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New human protein with function of improving mouse NIH/3T3 cell transformation and its encoding sequence

A technology of cell transformation and human protein, applied in anti-animal/human immunoglobulin, organic chemistry, animal/human peptide, etc., can solve the problem of lack of functional genes and high throughput

Inactive Publication Date: 2005-02-23
NEWORGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Human genomics research is currently a hot spot in the world. In addition to large-scale sequencing of human chromosome DNA and expressed sequence sequencing (EST), there is still a lack of high-throughput methods for screening functional genes starting from function.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1: Acquisition of cDNA gene and its promoting effect on mouse NIH / 3T3 cell clone formation

[0087] PP14212 was obtained by constructing a human placenta cDNA library using conventional methods; FP7030 and FP7915 were obtained from human fetal cDNA libraries constructed using conventional methods. Take 3, 6, and 10-month-old placental tissue (PP clone) or fetal tissue (FP clone), use Trizol reagent (GIBCO BRL company) to extract total RNA according to the manufacturer's instructions, and use mRNA purification kit (Pharmacia company) to extract mRNA . The cDNA library of the above mRNA was constructed using the pCMV-script TMXR cDNA library construction kit (Stratagene). The reverse transcriptase was changed to MMLV-RT-Superscript II (GIBCO BRL), and the reverse transcription reaction was carried out at 42°C. Transformed XL 10-Gold competent cells, obtained 1 × 10 6 cfu / μg titer cDNA library. In the first round, cDNA clones were randomly selected, and then...

Embodiment 2

[0090] Example 2: Obtain full-length gene by PCR from placenta or fetal cDNA:

[0091] Take 3, 6, and 9-month-old placental tissue (PP clone) or fetal tissue (FP clone), use Trizol reagent (GIBCO BRL company) to extract total RNA according to the manufacturer's instructions, and use mRNA purification kit (Pharmacia company) to extract mRNA . Use MMLV-RT-Superscript II (GIBCO BRL) and reverse transcriptase to perform reverse transcription at 42°C to obtain placental or fetal cDNA. Use specific primers for each gene (as shown in the table below), and perform 3'1 cycles at 97°C. 94°C 30″60°C 30″72°C 1′35 cycles, 72°C 10′1 cycle for PCR amplification to obtain the amplified products of each protein gene containing the complete open reading frame sequence. The amplified product was verified by sequencing and was consistent with the sequence measured in Example 1, and then the amplified product was transferred into host cells using conventional techniques to obtain recombinant pro...

Embodiment 3

[0094] Embodiment 3: cDNA clone sequence analysis

[0095] 1. PP14212 protein

[0096] A: Nucleotide sequence (SEQ ID NO: 1) Length: 1637 bases

[0097] B: Amino acid sequence (SEQ ID NO: 3) Length: 288 amino acids

[0098] C. Nucleotide and amino acid combination sequence (SEQ ID NO: 2) clone number and protein name: PP14212

[0099] Start code: 64 ATG Stop code: 928 TAG Protein molecular weight: 31640.56Da

[0100] 2. FP7030 protein

[0101] A: Nucleotide sequence (SEQ ID NO: 4) length: 3600 bases

[0102] B: Amino acid sequence (SEQ ID NO: 6) Length: 307 amino acids

[0103] C. Nucleotide and amino acid combination sequence (SEQ ID NO: 5) clone number and protein name: FP7030

[0104] Start codon: 585 ATG Stop codon: 1506 TGA Protein molecular weight: 32581.89Da

[0105] 3. FP7915 protein

[0106] A: Nucleotide sequence (SEQ ID NO: 7) length: 3491 bases

[0107] B: Amino acid sequence (SEQ ID NO: 9) Length: 178 amino acids

[0108] C. Nucleotide and amino acid com...

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Abstract

The present invention discloses a new kind of human protein with the functions of promoting 3T3 cell transformation, polynucleotides encoding this polypeptide and recombination process to produce the polypeptide. The present invention also discloses the agonist resisting the polypeptide and its treatment effect. The present invention also discloses the application of the polynucleotides encoding this new kind of human protein with the functions of promoting 3T3 cell transformation.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a new polynucleotide encoding a human protein with the function of promoting 3T3 cell transformation, and a polypeptide encoded by the polynucleotide. The present invention also relates to the use and preparation of such polynucleotides and polypeptides. Background technique [0002] Human genomics research is currently a hot spot in the world. In addition to large-scale sequencing of human chromosomal DNA and expressed sequence sequencing (EST), there is still a lack of high-throughput methods for screening functional genes starting from function. [0003] Cancer is one of the major diseases that endanger human health. In order to effectively treat and prevent tumors, people have paid more and more attention to gene therapy of tumors. Therefore, there is an urgent need in this field to develop and study human proteins and their agonists / inhibitors related to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H21/00C07K14/435C07K14/47C07K16/18C12N15/12C12N15/63C12N15/64C12P21/02
Inventor 顾健人杨胜利
Owner NEWORGEN
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