CN103320406A

CN103320406A - High-activity glutathion peroxidase GPX 1 mutant and its preparation method

Info

Publication number: CN103320406A
Application number: CN2013103027783A
Authority: CN
Inventors: 魏景艳; 宋健; 郭笑
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2013-07-18
Filing date: 2013-07-18
Publication date: 2013-09-25
Anticipated expiration: 2033-07-18
Also published as: CN103320406B; CN103898073B; CN103898074A; CN103898074B; CN103898073A

Abstract

A high-activity glutathion peroxidase GPX 1 mutant and its preparation method belong to the field of biotechnology. A mutant gene is obtained by a gene mutation or synthesis method, and then a catalytic group SeCys of GPX is introduced into a substrate binding part of the mutant through an auxotroph prokaryotic expression system or an auxotroph and SPP low-temperature combined expression system so as to endow the mutant with high GPX activity; or a target gene, along with a SeCys insertion sequence, is firstly assembled onto a secreting type mammalia cellular expression vector, and then a binding protein 2 of the SeCys insertion sequence is assembled onto an intracellular mammalia cellular expression vector, contransfection of the same lactation cell strain is carried out by the two vectors, and GPX is synthesized by the lactation cell in the presence of sodium selenite. The method provided by the invention is simple. The mutant provided by the invention has advantages of high activity, high yield and good stability. Yield decreasing and inactivation caused by renaturation of an inclusion body are avoided. Thus, natural GPX source limitation and unstable properties are solved.

Description

High vigor Selenoperoxidase GPX1 mutant and preparation method thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of high vigor Selenoperoxidase GPX1 mutant and preparation method thereof.

Background technology

The substrate that contains the Selenoperoxidase (GPX) of selenium is gsh (GSH), and catalytic group is seleno-cysteine (SeCys).In vivo, the same superoxide-dismutase of GPX (SOD), catalase (CAT) have consisted of body anti-oxidative defense system together.GPX plays an important role in this system, it is take substrate GSH as reductive agent, hydrogen peroxide and all kinds of hydroperoxide in the decomposer, thereby can remove activity in vivo oxygen (ROS), prevent lipid peroxidation, the various diseases that treatment is caused by active oxygen is such as aging, ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataract, tumour etc.Different from other antioxidase, GPX is except removing the ROS, and the lipid peroxide of can also degrading prevents the cell peroxide injury, and the function of the Cell protection of this uniqueness makes it occupy the position of particularly important in the antioxidase system.Yet, because source limited, the poor stability of natural GPX cause its artifact and the research of stand-in thereof to receive much concern.

Small molecule mimetics mainly contains PZ51(ebselen), AL3823A, BXT series product, their weakness is that vigor is low, only is about the thousandth of natural GPX.Macromolecular stand-in mainly contain abzyme (Chinese patent 94102481.4 and 96112628.0) and the GPX analogue enztme of the preparation take glutathione S-transferase (GST) as the albumen template, and its vigor is significantly higher than small molecule mimetics.Obviously, the macromole GPX analogue enztme of preparation has been received better effect take albumen with gsh (GSH) combining site as template, therefore the GPX analogue enztme technology of preparing of these high vigor of exploitation preparation has just become the study hotspot of academia, has broad application prospects for fields such as molecular biology, physiotechnology and medical science.The method of develop has so far: the catalytic group seleno-cysteine (SeCys) of introducing GPX with chemical method (Chinese patent 94102481.4,96112628.0) or auxotroph prokaryotic expression system (Chinese patent 200810050556.6) in non-GPX class template albumen.

Chinese patent 94102481.4,96112628.0,99104234.4,200810050556.6 the preparation method of disclosed all kinds of selenium-containing abzymes is exactly applied chemistry sudden change (modification) method is introduced GPX in antibody class template albumen catalytic group SeCys, following shortcoming is arranged: (1) is in preparation process, only this step of chemical mutation (modification) will be lost the zymoprotein of 20-40%, causes the analogue enztme productive rate significantly to descend; (2) preparation analogue enztme cycle long, complex operation, the time is long; (3) need to use phenylmethylsulfonyl fluoride, acetonitrile etc. in the chemical mutation process, these materials are virose material; (4) lack targeting, for large protein molecular, chemical reaction different loci of being everlasting is introduced a plurality of non-specific catalytic groups, so chemical mutation is introduced catalytic group and can't be reached the such specificity of transgenation method.

