New human protein with cancer inhibiting function and its encoding sequence
A human protein and sequence technology, applied in the field of polypeptides encoded by polynucleotides, the use and preparation of polynucleotides and polypeptides, and can solve problems such as lack of functional genes and high throughput
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Embodiment 1
[0085] Example 1: Obtaining of cDNA gene and its inhibitory effect on mouse NIH / 3T3 cell clone formation
[0086] FP3361, FP7072 and FP8080 were derived from human fetal cDNA libraries constructed by conventional methods. Fetal tissues aged 3, 6, and 9 months 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 (GIBCO BRL), and the reverse transcription reaction was carried out at 42°C. The XL 10-Gold competent cells were transformed, and a cDNA library with a titer of 1×106cfu / μg was obtained. 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 can...
Embodiment 2
[0089] Example 2: Obtain full-length gene by PCR from placenta or fetal cDNA:
[0090] Fetal tissues (FP clones) aged 3, 6, and 9 months 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-Superscript II (GIBCO BRL) and reverse transcriptase to perform reverse transcription at 42°C to obtain placental or fetal cDNA. Use the specific primers for each gene (as shown in the table below), at 97°C 3', 1 cycle. 94°C 30", 60°C 30", 72°C 1', a total of 35 cycles; 72°C 10', 1 cycle for PCR amplification to obtain the amplification 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 recombi...
Embodiment 3
[0093] Embodiment 3: cDNA clone sequence analysis
[0094] 1. FP3361 protein
[0095] A: Nucleotide sequence (SEQ ID NO: 1) length: 3046 bases
[0096] 1 GGTTTCACCA TGATGGTCAG GCTGGTCAAG ATCTCCTGAC CTCAAGTGAT CTGCCCGCCT
[0097] 61 CGGCCTCCCA AAGTGCTGGG ATTACAGACA TGAACCACAA CACCTGCCAA GCCCAGGTGT
[0098] 121 TTTGAATCCA GCACGGGCAA TGGCAAGACC CTGTCCCAAC AAACAAAAAA ACACTGGTGC
[0099] 181 CTGAGGCCCA CCCCGAGAGA TTCTGATTGA GTTGATTTGG ATTTGAATTA TCTTTAAAAT
[0100] 241 TTTGGATGTG AATTTTTTTTT TTTTTTCTTG GAATGGAGTT TCACTCTTGT TCCCCAGGCT
[0101] 301 GGAGTGCAAC GGGACGATCT TGGCTCACCG CAACCTCCGC CTACTGGGTT CAAGCCATTC
[0102] 361 TCCTGCCTCA GCTTCCCAAG TAGCTGGGAT TACAGGCATG TGCTACCATG CCCAGCTAAT
[0103] 421 TTTGTATTTT TAGTAGAGAC AGGGTTTCTC CATGTTGCTC AGGCTGGTCT TGAACTCCTG
[0104] 481 ACCTCAGGTG ATCCGCCCGC CTCGGACTCC CAAAGTGCTG GGATTACAGG CATGAGCCAC
[0105] 541 CATGCCTGGC CGGATGTGAA TTATCTTAAA AATTTTCAGG TAATTCTAAT GGGCCAAGGT
[0106] 601 TGAGAACCCC TGCTCTGGGC CC...
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