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New human protein having mouse NIH/3T3 cell conversion promoting function and its code 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: 2003-07-16
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] FP630, FP2234, FP6679, FP6779, FP14381 and FP15331 were obtained from human fetal cDNA libraries constructed according to conventional methods. Fetal tissue (FP clone) 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 (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 high-abundance cDNA clones and cDNA clones that had been proven to inhibit the growth of cancer c...

Embodiment 2

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

[0091] Fetal tissue (FP clone) 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 (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 for 1′35 cycles, and 72°C for 10′1 cycle to carry out 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 by conventional techniques to obtain recombinant proteins (SEQ ID NO: ...

Embodiment 3

[0093] Embodiment 3: cDNA clone sequence analysis

[0094] 1. FP630

[0095]A:核苷酸序列(SEQ ID NO:1)长度:1837个碱基1 GCTAAATCCC CTTGTAAATT TAACTGTTAG TCCAAAGAGG AACAGCTCTT TGGACACTAG 61 GAAAAAACCT TGTAGAGAGA GTAAAAAATT TAACACCCAT AGTAGGCCTA AAAGCAGCCA 121 CCAATTAAGA AAGCGTTCAA GCTCAACACC CACTACCTAA AAAACCCCAT CTCTACTAAA 181 AAAAAAAAAA TACAAAAAAT TAGCCAGGCA TGGTGGCGGG CGCCTGTAGT CCCAGCTACT 241 CCGGAGGCTG AGGCAGGAGA ATCGCTTGAA CCTGGGAGGC TGAGGTTGCA GTGAGCCGAG 301 ATCGCGCCAT TGCACTCCAG CCTCGACAAC AAGAGCCAAA CTCCGTCTCA AAAAAAAAAA 361 TTAAATAACA GCAAGCAACT GCATGCACGT CTGGGGGCGG TGTCCGGGGT GAGAAAGGCC 421 CCGCCAGCAA TCCATCCCAC AATCAGCGAT GGCTGAGGGG GTCTGGACCT CGCGGGACGG 481 GGCTGCACGC CCCCAAGCAA ATGCACAGCG CGGCTAAATT GGATTCGACA GCACCGGAAA 541 CGGCGACTCC CACTTGGGGC GCTGCGGACA CACGAGTCGA GGCTGCCTTC CAGGAAGCAA 601 ACAAAAAAAG GGGGGAAAAG GGGGGGAAAG AAAGAAAGAG AAAAAGGAGG GCGAGTGGCG 661 AGCAGGGGCC TCGGCCGCCA CCCACACGCC CCGAAGCGTG CTCGTCCCCC GCGCGGGGCT 721 CCCGGCCGCC GCCCTCGGCC ATCGGCTGC...

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Abstract

A novel human protein with the function of promoting mouse NIH / 3T3 cell conversion, the polynucleotide for coding the polypeptide, the process for preparing the said polypeptide by recombination, theantagon of the 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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Patent Type & Authority Applications(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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