Preparation method of recombinant ginseng superoxide dismutase
A technology of superoxide and dismutase, which is applied in the fields of genetic engineering and protein biochemistry, can solve the problems of high cost and low expression level, and achieve the effect of increasing industrial value, high expression level, and increased yield
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Embodiment 1
[0129] The construction of embodiment 1.rgSOD escherichia coli expression vector:
[0130] The secreted peptide gene refers to the secreted protein of Escherichia coli K12 strain and SEQ ID NO: 2 ( figure 2 ) shown in Membrane protein signal peptide design, and whole gene synthesis, its sequence is as shown in SEQ ID NO: 1 DNA fragment ( figure 1 ). The nucleotide sequence used to encode the signal peptide of the present invention and the deduced amino acid sequence of the signal peptide of the present invention are shown in Figure 7 . At the same time, an NdeI restriction site was introduced at its 5' end. The amino acid sequence of natural ginseng superoxide dismutase ( Figure 4 ) and the nucleotide sequence shown in SEQ ID NO: 3 are known ( image 3 ), such as NCBI's Genbank accession numbers are AF034630.1 and AAB87572 respectively, the acquisition of the target gene is based on the amino acid sequence of natural ginseng superoxide dismutase, using conventional gen...
Embodiment 2
[0132] The influence of embodiment 2 temperature on rgSOD expression level
[0133] Take a single clone, inoculate it into the LB primary seed solution, and cultivate it for 17-20hr; take three 50mL culture tubes, and inoculate them in three 50mL culture tubes with 5mL medium at a ratio of 1:50, and culture it for about 4hrs, 1mM / LIPTG induction, the induction temperature was 16°C, 25°C, and 37°C, respectively, the cells were collected, treated with lysozyme at 37°C for 2 hours, centrifuged at 15,000 rpm for 10 minutes, the supernatant was taken, and the expression was detected by SDS-PAGE. The experimental results show that the expression of rgSOD in shake flasks is almost the same at 16°C and 25°C, but the expression of secreted protein at 37°C is relatively small ( Figure 11 ).
Embodiment 3
[0134] The influence of embodiment 3IPTG concentration on rgSOD expression level
[0135] Take a single clone, inoculate it into the LB primary seed solution, and cultivate it for 17-20hr; inoculate it into five 50mL culture tubes containing 5mL medium at a ratio of 1:50, culture it at 37°C for about 4hrs, and cool it down to 20°C , add IPTG respectively, so that the concentration in the medium is 0.6mM, 0.4mM, 0.2mM, 0.1mM induction, collect the bacteria, treat with lysozyme at 37°C for 2 hours, centrifuge at 15000rpm for 10 minutes, take the supernatant, SDS-PAGE detection expression. Experimental results show that several gradient concentrations of IPTG induce rgSOD expression levels are similar, and it is more economical to induce with 0.1mM IPTG concentration, saving cost ( Figure 12 ).
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