Tranduced peptide-huamn erythrogenin fusion protein and its medicinal composition
A technology of erythropoietin and fusion protein, applied in the field of fusion protein, can solve the problem of high production cost, achieve the effects of low cost, improved transduction efficiency and simplified purification process
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Embodiment 1
[0029] Example 1 Extraction of Human Fetal Liver Total RNA
[0030] The RNAgentsR Total RNA Isolation System from Promega was used to extract total RNA from induced fetal liver.
Embodiment 2
[0031] Cloning of embodiment 2 hEPO gene
[0032] Design primers with reference to the EPO cDNA sequence in Genebank:
[0033] Primer 1: 5'-GGAATTCGCACCACCACGCCTAATC-3' (SEQ ID NO: 7)
[0034] Primer 2: 5'-GTCGACTCATCATCTGTCCCCTGTCCT-3' (SEQ ID NO: 8)
[0035] Using the RNA in Example 1 as a template, PCR amplification was performed according to the protocol recommended by the kit manufacturer to obtain full-length hEPO cDNA, with EcoRI and Sal I sites at the 5' and 3' ends, respectively.
[0036] Reverse transcription and PCR amplification: the reverse transcription PCR kit produced by Perkin ElmerCetus was used. Take 1-2ug of the extracted total cellular RNA, add it to a 20ul reverse transcription system, use oligo dT as a primer, and act at 42°C for 1h to synthesize the first strand of cDNA. The reverse transcription reaction was terminated at 99°C for 5 min. Add 20ul of the reverse transcription product into a 100ul PCR reaction system, which contains upstream and down...
Embodiment 3
[0037] Example 3 Construction and Identification of Recombinant hEPO Expression Plasmid
[0038] The RT-PCR product of hEPO in Example 2 was double digested with EcoRI and SalI, and then ligated with the expression vector pBV220 fragment recovered by the same digestion at a molar ratio of 4:1, transformed into competent Escherichia coli DH5α, and positive recombinants were screened pBV hEPO. Sent to Shanghai Shengong Company for sequencing.
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