Novel mutant EN-46 of human epidermal growth factor
An epidermal growth factor, EN-46 technology, applied in the fields of epidermal growth factor, growth factor/inducing factor, microorganism-based method, etc., can solve the problems of low final product yield, difficult quality control, mismatch, etc. Industrial production, easy extraction, separation and purification, and enhanced permeability
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Embodiment 1
[0095] Embodiment 1 adopts PCR random mutation technique to obtain mutant EN-46 nucleic acid molecule
[0096] Entrust Invitrogen to synthesize PCR primers (primer1 and primer2):
[0097] Primer1: 5'-CAT CAT ATG AAA AAG ACA GCT ATC-3'
[0098] Nd
[0099] Primer2: 5'-CAT CTG CAG TTA TTA ACG CAG TCC CAC-3’
[0100] PstI
[0101] Add to the 50μl PCR reaction system:
[0102] h 2 O 37μl
[0103]10XPCR buffer 5μl
[0104] 4dNTP (200μM each) 4μl
[0105] Primer1 (0.25μM) 1μl
[0106] Primer2 (0.25μM) 1μl
[0107] Template DNA (ompA-EGF) 1μl
[0108] Taq enzyme 1μl
[0109] PCR reaction:
[0110] Preheat at 94°C for 5 minutes, according to the program: 94°C, 30 seconds-50°C, 30 seconds-72°C, 30 seconds, 25 cycles of reaction.
[0111] PCR reaction
[0112]
Embodiment 2
[0113] Construction of embodiment 2 mutant EN-46 expression vector plasmid
[0114] The NdeI / PstI fragment of the PCR product was connected to the Y2K0422 (NdeI / PstI) expression vector. The obtained mutant gene EN-46 was placed between the strong promoter T7 and the terminator rrnBT1T2. Y2K0422 is a high-efficiency expression vector plasmid constructed and preserved by our company. This expression vector plasmid has a high copy number, and can inhibit the expression of non-target proteins after IPTG induction to obtain the highest expression of target proteins. The expression vector plasmid can also be replaced by a commercially available expression vector of the same type that has the same or equivalent characteristics as the Y2K0422 expression vector plasmid.
[0115] PCR product (NdeI / PstI) double restriction fragment
[0116]
[0117] Expression framework of mutant EN-46
[0118] in:
[0119] T7pro is T7 promoter
[0120] ompA is the leader peptide of Escherichia ...
Embodiment 3
[0124] Embodiment 3 expression vector plasmid transforms host bacterium, the cultivation and induced expression of host bacterium, and the engineering strain of screening high-efficiency expression
[0125] 1. Expression vector plasmid transformation host bacteria
[0126] The above constructed plasmid was transformed into Escherichia coli E. coli JM109 (DE3) by conventional methods, and the transformant containing the above expression framework was used to screen colonies with high expression of EN-46.
[0127] 2. Cultivation and induced expression of engineering bacteria and identification of expression product cells
[0128] Several transformant colonies were selected for expression experiments in test tubes. Ordinary LB medium for expression test (g / 1): peptone 10-15; yeast extract 5-15; NaCl 5-10; NaCl 2 HPO 4 0-10; ampicillin 0.1-0.2, initial pH 7.0.
[0129] Inoculate the test tube and incubate at 37°C to OD 600 When it reaches 3.0-4.0, add 100mM IPTG to induce ex...
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