Interfusion protein human serum protein and erythropoietin
A technology of erythropoietin and human serum albumin, applied in the field of biochemistry, can solve the problems of low molecular weight, short half-life, toxic and side effects, etc.
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Embodiment 1
[0044] Cloning of Example 1HSA cDNA
[0045] Take normal human liver tissue, use RNA extraction kit (TaKaRa company) to isolate total RNA, and use general RT-PCR kit (TaKaRa company) to synthesize cDNA of HSA in one step. The primers used were:
[0046] hsa1: 5'-GACAAGCTTATGAAGTGGGTAACCTTTTATTTC-3'
[0047] hsa2: 5'-GAAGGATCCTAAGCCTAAGGCAGCTTGACTTG-3'
[0048] The 5' end of primer hsa1 contains a HindIII site, and the 3' end of hsa2 contains a BamHI site.
[0049] RT-PCR one-step method is:
[0050] Add 2 μl reverse transcriptase, 2 μl Taq enzyme, 10 μmol / L hsa1 and hsa2 primers 4 μl each, 2.5 mmol / LdNTP 10 μl, 25 mmol / LMgcl in 100 μl reaction system 2 20 μl, 10 μl of 10x reaction buffer, 2 μl of RNA. Using an Eppendorf Mastercycler Personal PCR instrument, the RT-PCR conditions were: reverse transcription at 50°C for 30 minutes; pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 3 minutes;...
Embodiment 2
[0051] Embodiment 2 Fusion of connecting peptide cDNA and EPO cDNA
[0052] Take the EPO engineering bacteria of our company, use the DNA extraction kit (TaKaRa company) to extract DNA, and PCR amplify the cDNA of EPO. The primers used were:
[0053] epo1: 5'-GCCCCACCACGCCTCATCTG-3'
[0054] epo2: 5'-GTTGCGGCCGCTCATCTGTCCCCTGTCCTGCAAG-3'
[0055] The 5' end of the primer epo2 contains a NotI site, which is convenient for cloning into the expression vector.
[0056] PCR reaction:
[0057] Add 0.5 μl pfu enzyme to 100 μl reaction system, 4 μl each of 10 μmol / L epo1 and epo2 primers, 8 μl 2.5 mmol / L dNTP, 10 μl 10x reaction buffer, 2 μl DNA, H 2 O to make up 100 μl. Using Eppendorf Mastercycler Personal, the RT-PCR conditions were: 94°C pre-denaturation for 2 minutes; 94°C denaturation for 45 seconds, 57°C annealing for 45 seconds, 72°C extension for 1 minute, and after 30 cycles, 72°C extension for 5 minutes. Analyzing the reactant by gel electrophoresis showed a band of e...
Embodiment 3
[0064] Example 3 Fusion of HSA cDNA and EPO cDNA
[0065] The HSA gene and the EPO gene with Linker are fused by conventional DNA recombination method:
[0066] Digest the pBS-LEPO plasmid with BamHI and NotI, cut off the Linker-EPO fragment, perform gel electrophoresis on the digested product, cut the 600bp fragment, recover and purify it with a DNA Fragment Gel Recovery Kit (OMIGA Company), and then clone into In the pBS-HSA plasmid, the HSA gene, Linker and EPO genes are fused into a complete gene. The plasmid was extracted by conventional alkaline lysis method to identify the recombinant, and the obtained recombinant was named pBS-HLE. The complete gene encoding the HSA-L-EPO fusion protein is then inserted into the HindIII and NotI sites of a mammalian expression vector such as pcDNA3 (Invitrogen). The final expression plasmid (termed pcDNA3-HLE) contains the CMV early gene promoter-enhancer required for high-level expression in mammalian cells. This plasmid also conta...
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