Coding polynucleotide of human protein with function of suppressing cancer cell growth
A polynucleotide and encoding 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 gene 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] PP2030, PP2243, PP6569, PP7080, PP7302, PP7759, PP8304, PP8675, PP9455, PP9748, PP9932 and PP10141 were obtained by constructing a human placenta cDNA library by conventional methods. Placental tissues at 3, 6, and 9 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 (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 c...
Embodiment 2
[0088] Example 2: Obtain full-length gene and recombinant protein expression by PCR from placenta cDNA
[0089] Human placental 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). Use MMLV-RT-SuperscriptII (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 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 rec...
Embodiment 3
[0091] Embodiment 3: cDNA clone sequence analysis
[0092] 1. PP2030
[0093] A: Nucleotide sequence (SEQ ID NO: 1) Length: 2144 bases
[0094] 1 GCAACATGGC GGCTGCCGTG GTGCAGCGCC CGGGCTGAGC GACAGCAAGT GCAGCGGGCT
[0095] 61 CCTACCCCGG GTGAGGGGTG GCCTCCGCGT TGGATCGTGC CCTCTTCAGC CCGCTCCTGT
[0096] 121 CCCCGACATC ACGTGTATTC CGCACGTCCC CTCCGCGCTG TGTGTCTACT GAGACGGGGA
[0097] 181 GGCGTGACAG GGCCCGGGTC CCTTCTCAGT GGTGCTCTGT GCTTCAGGGC AAGCTCCCCG
[0098] 241 TCTCCGGGCG CACTTCCCTC GCCTGTGTTC GGTCCATCCT CCTTTCTCCA GCCTCCTCCC
[0099] 301 CTCGCAGGCG GATGACCCGG ACGACGGGCC AGTGCCTGGC ACCCCGGGGTGCCAGGGTC
[0100] 361 CACGGGGAAC CCGAAGTCCG AGGAGCCCGA GGTCCCGGAC CAGGAGGGGC TGCAGCGCAT
[0101] 421 CACCGGCCTG TCTCCCGGCC GTTCGGCTCT CATAGTGGCG GTGCTGTGCT ACATCAATCT
[0102] 481 CCTGAACTAC ATGGACCGCT TCACCGTGGC TGGCGTCCTT CCCGACATCG AGCAAGTTCT
[0103] 541 TCAACATCGG GGACAGTAGC TCTGGGCTCA TCCAGACCGT GTTCATCTCC AGTTACATGG
[0104] 601 TGTTGGCACC TGTGTTTGGC TACCTGGGTG AC...
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