Saccharomycetes for expressing recombinant human serum albumin, construction method and application thereof, and method for expressing recombinant human serum albumin
A human albumin and yeast technology, applied in the biological field, can solve the problems of low expression amount and long construction period of expression strains, and achieve the effects of correct protein structure, low cost and increased expression amount
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Embodiment 1
[0046] Example 1: Obtaining of Human Albumin Coding Sequence
[0047] Referring to the "Homo sapiens serum albumin precursor, mRNA, complete cds" sequence published by GenBank (accession number: AY728024.1), Shanghai Sangon Bioengineering Co., Ltd. was entrusted to synthesize the human albumin coding sequence of the present invention (see SEQ ID for the synthetic sequence NO: 1). Wherein, the 5' end of the coding sequence includes a BamHI restriction site GGATCC, and then connects the human albumin signal peptide coding sequence (SEQ ID NO: 2), and then connects the human serum albumin mature peptide coding sequence (SEQ ID NO : 3), there is an EcoRI restriction site GAATTC at the 3' end of the human albumin mature peptide coding sequence. Insert the synthesized DNA sequence into the pUC57 vector and name it pUC57-Alb (vector structure diagram is attached figure 1 shown).
Embodiment 2
[0048] Example 2: Construction of recombinant expression vector pPIC9K-AlbN
[0049] Use BamHI and EcoRI endonucleases to excise the nucleic acid fragments of pUC57-Alb with the coding sequence of human albumin signal peptide and the coding sequence of human albumin mature peptide, separate by 1% agarose gel electrophoresis, and recover by DNA gel The kit recovers nucleic acid fragments of about 1800bp. Use BamHI and EcoRI endonucleases to excise the α-factor signal peptide coding sequence and the multiple cloning site sequence in the pPIC9K expression vector (Invitrogen, with its own α-factor signal peptide coding sequence, his4 gene selection marker, and kanamycin resistance gene) , separated by 1% agarose gel electrophoresis, and a 9000bp vector backbone was recovered with a DNA gel extraction kit. Ligate and transform the above recovered vector backbone and nucleic acid fragments into Escherichia coli DH5α competent cells, and use LB plates containing 100 μg / ml ampicillin...
Embodiment 3
[0050] Embodiment 3: Construction of recombinant expression vector pPIC9-AlbN
[0051] Use BamHI and EcoRI endonucleases to excise the nucleic acid fragments of pUC57-Alb with the coding sequence of human albumin signal peptide and the coding sequence of human albumin mature peptide, separate by 1% agarose gel electrophoresis, and recover by DNA gel The kit recovers nucleic acid fragments of about 1800bp. Use BamHI and EcoRI endonucleases to excise the α-factor signal peptide coding sequence and multiple cloning site sequence in the pPIC9 expression vector (Invitrogen, with α-factor signal peptide sequence and his4 gene selection marker), and use 1% agarose gel electrophoresis Separate and recover the 9000bp vector backbone with a DNA gel recovery kit. The vector backbone and nucleic acid fragments recovered above were ligated and transformed into Escherichia coli DH5α competent cells, and positive clones were screened with LB plates containing 100 μg / ml ampicillin resistance...
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