Human lysozyme-glutathione transferase fusion protein, expression vector and application thereof
A technology of fusion protein and human lysozyme, applied in the biological field, can solve the problems of insufficient production and application of commercial egg white lysozyme
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Embodiment 1
[0027] Example 1 Optimization of human lysozyme gene sequence
[0028] The intrinsic characteristics of exogenous genes, such as codon bias, are important factors affecting the effective expression of exogenous proteins. The present invention takes the human lysozyme gene (hLYS, GenBank: J03801.1) as the starting template, and redesigns the gene sequence according to the codon preference of Pichia pastoris shown in Table 1 (codon optimization, adding GST tags, etc. , such as low-frequency codons are optimized to high-frequency codons), and artificially synthesized, so that the hLYS gene can be more efficiently transcribed, translated, and expressed in the recombinant Pichia pastoris expression system.
[0029] Table 1 Codon preference of Pichia pastoris
[0030]
[0031] After sequence determination, the base sequence of the optimized artificially synthesized hLYS gene is shown in SEQ ID No.1 (this gene can also be purchased directly from BGI). figure 1 For sequence comp...
Embodiment 2
[0032] Embodiment 2 Synthesis of human lysozyme-glutathione transferase fusion protein
[0033] In order to increase the expression efficiency of the target protein, constructing fusion protein has become an effective method. The invention further constructs a novel human lysozyme-glutathione transferase fusion protein by means of genetic engineering.
[0034] The upstream and downstream primers were designed for the human lysozyme gene and glutathione transferase gene (GenBank: U13853.1, 258-977bp) before and after the above optimization, and amplified by PCR.
[0035] P1: 5'-GGC GA ATTC ATG TCCCCTATACTAGGTTATTG-3'
[0036] P2: 5'-GTCACGATGCGGCCGCTCGAGTCGAAAGGTTTTCGAAAGATGTGAATTGG-3'
[0037] P3: 5'-GGC GCGGCCGC TTAAACACCACAACCTTGAACGTAT-3'
[0038] PCR was amplified by Taq enzyme, and the system composition was: DNA 5 μL, 10×PCR Buffer (Mg 2+Plus) 5 μL, dNTPs (2 mmol / L) 5 μL, upstream primer (10 μmol / L) 2 μL, downstream primer (10 μmol / L) 2 μL, Taq polymerase (5U / μL) ...
Embodiment 3
[0042] Example 3 Construction of recombinant expression vector pPIC9K-hLYS
[0043] The fusion protein synthesized above was connected with the cloning vector pMD19T to construct the recombinant plasmid pMD19T-hLYS, which was transformed into Escherichia coli TOP10 competent cells, and the recombinant plasmid pMD19T-hLYS was used as a template to obtain the target gene fragment hLYS by PCR amplification and enzyme digestion, and the real The nuclear vector pPICZαA was connected to construct the recombinant expression vector pPIC9K-hLYS. Specific steps are as follows:
[0044] 1) In vitro connection of the fusion protein and the cloning vector pMD19T: the pMD19-T vector cloning kit produced by TaKaRa Company was used. According to the instructions, prepare the following ligation reaction solution in a microcentrifuge tube, 10 μL of the whole volume, 0.5 μL of pMD19-T Vector, 4.5 μL of the target DNA fragment, 15 μL of Ligation solution, and react overnight at 16°C.
[0045] 2...
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