Method for recombining, expressing and producing human thymosin in yeast
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A technology of thymosin, a mature person, applied in the field of genetic engineering
Active Publication Date: 2013-02-13
冯鹏波
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[0011] Although there have been reports of fusing human thymosin β4 or α1 to the whole human albumin molecule, no matter in yeast, bacteria, or other expression systems, there has been no production of thymosin β4 and α1 by fusing the amino-terminal part of human serum albumin. to report
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Embodiment 1
[0041] Embodiment 1: the production of recombinant human thymosin β4
[0042] Step 1. Design and biosynthesis of the N-terminal partial human serum albumin (N-HSA) gene
[0043] According to the human serum albumin gene whose sequence number is recorded as NM_000477 in GenBank or P02768 (SEQ ID No.1) in Uniprot, 2 primers were designed, and the restriction sites and protective bases of XhoI and PstI were introduced respectively. This DNA fragment contains XhoI and PstI restriction sites and encodes 186 amino acid residues of amino-terminal human serum albumin.
[0044] Upstream primer sequence: 5'-GATCAGTCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGC-3';
[0045] Downstream primer sequence: 5'-GCAGTCACTGCAGCCGAAGTTCATCGAGCTTTGGC-3';
[0046] The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The PCR method is: add 5 μl 10×Pfu buffer (containing MgSO4), 40 μl water, 1 μl (100ng / μl) to 50 μl system
[0047] For cDNA library, 1 μl of upstream and downstream primers ...
Embodiment 2
[0061] Embodiment 2: the production of recombinant human thymosin α1
[0062] Step 1. Design and biosynthesis of the N-terminal partial human serum albumin (N-HSA) gene
[0063] According to the human serum albumin gene whose sequence number is recorded as NM_000477 in GenBank or P02768 (SEQ ID No.1) in Uniprot, 2 primers were designed, and the restriction sites and protective bases of XhoI and PstI were introduced respectively. This DNA fragment contains XhoI and PstI restriction sites and encodes 186 amino acid residues of amino-terminal human serum albumin.
[0064] Upstream primer sequence: 5'-GATCAGTCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGC-3';
[0065] Downstream primer sequence: 5'-GCAGTCACTGCAGCCGAAGTTCATCGAGCTTTGGC-3';
[0066] The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The PCR method is: add 5 μl 10×Pfu buffer (containing MgSO4) to 50 μl system, 40 μl water, 1 μl (100 ng / μl) cDNA library, 1 μl (5 μmol / L) of upstream and downstream primers, 1...
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Abstract
The invention provides a method for massively producing human thymosin with low cost. The method is used for implementing restriction enzyme digestion to obtain a fusion gene sequence with a structure as follows: A-X-C, wherein A is a nucleotide sequence of site (1-150)-(1-372) amino acids at the N- end of coded mature human serum albumin; X is the nucleotide sequence of connecting peptide with enterokinase or tobacco etch virus (TEV) protease cutting site contained in a code; and C is a human thymosin gene. The method comprises steps as follows: connecting the fusion gene sequence to an expression carrier; transforming and introducing the expression carrier into saccharomycetes; carrying out induction expression to obtain soluble human serum albumin-thymosin fusion protein; then adding the TEV protease for cutting; and separating and purifying to obtain the recombined human thymosin. The human thymosin production method provided by the invention has the advantages that consistency of quality of products can be ensured, no limitation is generated from sources of raw materials, cost is low, an expression index is high, and mass production can be achieved and the like.
Description
technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for producing human thymosin through recombinant expression of yeast. Background technique [0002] Peptides have a very important and irreplaceable regulatory effect on the human body, which involves almost all physiological activities of the human body. For the immune system of the human body, peptides, especially thymosin or thymosins, are more important and direct. Thymosin is an immune drug that was used earlier (Goldstein AL.2007.History of the discovery of the thymosins.Ann N YAcad Sci.1112:1-13). Thymosin is secreted by thymus epithelial cells, a class of polypeptide hormone mixture containing more than 40 components, first discovered by Goldstein et al. in 1966 from fetal bovine thymus protein extract, and its level is closely related to the body's immune function. According to the positions of these polypeptides on the isoelectric focusing electro...
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