The another kind of preparation method that Chinese patent 200810050556.6 also discloses people source strand selenium-containing abzyme introduces catalytic group with auxotroph prokaryotic expression system (intestinal bacteria) and transgenation method, but it only limits to the preparation of people source strand selenium-containing abzyme, does not comprise the preparation method of Selenoperoxidase (GPX) mutant and other GPX analogue enztme.Have the glutathione S-transferase (GST) of GPX activity the preparation method (Yu, H.J., Liu, J.Q.,

, A., Li, J., Luo, G.M., and Shen, J.C.J.Biol.Chem.2005,280,11930-11935) also appear in the newspapers, although this method also adopts auxotroph prokaryotic expression system and transgenation method to introduce catalytic group, but it only limits to prepare the GPX analogue enztme take GST as template albumen, does not comprise preparing the GPX mutant take natural GPX as template.Introducing method that catalytic group prepares analogue enztme with the auxotroph prokaryotic expression system in non-GPX class template albumen is catalytic group to be incorporated into its that same substrate GSH combining site arranged with GPX to plant and make it produce the GPX vigor in albumen.

Yet because these template albumen do not possess the catalytic group of natural GPX self, therefore be difficult to find ideal and the position of catalytic group accurately; Simultaneously owing to not resembling desirable catalysis triplet the natural GPX in these template albumen, so the catalytic efficiency of this analoglike enzyme is not high yet.If prepare the GPX mutant take natural GPX as template with gene engineering method then can overcome these shortcomings.Because the codon UGA of the catalytic group SeCys of coding GPX is terminator codon, in common prokaryotic expression system, need in the open reading frame of GPX gene, the codon UGA downstream of next-door neighbour SeCys introduces neck ring structure and UGA could be translated into SeCys rather than terminator codon, and the introducing of neck ring will inevitably cause the change of GPX space conformation in the open reading frame, and then affects enzymic activity.Therefore common prokaryotic expression system is unsuitable for directly expressing the seleno-protein that contains with GPX activity.

Summary of the invention

The object of the present invention is to provide a kind of high vigor Selenoperoxidase GPX1 mutant and preparation method thereof.

Using gene engineering technique, cell culture technology, auxotroph prokaryotic expression technology and SPP(Single protein production, single protein production) preparation of system hypothermia expression technology has the GPX1 mutant of high GPX vigor.It is the method for in mammalian cell strain or auxotroph prokaryotic expression system and SPP low temperature expression system thereof, directly expressing the GPX1 mutant protein with enzymatic activity high with genetic engineering technique.The novel artificial enzyme that present method does not need chemically modified to prepare to have high GPX vigor.The inventive method has broad application prospects aspect bio-pharmaceuticals.

The present invention with people GPX1(referring to NCBI, NM_000581.2) be template, fit rite-directed mutagenesis by computer mould, obtain a kind of novel high vigor Selenoperoxidase GPX1 mutant, it is comprised of 203 amino acid, compare with people GPX1, it does not contain halfcystine, has higher GPX vigor and better stable, it is that all cysteine mutation among the GPX1 are become Serine, but the 49th remains seleno-cysteine (SeCys, single-letter is abbreviated as U, is write as Xaa in the sequence table).(SEQ ID No:1) is as follows for the aminoacid sequence of described GPX1 mutant:

MSAARLAAAAAAAQSVYAFSARPLAGGEPVSLGSLRGKVLLIENVASLUGTTVRDYTQMN?ELQRRLGPRGLVVLGFPSNQFGHQENAKNEEILNSLKYVRPGGGFEPNFMLFEKSEVNGAGAHP?LFAFLREALPAPSDDATALMTDPKLITWSPVSRNDVAWNFEKFLVGPDGVPLRRYSRRFQTIDIEP?DIEALLSQGPSSA

Further, the concrete aminoacid sequence of described GPX1 mutant also comprises, any one or a plurality of L-Ala (Ala) in the above-mentioned aminoacid sequence are sported formed any one the novel GPX1 mutant of Serine (Ser).Such as 87 L-Ala (Ala) are sported Serine (Ser), formed sequence is SEQ ID No:2(embodiment 9).

Further, the concrete aminoacid sequence of described GPX1 mutant also comprises, with the 2nd, 78,115,156,202 5 Serines in the above-mentioned aminoacid sequence any one or a plurality of replaced by seleno-cysteine after formed any one novel GPX1 mutant.Replaced rear formed sequence SEQ ID No:3(embodiment 10 such as the 2nd Serine by seleno-cysteine (being write as Xaa in the sequence table)).

