpressed, telomeric DNA shortens. The inventors further proved that ...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K14/47C12N15/12C12N15/63A61K38/17A61P35/00C12Q1/68G01N33/68
Inventor 周金秋张登宏黄妤徐露霞
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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