Codon optimization of artificially designed structure and preparation and application of chemically synthesized gene rdh and its expression product polypeptide g1
A technology of structure design and chemical synthesis, applied in chemical instruments and methods, botanical equipment and methods, applications, etc., can solve the problem of no advanced structure of polypeptide G1 molecule, achieve good application effect, broad application prospect, and reduce product cost Effect
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Embodiment 1
[0050] PCR amplification and purification of gene rdh:
[0051] According to the nucleotide sequence of the gene rdh shown in SEQ ID No.1, a pair of PCR amplification primers are designed, namely:
[0052] Upstream primer P1: 5'-TCGCG^GATCCATGGGAGCTTCTCTGCAAAT-3'
[0053] Downstream primer P2: 5'-TCGAGTGC^GGCCGCTTAGCTGGAGAGCTTCTTCAACC-3'.
[0054] Among them, G^GATCC is the BamH1 restriction enzyme cut point of the primer, and 5'GC^GGCCGC 3' is the NotI restriction enzyme cut point. PCR amplification was performed using rdh as a template. Amplification conditions: pre-deformation at 95°C for 2min30sec, followed by 30 cycles of programmed amplification at 95°C for 30sec, 58°C for 30sec, and 68°C for 1min. The amplified product was purified with a PCR product purification kit.
Embodiment 2
[0056] Construction of rdh gene clone strain:
[0057] The PCR amplification and purification product in Example 1 was ligated with the pEV cloning vector digested with the restriction enzyme EcoRV to obtain the pEV-rdh plasmid. Then transform Escherichia coli E.coli DH5a with this recombinant plasmid, by adding 100mg / L ampicillin (Amp) in the LB solid medium 37 ℃ culture growth, screen out the positive clone strain E.coli DH5a-pEV by selection pressure -rdh. The plasmid pEV-rdh can be continuously extracted through the cultivation of the bacterial strain.
Embodiment 3
[0059] Construction of rdh gene expression engineering strain:
[0060] Plasmids pEV-rdh and pET28a were digested with BamH1 and NotI, and the rdh gene was cloned into pET28a to obtain recombinant plasmid pET28a-rdh. Then use this recombinant plasmid to transform E.coli JM109 (DE3), and use the LB medium added with 50mg / L kanamycin (Km) to screen to obtain the expression engineering strain E.coliJM109 (DE3)-pET28a-rdh of positive clone.
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