New human protein having cancer cell growth in hibiting function and its code sequence
A human protein and function technology, applied in the use and preparation of polynucleotides and polypeptides, in the field of polypeptides encoded by polynucleotides, can solve problems such as lack of functional genes and high throughput
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Embodiment 1
[0084] Example 1: Obtaining of cDNA gene and its inhibitory effect on cancer cell clone formation
[0085]FP3420, FP7019, FP12591, FP13812 and FP15256 were from human fetal cDNA libraries constructed by 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 cDNA library for cfu / μg cDNA titer. 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 cells were used as probes to h...
Embodiment 2
[0088] Example 2: Obtain full-length gene by PCR from placenta or fetal cDNA:
[0089] Take placental tissue (PP clone) or fetal tissue (FP clone) at 3, 6, and 9 months old, use Trizol reagent (GIBCOBRL 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 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 is verified by sequencing and is consistent with the sequence measured in Example 1, and then the amplified product is transferred to a host cell using conventional techniques to obtain a recombinant protein...
Embodiment 3
[0091] Embodiment 3: cDNA clone sequence analysis
[0092] 1. FP3420
[0093]A:核苷酸序列(SEQ ID NO:1)长度:2554个碱基1 GCTGTGTTGT CTAGGCTGGT CTCAAACTCT GGGGCTCAAG TGATCCTCCC ACCTTGGCCT 61 CCCAAAGTGC TGGGATTGCA GGCATGGGCC ACCACAACCG GCTCTGCTTT TACTTTTTAA 121 ATGTAGCTAC TAATAAATCA AAAACTACAT ATGTAGCTCC CATTACATGT TCACTGAACA 181 GCACTGGTCC AGGTATAGAG GAAAGAAGGC CAGGCAGCGG GAAGGAAGGG AATGGCTGGG 241 TACAGCAGGC ATGGGGCGGG CAGACTTGAG AAGCCTTGGT GACAGTGTGC ACATAGGAGT 301 CAGGGTGGAG GTGATGTGAA GGGTAGGCAC AGAGGCCAGG GGGAGGAAGG TGGTGTCATT 361 CACTTATGCA GGAGGATGGG GAGGAGCAGA CTCGAGGGAA GAGGATGGCC TCAGTTTGGG 421 GAAAGGTGAA GAGGGCAGTG GGGGAGCACT CCTGTGAGGA TGCCCAACAG ACGTCAGGCT 481 TCTTGTCCAG ACCTGCCCCC TGAGTCTGAC CTTCGCTTGT CCCATGCTGC CTCCCTTCTC 541 AGCCTGGGGC TGGTCTCACC CAAGCCAGGG GGGCAGAACC GTGCTCGGGC TCCGTTGCCC 601 CTTGTCTGCC ATTCACAGTG ATCCTGCTGG AGCTTGGACA CCGGGGTCCG GGCTCAGGTG 661 GATGCCTTGG CCAGAGAAGG GAGTTCTCGC AGAGACGCAT GCCAACAGCC ACAATAAGTC 721 AGACAATTCT GTTTTTCCAA CAGAGGGA...
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