Escherichia expression system for secreting and expressing recombinant human epidermal growth factor
A technology for secreting expression and expression plasmid, which can be used in the field of bioengineering and can solve problems such as low content
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Embodiment 1
[0039] Example 1 Chemically synthesized signal peptide phoAspL10 sequence and human epidermal growth factor (hEGF) tandem gene (SEQ ID NO: 2)
[0040] ↓Start codon
[0041] 5'GAT ACC AAA CAA AGC ACT CTG CTG CTG CTG CTG CTG CTT CTG CTG
[0042]|← Newly designed signal peptide sequence (the underlined part is
[0043] CTG CTG ACC CCT GTG ACA AAA GCG AAT TCCGAC TCT GAA TGC CCG
[0044] ten leucines in a row) →|←
[0045] CTG TCT CAC GAC GGT TAC TGC CTA CAC GAT GGT GTT TGC ATG TAT
[0046] human epidermal growth factor (hEGF) gene
[0047] ATC GAA GCT CTG GAC AAA TAC GCG TGC AAC TGT GTT GTT GGT TAC
[0048] ATC GGT GAA CGT TGC CAG TAC CGT GAC CTG AAA TGG TGG GAA CTG
[0049] ↓Stop codon
[0050] CGT GCTT GGG3'
[0051] →| HindIII restriction site (underlined part)
[0052] When designing and synthesizing the above-mentioned tandem genes, we fully followed the following principles: the codons preferred by Escherichia coli were sele...
Embodiment 2
[0054] The construction of embodiment 2 expression plasmid ( figure 1 )
[0055] 2.1 Enzyme digestion treatment of the starting vector pBLGlu2
[0057] Prepare the following reaction mixture:
[0058] 10 μg of plasmid pBLGlu2;
[0059] 20 μl 10×H buffer (TaKaRa);
[0060] 5 μl EcoRI restriction endonuclease (15U / μl, TaKaRa company);
[0061] Make up 200 μl with sterilized double distilled water.
[0062] The above reaction mixture was reacted at 37° C. for 4 hours.
[0063] 2.1.2 Recovery of the vector after digestion
[0064] Add 20μl 3M sodium acetate and 400μl absolute ethanol to the reaction mixture obtained in 2.1.1, mix well, centrifuge at 12,000rpm for 5 minutes, discard the supernatant, then add 400μl 70% ethanol, mix well, 12,000rpm Centrifuge for 2 minutes, discard the supernatant, and vacuum dry.
[0065] 2.1.3 Digestion with Mung Bean Nuclease
[0066] Prepare the following reaction mixture:
[0067] The carrier recovered i...
Embodiment 3
[0123] Example 3 Construction and screening of genetically engineered strains
[0124] 3.1 Construction of genetically engineered strains
[0125] Prepare competent cells of Escherichia coli BL21(DE3) according to the method in 2.4, transform the expression plasmid pBL10EGF into the competent cells of BL21(DE3) according to the method in 2.5, and take 100 μl of LB coated with 100 μg / ml ampicillin Plates (agar content 1.5%) were cultured upside down at 37°C overnight. 3.2 Screening of genetic engineering strains
[0126] 3.2.1 Induced expression of genetically engineered strains
[0127] Pick the single colony obtained in 3.1 and put it into 3 ml of LB medium (the concentration of ampicillin is 100 μg / ml), cultivate overnight at 37°C and 200 rpm, and then transfer it into LB medium at a ratio of 1:10, and then After continuing to cultivate for 4 hours, the temperature was raised to 42° C., and the cultivation was terminated after 6 hours. Take 1ml of the culture supernatant...
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