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Yak copper zinc superoxide dismutase recombinant expression protein

A positive recombinant plasmid and yak technology, applied in the field of genetic engineering, can solve problems such as shortage, increased work difficulty and production cost, inclusion body precipitation, etc.

Inactive Publication Date: 2010-09-01
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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AI Technical Summary

Problems solved by technology

Many exogenous SOD gene expression systems have adopted the strategy of intracellular expression. E. coli Although intracellular expression has the advantages of high yield and easy operation, due to the lack of enzymes required for post-translational modification in eukaryotes, or the lack of certain enzymes and cofactors required in the protein folding process, a large number of intermediates Accumulation, easy to form inclusion body precipitation
Insoluble and biologically inactive inclusion bodies must go through tedious processes such as denaturation and renaturation to obtain their natural structure and biological activity, which increases the difficulty of work and production costs

Method used

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  • Yak copper zinc superoxide dismutase recombinant expression protein
  • Yak copper zinc superoxide dismutase recombinant expression protein
  • Yak copper zinc superoxide dismutase recombinant expression protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1, yak Cu,Zn-SOD Construction of cDNA-pET22b(+) recombinant vector

[0088] 1. Primer design

[0089] according to the cow Cu,Zn-SOD cDNA sequence (NM174615), using DNAsisforWindowsVer2.5 to design a pair of PCR primers, namely upstream primer P1 and downstream primer P2 ：

[0090]

[0091] The underlined part in the above upstream primer P1 sequence is a restriction endonuclease Nco I restriction site;

[0092] The underlined part in the downstream primer P2 sequence is a restriction endonuclease Hind Ⅲ Restriction site;

[0093] The box part in the primer sequence is the base protected by the enzyme cutting site;

[0094] 2. RT-PCR amplification of yak Cu,Zn-SOD cDNA fragment

[0095] (1) RNA extraction from yak liver

[0096] According to the total RNA extraction method provided by SIGMA, the total RNA of yak liver was extracted according to the following steps.

[0097] Take 50-100 mg of yak liver tissue stored at -70°C, shred it, tran...

Embodiment 2

[0179] Embodiment 2, yak Cu,Zn-SOD- pET22b(+) -E. coli Expression of BL21(DE3) genetic engineering bacteria

[0180] 1. Build Yak Cu,Zn-SOD- pET22b(+) -E. coli BL21(DE3) Genetically Engineered Bacteria

[0181] Will Cu,Zn-SOD - pET22b (+) recombinant vector is transformed into Escherichia coli BL21 (DE3) host bacteria, the specific operation steps are the same as the above-mentioned embodiment 1 step three yak Cu,Zn-SOD The transformation operation in cDNA cloning is the same.

[0182] 2. IPTG induction Cu,Zn-SOD -pET22b(+) -E. coli Expression of BL21(DE3) genetic engineering bacteria

[0183] (1) Pick a single colony of recombinant bacteria and culture it overnight in LB medium, and use the bacterial sample containing empty vector as a control;

[0184] (2) The next day, the activated seed bacteria of the experimental group and the control group were inoculated into the LB medium containing ampicillin at a volume ratio of 1:40, and cultured at 37°C with sha...

Embodiment 3

[0203] Embodiment 3, yak Cu, Zn-SOD protein purification

[0204] one, Purification of Cu,Zn-SOD Protein by Inclusion Body Washing

[0205] (1) The expression bacteria induced by IPTG were centrifuged at 12000r / min at 4°C for 15min to collect the bacteria;

[0206] (2) Add cell lysate (20mMNa 3 PO 4 , 500mMNaCl, pH 7.4, 1g of wet weight of cells and 10ml of lysate) resuspended cells, washed twice;

[0207] (3) Place the bacterial cell suspension on ice for 20 minutes by ultrasonication, break for 5 seconds, break for 9 seconds, centrifuge at 12,000 r / min at 4°C for 15 minutes, and collect the precipitate;

[0208] (4) Add inclusion body washing solution Ⅰ (20mM Na 3 PO 4 , 500mMNaCl, 0.1%TritonX-100, pH7.4, 1g of wet weight of bacteria and 20ml of inclusion body washing solution) resuspend the precipitate, let stand at 4°C for 20min, and wash 5 times repeatedly;

[0209] (5) In the same way, use inclusion body washing solution II (20mM Na 3 PO 4 , 500mMNaCl, 2MUrea...

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Abstract

The invention provides a construction method of a yak copper zinc superoxide dismutase recombinant expression system and a recombinant expression protein. The method comprises the following steps of: cloning yak liver Cu, Zn-SOD cDNA by means of reverse transcription polymerase chain reaction; connecting the Cu, Zn-SOD cDNA to a pronucleus expression vector pET22b(+); building a recombinant pronucleus expression vector; converting the vector into a colon bacillus to build a gene engineering bacteria; performing IPTG induction expression to the gene engineering bacteria, and purifying Cu, Zn-SOD cDNA protein; and dialyzing the renaturation of the obtained protein by means of urea concentration gradient, and testing the enzymatic activity by means of pyrogallol automatic oxidation. The invention provides the effective and stable construction method of the yak Cu, Zn-SOD recombinant expression system and the recombinant expressed yak Cu, Zn-SOD protein. The invention enriches the SOD resources, reduces the production cost, and lays the foundation of the development and use of the SOD in the fields of autoimmune diseases treatment, pharmacy, health care products, food and cosmetics, etc.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing a yak Cu,Zn-SOD -pET22b(+)- E. coli BL21 (DE3) genetic engineering bacteria and IPTG induced expression and recombinant protein purification technology. Background technique [0002] Superoxide dismutase (superoxide dismutase, SOD) is a class of redox enzymes containing different metal ions, which widely exist in various organisms in nature. According to the different metal cofactors, SOD can be divided into three types: copper Zinc superoxide dismutase (Cu,Zn-SOD), manganese superoxide dismutase (Mn-SOD) and iron superoxide dismutase (Fe-SOD). Cu, Zn-SOD mainly exists in the eukaryotic cytoplasm, and also exists in the nucleus, lysosome, and peroxisome; Mn-SOD exists in both the mitochondria of eukaryotes and the cytoplasm of prokaryotes ; Fe-SOD mainly exists in chloroplasts and prokaryotes. Mn-SOD and Fe-SOD appeared in the early stag...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/63C12N15/53C12N1/21C12N9/08C12R1/19
Inventor 徐亚欧毛德才毛亮袁忠杨德孝郑玉才
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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