Method for preparing fusion protein contg. human interferon-alpha 2 and human seralbumin and its products
A human serum albumin, fusion protein technology, applied in the field of long-acting fusion protein drugs, can solve the problems of increasing patient pain, treatment costs, toxic side effects, and high plasma clearance rate
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Embodiment 1
[0027] Example 1: Cloning of IFNα2 cDNA:
[0028] Take normal human peripheral blood plus lymphocyte separation medium, separate human blood leukocytes, and dilute them with DMEM medium containing 5% fetal bovine serum to a cell concentration of 5×10 7 cells / ml, add Newcastle disease virus to 100 hemagglutination units / ml, 37°C, 5% CO 2 , incubate for 1 hour to make the virus adsorb on the cells, centrifuge at 1000×g, discard the supernatant, add fresh DMEM medium with 5% fetal bovine serum, 37°C, 5% CO 2 , and cultured for 18 hours. Total RNA was isolated with an RNA extraction kit, and then reverse-transcribed into cDNA with a one-step RT-PCR kit (purchased from Dalian Bao Biological Co., Ltd.) and IFN α2 was amplified from it. The primers used were as follows:
[0029] Pa2b1: 5'-AGA GTC GAC ATGTGTGATCTGCCTCAA-3'
[0030] Pa2b2: 5'-GCC AAGCTT TCATTCCTTACTTCTTAAACT-3'
[0031] The one-step RT-PCR reaction system was prepared as follows:
[0032] 10×one step R...
Embodiment 2
[0035] Example 2: Cloning of HSA cDNA:
[0036] HSA cDNA was amplified from human fetal liver cDNA library by PCR. The primers used were:
[0037] HSA1: 5'-AGA GTC GAC GATGCACACAAGAGTGAGGTTGCTC-3'
[0038] HSA2: 5'-GCC AAGCTT TTATAAGCCTAAGGCAGCTTGACTT-3'
[0039] The PCR method is as follows: add to the 50 μl reaction system: 3 μl of primers for 10 μmol / L HSA1 and HSA2, 5 μl of 2 mmol / L dNTP, 5 μl of 10×pfu Buffer, 0.5 μl of pfu DNA polymerase at 5 U / μl (dNTP, 10 ×pfu Buffer and pfu DNA polymerase were purchased from Shanghai Bioengineering Technology Service Company, the same below), human fetal liver cDNA library 1 μg, added double distilled water to make up 50 μl. PTC-100 at MJResearch TM On the PCR instrument, the PCR conditions are: fully denature at 95°C for 5 minutes, denature at 94°C for 1 minute, anneal at 60°C for 1 minute, extend at 72°C for 90 seconds, and cycle 30 times.
[0040] The reaction product was analyzed by agarose gel electrophoresis, and a band ...
Embodiment 3
[0041] Example 3: Cloning of fusion fragments of HSA cDNA and IFN α2 cDNA:
[0042] PCR amplification of HSA cDNA: the primers used are as follows:
[0043] Pf1: 5'-CCCTCC GAATTC AAAAGAGATGCACACAAGAGTGAGGTTGCT-3'
[0044] Pf2: 5'-TGGGTTTGAGGCAGATCACATAAGCCTAAGGCAGCTTGAC-3'
[0045] The PCR method is as follows: Add 2.5 μl of 10 μmol / L Pf3 and Pf4 primers to the 50 μl reaction system, 5 μl of 2 mmol / L dNTP, 5 μl of 10×pfu Buffer, 0.5 μl of 5 U / μl pfu DNA polymerase, pBlue- HSA 1ng, add double distilled water to make up 50μl. PTC-100 at MJ Research TM On the PCR instrument, the PCR conditions are: fully denature at 95°C for 5 minutes, anneal at 60°C for 1 minute, extend at 72°C for 4 minutes, denature at 94°C for 1 minute, and cycle 30 times.
[0046] PCR amplification of IFN α2 cDNA: The primers used are as follows:
[0047] Pf3: 5'-GTCAAGCTGCCTTAGGCTTATGTGATCTGCCTCAAACCCA-3'
[0048] Pf4: 5'-CATAAG GCGGCCGC TCATTCCTTACTTCTTAAACTTTC-3'
[0049] The PCR method is as f...
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