Further, the concrete aminoacid sequence of described GPX1 mutant also comprises, owing to having used the pColdI(TAKARA company of being convenient to the target protein purifying) etc. secretor type protokaryon or carrier for expression of eukaryon and introduce Histidine purification tag on this carrier and other formed any one novel GPX1 mutant such as amino acid at the aminoterminal of above-mentioned aminoacid sequence or carboxyl terminal.Such as introducing pColdI(TAKARA company at the aminoterminal of sequence SEQ ID No:1, SEQ ID No:2 and SEQ ID No:3) formed sequence SEQ ID No:4, SEQ ID No:5 and SEQ ID No:6(embodiment 11 behind the Histidine purification tag of prokaryotic expression carrier and the amino acid of factor Xa cleavage site).

The method of the high vigor Selenoperoxidase of preparation of the present invention GPX1 mutant specifically comprises following method:

Method 1:

First synthetic target gene and be assembled on the secretor type prokaryotic expression carrier or first the GPX1 genome installed on the secretor type prokaryotic expression carrier, obtain target gene with transgenation (or amino acid substitution) method, again by the auxotroph prokaryotic expression system or by auxotroph protokaryon and SPP low temperature associating expression system, the catalytic group seleno-cysteine (SeCys) of GPX is incorporated into the substrate binding site of GPX1 mutant, thereby on this albumen the substrate binding site of existing GPX, catalytic group and catalysis triplet that GPX is arranged again, the result gives the activity of its high GPX, has just produced the Selenoperoxidase GPX1 mutant of high vigor.

Method 2:

Or the gene of the GPX1 mutant of will encoding first is assembled on the secretor type cells of mamma animals expression vector together with selenocysteine insertion sequence, again with selenocysteine insertion sequence in conjunction with albumen 2(SBP2) be assembled in the born of the same parents on the type cells of mamma animals expression vector, with two kinds of same cells of mamma animals strains of carrier cotransfection, then prepare the GPX1 mutant protein with screening the positive cell strain that contains GPX1 mutant and two kinds of genes of SBP2 when obtaining, just at the external genetically engineered Selenoperoxidase GPX1 mutant that has produced high vigor, thereby solve the limited problem in natural GPX source.

Concrete preparation process of the present invention:

First method of the present invention is the gene that can express first GPX1 mutant protein of the present invention in biotech firm with the dna synthesizer synthetic; guarantee not contain in the gene ACA sequence, unite preparation GPX1 mutant protein with single protein production system and auxotroph prokaryotic expression system again.

1), the structure of expression vector: according to the aminoacid sequence of GPX1 mutant of the present invention, can in auxotrophic strain, express the gene of GPX1 mutant protein with the dna synthesizer synthetic in biotech firm, guarantee that 5 of target gene ' end contains initiator codon (ATG), 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 49 seleno-cysteines (SeCys) of target gene replaces with the codon of halfcystine (Cys) (can be TGC etc.), and full length gene does not contain the ACA sequence; Concrete gene order can be (referring to NCBI with the GPX1 gene, NM_000581.2) in the 2nd, 78,115, the encoding sequence of 156 and 202 halfcystine replaces with the codon of Serine, the codon of the 49th seleno-cysteine replaces with the codon of halfcystine, and according to the degeneracy of codon, under the prerequisite that does not change aminoacid sequence, ACA sequences all in the gene is all replaced with the resulting encoding gene of non-ACA sequence, also can be that other anyly can express the GPX1 mutant protein in auxotrophic strain, and total length does not contain the encoding gene (because the degeneracy of codon, same aminoacid sequence can have multiple different encoding gene) of ACA sequence; Contain the GPX1 mutant gene of specific restriction enzyme site and secretor type prokaryotic expression carrier (such as pCold series etc.) with identical restriction endonuclease cutting two ends, the GPX1 mutant gene is assembled on the secretor type prokaryotic expression carrier by specific restriction enzyme site with dna ligase again; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the GPX1 mutant gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2), the screening of positive transformant and protein expression and purifying:

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains the GPX1 mutant gene that makes up in the step 1), be coated with the nutrient agar plate that contains Cys, the screening positive strain; PMazF(TAKARA, the Cat#3367 that will contain again express nucleic acid restriction endonuclease MazF) the Plasmid Transformation positive strain, with the M9 solid medium screening positive transformant that contains two resistances; With positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, through 4-25 ℃ of abduction delivering of IPTG low temperature, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX1 mutant that contains SeCys at the combining site of substrate GSH, zymoprotein is expressed and is secreted in the pericentral siphon chamber of thalline with soluble form, and MazF can and cut off single stranded RNA particular sequence ACA by identification, suppresses the expression of host protein; Cultivate in a small amount first the strain of screening high expression level, again enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, the ice bath with the liquid low-temperature centrifugation, are removed bacterial sediment, are obtained supernatant liquor; With gsh (GSH) affinitive layer purification GPX1 mutant protein, namely get GPX1 mutant protein sterling after the dialysis freeze-drying.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot).

