Construction and application of efficient expression system of high-stable superoxide dismutase
A superoxide, high-efficiency expression technology, applied in the field of enzyme engineering, can solve the problems of not producing enough superoxide dismutase in time, unsatisfactory anti-oxidation effect, unstable activity, etc., and achieves vitality that is not easy to lose and high stability , express the effect of large output
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0069] The construction of the high-efficiency expression system of highly stable superoxide dismutase is characterized in that it has a protein sequence of highly stable superoxide dismutase as shown in any one of SEQ ID NO.1-7.
[0070] The gene sequence corresponding to the protein sequence coding for highly stable superoxide dismutase is shown as one of SEQ ID NO.8-15.
[0071] Specifically, the gene sequence corresponding to the protein sequence SEQ ID NO.1 encoding a highly stable superoxide dismutase 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 ...
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: use restriction enzymes NdeI and HindIII as the restriction enzyme cutting sites of upstream and downstream primers, and use the 5' end and 3' end of the sequence of SEQ ID NO.8-15 as the starting point of amplification to design specific sex primers. Preferably, the primer design sequence is: upstream primer 5'-GGCATATGATGGGTGTTCATAAATTAG-3'; downstream primer: 5'-GGCCAAGCTTCTTAATGAAGTCTTTTAAG-3';
[0105] Gene amplification: use the pfu high-fidelity DNA polymerase amplification system, add the above primers and templates to form a 50 μl reaction system, and amplify in a PCR instrument for 35 cycles;
[0106] Double enzyme digestion: The target gene and the vector pET30a were double digested with NdeI and HindIII, ...
Embodiment 3
[0117] When the inventor explored and researched the present invention, he also compared other types of products, as follows:
[0118] After the present invention and three commercially available products of the same kind were formulated into solutions with the same protein concentration (0.1 mg / ml), the activity was detected with a "superoxide dismutase detection kit (NBT method)". as attached image 3 shown; attached image 3 Among them, the substrate will turn purple after being autoxidized, and superoxide dismutase can prevent the autoxidation 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 kinds of commercially available similar products are formulated into the solution of identical protein concentration (0.1mg / ml), detect activity with " superoxide dismutase detection kit (NBT method) ", as attached Figure 4 Shown, adopt the histogram to...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More