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

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

Inactive Publication Date: 2004-02-11
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] FP17659, FP17720, FP17859, FP17889, FP17926, FP18346, FP18407 and FP18717 were obtained from human fetal cDNA libraries constructed by conventional methods. Fetal tissue was taken, total RNA was extracted with Trizol reagent (GIBCO BRL company) according to the manufacturer's instructions, and mRNA was extracted with mRNA purification kit (Pharmacia company). The cDNA library of the above mRNA was constructed with the pCMV-script TMXR cDNA library construction kit (Stratagene). The reverse transcriptase was changed to MMLV-RT-Superscript II (GIBCOBRL), 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 high-abundance cDNA clones and cDNA clones that had been proven to inhibit the growth of cancer ...

Embodiment 2

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

[0091] Fetal tissue was taken, total RNA was extracted with Trizol reagent (GIBCO BRL company) according to the manufacturer's instructions, and mRNA was extracted with mRNA purification kit (Pharmacia company). Use MMLV-RT-Superscript II (GIBCOBRL) 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 31 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 to a host cell using conventional techniques to obtain a recombinant protein (SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23).

[00...

Embodiment 3

[0094] Embodiment 3: cDNA clone sequence analysis

[0095] 1. FP17659A: Nucleotide sequence (SEQ ID NO: 1) length: 1974 bases

[0096] B: Amino acid sequence (SEQ ID NO: 3) Length: 354 amino acids

[0097] C. Nucleotide and amino acid combination sequence (SEQ ID NO: 2) clone number and protein name: FP17659 start codon: 193 ATG stop codon: 1255 TGA protein molecular weight: 37674.30KDa2.FP17720

[0098] A: Nucleotide sequence (SEQ ID NO: 4) length: 3190 bases

[0099] B: Amino acid sequence (SEQ ID NO: 6) Length: 127 amino acids

[0100] C. Nucleotide and amino acid combination sequence (SEQ ID NO: 5) clone number and protein name: FP17720 start codon: 1815 ATG stop codon: 2196 TAA protein molecular weight: 13775.99KDa3.FP17859

[0101] A: Nucleotide sequence (SEQ ID NO: 7) length: 2267 bases

[0102] B: Amino acid sequence (SEQ ID NO: 9) Length: 95 amino acids

[0103] C. Nucleotide and amino acid combination sequence (SEQ ID NO: 8) clone number and protein name: FP1785...

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Abstract

The present invention discloses a new kind of human protein with 3T3 cell transformation improving function, 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 human protein with 3T3 cell transformation improving function.

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 Applications(China)
IPC IPC(8): C07H21/00C07K14/435C07K14/47C07K16/18C12N15/11C12N15/12C12N15/63C12N15/64C12P21/02
Inventor 顾健人杨胜利
Owner NEWORGEN
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