The method is by inducing the auxotroph prokaryotic expression system to introduce catalytic group at the substrate binding site of GPX1 mutant under the low temperature, thereby produces the GPX1 mutant of high vigor.

Described is to use pH7.5 with gsh (GSH) affinitive layer purification GPX1 mutant protein, and 50mmol/L Tris-Cl balance and wash-out foreign protein are with the buffer solution elution target protein that contains 10mmol/L GSH.

Described with the liquid low-temperature centrifugation, can be at 4 ℃, the centrifugal 15-30min of 8000-12000g.

Second method of the present invention is the GPX1 gene that increases first, then take it as template, obtain to express the gene of GPX1 mutant protein of the present invention with the transgenation method, and the ACA sequence in the gene that under guaranteeing the constant prerequisite of its aminoacid sequence, suddenlys change, obtain not contain the GPX1 mutant gene of ACA, unite preparation GPX1 mutant protein with single protein production system and auxotroph prokaryotic expression system again.

1), the structure of expression vector:

According to the gene order of disclosed GPX1 in the gene library (referring to NCBI, NM_000581.2) design primer, its encoding gene increases, when the design primer, guarantee that 5 of gene ' end contains initiator codon (ATG), 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the codon of the 2nd and 202 Cys replaces to the codon of Ser, other aminoacid sequence is constant; After identical restriction endonuclease cut vector and target gene, by specific restriction enzyme site the GPX1 genome is installed to (such as pCold series etc.) on the secretor type prokaryotic expression carrier with dna ligase again; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to 49 seleno-cysteines that will sport halfcystine among the GPX1 and the amino acid whose gene order near it, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the seleno-cysteine in the GPX1 gene that is structured on the prokaryotic expression carrier is become the codon of halfcystine; In like manner, method with above-mentioned rite-directed mutagenesis is being guaranteed not introduce under the prerequisite of ACA sequence, the sequence encoding mutant of other halfcystine in the GPX1 gene is become the codon of Serine, again according to the degeneracy of codon, under the prerequisite that does not change aminoacid sequence, all ACA series jumps in the GPX1 gene are fallen; Determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the gene after guaranteeing to suddenly change can be expressed GPX1 mutant protein of the present invention in auxotrophic strain; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2) expression and purification of the screening of positive transformant and mutant protein

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains the GPX1 mutant gene that makes up in the step 1), be coated with the nutrient agar plate that contains halfcystine, the screening positive strain; Plasmid pMazF with express nucleic acid restriction endonuclease MazF transforms positive strain again, with the M9 solid medium screening positive transformant that contains two resistances; With positive transformant spread cultivation support after, containing seleno-cysteine, in the substratum of essential growth factor and nutrient substance, through 4-25 ℃ of abduction delivering of isopropylthio-β-D-galactoside low temperature, auxotrophic strain-BL21 (DE3) Cys can synthesize seleno-cysteine with the codon of halfcystine, directly give expression to the restructuring GPX1 mutant that contains seleno-cysteine at the combining site of substrate gsh, zymoprotein is expressed and is secreted in the pericentral siphon chamber of thalline with soluble form, and MazF can and cut off single stranded RNA particular sequence ACA by identification, suppresses the expression of host protein; Cultivate in a small amount first the strain of screening high expression level, again enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, the ice bath, with the liquid low-temperature centrifugation, removal bacterial sediment, acquisition contain the supernatant liquor of GPX1 mutant; With gsh (GSH) affinitive layer purification GPX, namely get the zymoprotein sterling after the dialysis freeze-drying.

The method has been introduced catalytic group by inducing the auxotroph prokaryotic expression system to unite the single protein production of SPP system under the low temperature at the substrate binding site of GPX1 mutant, thereby produces the GPX1 mutant protein of high vigor.

