Tandem DNA for multiple active peptides, construction method and expression vectors
A construction method and expression vector technology, applied in the field of bioengineering, can solve problems such as simultaneous expression that has not been reported, and achieve the effect of increasing the ratio
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Embodiment １
[0020] Example 1: Tandem DNA design and synthesis of two milk-derived hypotensive peptides CEI-12 and CEI-7
[0021] a. Tandem peptide sequence design of two milk-derived hypotensive peptides CEI-12 and CEI-7
[0022] The milk-derived hypotensive peptides CEI-12 (SEQ ID NO3) and CEI-7 (SEQ ID NO4) involved in the present invention correspond to the residue sequence of 23-34 of milk α-casein and 177 residues of milk β-casein, respectively. -183 residue sequence. The amino acid residues in the N segments of CEI-12 and CEI-7 are Lys and Arg, respectively, which are both trypsin cleavage sites. The amino acid sequences of CEI-12 and CEI-7 are alternately concatenated three times. Asp-Asp-Asp-Asp-Lys sequence is added to the N section, and the sequence is shown in SEQ ID NO2.
[0023] b. Gene design of two milk-derived hypotensive peptides CEI-12 and CEI-7 tandem peptides
[0024] The obtained amino acid sequence (SEQ ID NO2) was based on Escherichia coli ( Escherichia coli ) ...
Embodiment 2
[0025] Example 2: Construction of expression vector pUC18-CEI-12,7 and construction of Escherichia coli BL21-MF3A3 engineering bacteria
[0026] a. The nucleotide sequence SEQ ID NO1 designed in Example 1 was synthesized by Shanghai Xuguan Biotechnology Development Co., Ltd., cloned into pUC18 (purchased from Novagen), and the restriction site was Kpn I and Bam HI, named the recombinant vector pUC18-CEI-12,7, and transformed it into Escherichia coli ( Escherichia coli ) DH5α (purchased from Novagen).
[0027] b. Extract the plasmid from Escherichia coli DH5α containing pUC18-CEI-12,7, and carry out Kpn I and Bam HI (purchased from TaKaRa Company) was digested with double enzymes, and a fragment of about 200bp was recovered.
[0028] c. Perform expression vector pET-30a (purchased from Novagen) Kpn I and Bam HI (purchased from TaKaRa Company) was digested with double enzymes to recover large fragments.
[0029] d. Connect the recovered fragments with cohesive ends...
Embodiment 3
[0032]Example 3: Expression and detection of two milk-derived hypotensive peptides CEI-12 and CEI-7 tandem polypeptides
[0033] The Escherichia coli BL21-MF3A3 obtained in Example 2 was inoculated into 50 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L) containing Kanamycin Kan (50 μg / mL) in a volume ratio of 1% inoculum , NaCl 10g / L, pH7.0), 37°C, 200r·min -1 Cultivate in a constant temperature shaker until the OD600 is 1.5, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1mmol L –1 , induced at 28°C for 12 h, 5000r min -1 The bacteria were collected by centrifugation, frozen and thawed three times repeatedly, and ultrasonically disrupted in PBS buffer solution (pH 7.0) (300W, 2s / 2s, 2min), 8000r min -1 The precipitate was collected by centrifugation, dissolved in 8mL upper column buffer solution, purified by an activated Ni-column (purchased from Novagen) to obtain His-tagged recombinant protein, and subjected to SDS-PAGE electrophoresis,...
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