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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

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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The activity of SOD can be achieved by using eukaryotic expression system, but unfortunately, because of factors such as low expression level and high cost, almost no one uses these expression systems to produce SOD at present

Method used

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  • Preparation method of recombinant ginseng superoxide dismutase
  • Preparation method of recombinant ginseng superoxide dismutase
  • Preparation method of recombinant ginseng superoxide dismutase

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Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides a nucleotide sequence of a signal peptide suitable for escherichia coli to carry out secretory expression on recombinant ginseng superoxide dismutase (rgSOD), an SOD nucleotide sequence for coding gSOD, a carrier containing the nucleotide sequences, an engineering cell containing the carrier and a method for purifying rgSOD 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 rgSOD pure product can be efficiently, simply and conveniently obtained at a low cost. The invention also provides application of rgSOD prepared by the method to preparation of medicines, food, health care products and cosmetics for treating human aging related diseases or pathologic symptoms.

Description

technical field [0001] The present invention relates to Escherichia coli secreting and expressing the nucleotide sequence encoding the signal peptide of recombinant ginseng superoxide dismutase and the nucleotide sequence encoding ginseng SOD, the vector containing the above nucleotide sequence, the engineered cell containing the aforementioned vector and obtained from The method for purifying rcSOD by engineering cells and its application belong to the fields of genetic engineering and protein biochemistry. Background technique [0002] Superoxide dismutase (Superoxide Dismutase, EC1.15.1.1, SOD) is a metalloenzyme widely present in animals, plants and microorganisms. Can catalyze superoxide radicals (O 2- ) undergoes a disproportionation reaction, which is O in the body 2- (Li Ruizhen, Yang Qingjian, Chen Yirui. Determination of Superoxide Dismutase (SOD) Activity and Its Application Research. Hainan Qiongzhou University Journal, October 28, 2004: 34-36). thereby cleari...

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 刘爽邓小霞庞玉王子华孙晓悦
Owner 吉林金梓源生物科技股份有限公司
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