Construction and application of highly stable superoxide dismutase high-efficiency expression system
A superoxide and dismutase technology, applied in the field of enzyme engineering, can solve the problems of not producing enough superoxide dismutase in time, unsatisfactory antioxidant effect, unstable activity, etc. , the effect of expressing large yield
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Embodiment 1
[0069] The construction of a high-efficiency expression system of high-stable superoxide dismutase is characterized in that the protein sequence of high-stable superoxide dismutase is shown in any one of SEQ ID NO.1-7.
[0070] The gene sequence corresponding to the protein sequence encoding a highly stable superoxide dismutase is shown in one of SEQ ID NO. 8-15.
[0071] Specifically, the gene sequence corresponding to SEQ ID NO.1 encoding a highly stable superoxide dismutase protein sequence is SEQ ID NO.8 or SEQ ID NO.9, and the gene sequence corresponding to SEQ ID NO.2 is SEQ ID NO.2 The gene sequence corresponding to ID NO.10 and SEQ ID NO.3 is SEQ ID NO.11, the gene sequence corresponding to SEQ ID NO.4 is SEQ ID NO.12, and the gene sequence corresponding to SEQ ID NO.5 is SEQ ID NO.5 The gene sequence corresponding to ID NO.13 and SEQ ID NO.6 is SEQ ID NO.14, and the gene sequence corresponding to SEQ ID NO.7 is SEQ ID NO.15;
[0072] Wherein, in SEQ ID NO.9-15, R=A o...
Embodiment 2
[0102] The construction method of the high-efficiency expression system of highly stable superoxide dismutase comprises the following steps:
[0103] (1) Template synthesis: Synthesize according to any one of the sequences SEQ ID NO. 8-15;
[0104] Primer design: The restriction endonucleases NdeI and HindIII were used as the restriction endonuclease cleavage sites of the upstream and downstream primers, and the 5' and 3' ends of the sequences of SEQ ID NO. 8-15 were used as the amplification starting point to design specific Sex primers. Preferably, the primer design sequence is: upstream primer 5'-GGCATATGATGGGTGTTCATAAATTAG-3'; downstream primer: 5'-GGCCAAGCTTCTTAATGAAGTCTTTTAAG-3';
[0105] Gene amplification: using pfu high-fidelity DNA polymerase amplification system, adding the above primers and templates to form a 50 μl reaction system, and amplifying 35 cycles in a PCR instrument;
[0106] Double enzyme digestion: The target gene and the vector pET30a were double di...
Embodiment 3
[0117] When the present inventor was exploring and researching the present invention, with regard to other types of products, he also made a comparison, as follows:
[0118] After the present invention and three commercially available products of the same kind were prepared into a solution with the same protein concentration (0.1 mg / ml), the activity was detected by "superoxide dismutase detection kit (NBT method)". as attached image 3 shown; attached image 3 In , the substrate will turn purple after auto-oxidation, and superoxide dismutase can prevent the auto-oxidation of this substrate, so the lighter the color, the higher the enzyme activity, and the darker the color, the lower the enzyme activity.
[0119] After the present invention and three commercially available products of the same kind were prepared into solutions with the same protein concentration (0.1 mg / ml), the activity was detected with the "superoxide dismutase detection kit (NBT method)", as shown in the ...
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