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Human Protein for promoting transform of 3T3 cell and its coding sequence

A technology of cell transformation and human protein, which is applied to the use and preparation of polynucleotides and polypeptides, and the field of polypeptides encoded by polynucleotides, which can solve the problems of lack of functional genes and high throughput

Inactive Publication Date: 2005-07-06
SHANGHAI INST OF ONCOLOGY
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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] Example 1: Acquisition of cDNA gene and promotion of 3T3 cell clone formation

[0087] PP12719, PP13181, PP13191, PP13479, PP13439, PP13842, PP14673 and PP14776 were obtained by constructing a human placenta cDNA library by conventional methods. Placental tissues at 3, 6, and 10 months old were taken, and total RNA was extracted with Trizol reagent (GIBCO BRL Company) according to the manufacturer's instructions, and mRNA was extracted with an 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 ...

Embodiment 2

[0090] Embodiment 2: PCR obtains full-length gene from placenta cDNA:

[0091] Placental tissues at 3, 6, and 10 months old were taken, and total RNA was extracted with Trizol reagent (GIBCO BRL Company) according to the manufacturer's instructions, and mRNA was extracted with an mRNA purification kit (Pharmacia Company). Use MMLV-RT-SuperscriptII (GIBCO BRL) and reverse transcriptase to perform reverse transcription at 42°C to obtain placental cDNA. Using the transmutation primers of each gene (as shown in the table below), follow 97°C for 3 minutes, 1 cycle; 94°C for 30 seconds → 60°C for 30 seconds → 72°C for 1 minute, a total of 35 cycles; 72°C for 10 minutes, 1 cycle. PCR amplification was carried out for one cycle 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 transferr...

Embodiment 3

[0093] Embodiment 3: cDNA clone sequence analysis

[0094] 1. PP12719

[0095] A: Nucleotide sequence (SEQ ID NO: 1) Length: 1179

[0096] 1 GTGGGATTAC AGGCGTGAGC CACCACCACA CCCAGCCCTG GGAATGGAGT CTTGATTCTT

[0097] 61 CTCTGCCCCT CACTGATTCT TCCAAACTGG AAACCCTGAA GCTGAGAGCC CAGCATGGTT

[0098] 121 CCTGGCAAAC AGCAGGCACT CAAATATTGA TTGGTTTACT GTATGACTAG TAGAGACCCC

[0099] 181 AACGAGCAAA ACTGTGGCCT AATAAAATTC TGGCTCCTCT CCCAGACTTC CCCTCCCTTT

[0100] 241 GAGAAATGCC AGAAGCTTCT TAGGGAGGCT CTTGCCAACC TAGACATCAC AGGCACTCAT

[0101] 301 GGGGCAGCTC CAGCCTCTTC CTCCTGTCAT CACCATAATG CATCCATATC TACAATATGG

[0102] 361 CAAATTTCAT ATCCTTCCAA CCTCTTTCCT GCATTATTGA TGGGCTGTGT GCACTTTTTTA

[0103] 421 AAAAATCAAT TAGATCAGGG CGTGGAGCTG GAGTTCAAAG AAGCCTTTAA AAGTCTGCTC

[0104] 481 TTCTGTTTTG CTGTTTTGAA TAGGCACAGA TAAAGCTTTC CCTCTGGTTT GAATAAGCCA

[0105] 541 AGCTCAGTGC TAGGTTGGCT CTGATTGGCC AGGACTAGGA AAATGCGGTT AAGATGCAAA

[0106] 601 CACAAGCAAA TATAACCCAG TATCTCTGTG GCCA...

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Abstract

A novel human protein with the function of promoting 3T3 cell transform, the polynucleotide for coding the peptide, the process for preparing said polypeptide by recombination, the antagon of said polypeptide and its medical action, and the application of said polynucleotide are disclosed.

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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IPC IPC(8): C07H21/00C07K14/47C07K16/18C12N15/11C12N15/12C12N15/63C12P21/02
Inventor 顾健人
Owner SHANGHAI INST OF ONCOLOGY
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