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Preparation method of recombinant Cordyceps militaris superoxide dismutase

A technology of superoxide and Cordyceps militaris, which is applied in the fields of genetic engineering and protein biochemistry, can solve the problems of shortening the growth period of the second peak, harmful growth of Saccharomyces cerevisiae, and reducing the growth period of the second peak, so as to achieve industrialization value improvement, The effect of simplifying the purification process and improving the yield

Active Publication Date: 2018-05-25
吉林金梓源生物科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The knockout or deletion mutation of SOD1 is very harmful to the growth of Saccharomyces cerevisiae in anaerobic environment, and leads to a significant shortening of the post-diauxic lifespan; while the knockout or deletion mutation of SOD2 can cause growth inhibition and the same post-reduction bimodal growth phase

Method used

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  • Preparation method of recombinant Cordyceps militaris superoxide dismutase
  • Preparation method of recombinant Cordyceps militaris superoxide dismutase
  • Preparation method of recombinant Cordyceps militaris superoxide dismutase

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Embodiment 1

[0128] The construction of embodiment 1.rcSOD escherichia coli expression vector:

[0129] The secreted peptide gene refers to the secreted protein of Escherichia coli K12 strain and SEQ ID NO: 2 ( figure 2 ) shown in the membrane protein signal peptide design, and the whole gene synthesis, its sequence is as shown in SEQ ID NO: DNA fragment shown in 1 ( 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 Cordyceps militaris superoxide dismutase ( Figure 4 ) and the nucleotide sequence shown in SEQ ID NO: 3 ( image 3 ) is known, such as the Genbank accession numbers of NCBI are AY195841.1 and AAO40743.1 respectively, the acquisition of the target gene is based on the amino acid sequence of natural Cordyceps militar...

Embodiment 2

[0131] The influence of embodiment 2 temperature on rcSOD expression level

[0132] 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 rcSOD 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

[0133] The impact of embodiment 3IPTG concentration on the expression level of rcSOD

[0134] 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. The experimental results show that the expression of rcSOD induced by several gradient concentrations of IPTG is almost the same, and it is more economical and cost-effective to induce with 0.1mM IPTG concentration ( Figure 12 ).

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Abstract

The invention provides a nucleotide sequence of a signal peptide suitable for escherichia coli to carry out secretory expression on recombinant cordyceps sinensis superoxide dismutase (rcSOD), an SOD nucleotide sequence for coding cordyceps sinensis SOD, a carrier containing the nucleotide sequences, an engineering cell containing the carrier and a method for purifying rcSOD from the engineering cell. An optimized SOD gene is very suitable for being expressed by escherichia coli and has the characteristics of high expression, high stability and high secretion. The rcSOD pure product can be efficiently, simply and conveniently obtained at a low cost. The invention also provides application of rcSOD prepared by the method to preparation of medicines, food, health care products and cosmetics for treating human SOD related diseases or pathologic symptoms.

Description

technical field [0001] The present invention relates to the nucleotide sequence encoding the signal peptide of the recombinant Cordyceps militaris superoxide dismutase secreted and expressed by Escherichia coli and the nucleotide sequence encoding the SOD of Cordyceps militaris, the vector containing the above-mentioned nucleotide sequence, and the engineering cell containing the above-mentioned vector The method for purifying rcSOD from the engineered cell and its use belong to the fields of genetic engineering and protein biochemistry. Background technique [0002] Superoxide dismutase (SOD) is an enzyme that catalyzes the conversion of superoxide into oxygen and hydrogen peroxide through a dismutation reaction. It widely exists in various animals, plants, and microorganisms, and is an important antioxidant that protects cells exposed to oxygen. According to the different metal prosthetic groups it contains, it can be divided into three types. The first one is copper (Cu)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0089C12Y115/01001
Inventor 刘爽邓小霞孙晓悦庞玉王子华
Owner 吉林金梓源生物科技股份有限公司
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