Described is to use pH7.5 with gsh (GSH) affinitive layer purification GPX1 mutant, and 50mmol/L Tris-Cl balance and wash-out foreign protein are with the buffer solution elution target protein that contains 10mmol/L GSH.

The third method of the present invention is can express GPX1 mutant protein gene of the present invention in biotech firm with the dna synthesizer synthetic first, prepares the GPX1 mutant protein with the auxotroph prokaryotic expression system again.

1), the structure of expression vector:

Aminoacid sequence according to GPX1 mutant of the present invention, can in auxotrophic strain, express the gene of GPX1 mutant protein with the dna synthesizer synthetic in biotech firm, guarantee that 5 of target gene ' end contains initiator codon (ATG), 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantees that the encoding sequence of 49 seleno-cysteines (SeCys) of target gene replaces with the codon of halfcystine (Cys) (can be TGC etc.); Concrete gene order can be (referring to NCBI with the GPX1 gene, NM_000581.2) encoding sequence of the 2nd, 78,115,156 and 202 halfcystine replaces with the codon of Serine in, the codon of the 49th seleno-cysteine replaces with the resulting encoding gene of codon of halfcystine, it also can be other any encoding gene (because the degeneracy of codon, same aminoacid sequence can have multiple different encoding gene) that can in auxotrophic strain, express the GPX1 mutant protein; Contain the GPX1 mutant gene of specific restriction enzyme site and secretor type prokaryotic expression carrier (such as pCold series etc.) with identical restriction endonuclease cutting two ends, the GPX1 mutant gene is assembled on the secretor type prokaryotic expression carrier by specific restriction enzyme site with dna ligase again; Containing in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the GPX1 mutant gene, is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains the GPX1 mutant gene that makes up in the step 1), be coated with the nutrient agar plate that contains Cys, the screening positive transformant; With positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, (IPTG) induces through isopropylthio-β-D-galactoside, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, the last GPX1 mutant protein that contains SeCys at the combining site of substrate GSH that directly gives expression to, zymoprotein is expressed and is secreted in the pericentral siphon chamber of thalline with soluble form; Cultivate in a small amount first the strain of screening high expression level, again enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, the ice bath with the liquid low-temperature centrifugation, are removed bacterial sediment, are obtained supernatant liquor; With gsh (GSH) affinitive layer purification GPX1 mutant protein, namely get GPX1 mutant protein sterling after the dialysis freeze-drying.

The method has been introduced catalytic group by transgenation and auxotroph prokaryotic expression system at the substrate binding site of GPX1 mutant, thereby produces the GPX1 mutant protein of high vigor.

The 4th kind of method of the present invention is the GPX1 gene that increases first, then take it as template, obtains to express the gene of GPX1 mutant protein of the present invention with the transgenation method, prepares the GPX1 mutant protein with the auxotroph prokaryotic expression system again.

1), the structure of expression vector:

According to the gene order of disclosed GPX1 in the gene library (referring to NCBI, NM_000581.2) design primer, its encoding gene increases, when the design primer, guarantee that 5 of gene ' end contains initiator codon (ATG), 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the codon of the 2nd and 202 Cys replaces to the codon of Ser, other aminoacid sequence is constant; After identical restriction endonuclease cut vector and target gene, by specific restriction enzyme site the GPX1 genome is installed to (such as pCold series etc.) on the secretor type prokaryotic expression carrier with dna ligase again; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to 49 SeCys that will sport halfcystine (Cys) among the GPX1 and its neighbour amino acid whose gene order, centered by the codon of mutating acid, the long 25-50bp of primer; With rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit (operation of test kit specification sheets is pressed by Invitrogen company), the sequence encoding mutant of the SeCys in the GPX1 gene that is structured on the prokaryotic expression carrier is become the codon (such as TGC) of Cys; In like manner, with the method for above-mentioned rite-directed mutagenesis the sequence encoding mutant of other halfcystine in the GPX1 gene is become the codon of Serine; Determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the gene after guaranteeing to suddenly change can be expressed GPX1 mutant protein of the present invention in auxotrophic strain; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

Lung biopsy: prepare the GPX1 mutant protein with synthetic target gene and mammalian cell expression system

1), the structure of expression vector:

Aminoacid sequence according to GPX1 mutant of the present invention, encoding gene in biotech firm with dna synthesizer synthetic GPX1 mutant, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end is introduced whole base sequences of 3 of GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A in the terminator codon downstream, concrete sequence is referring to NCBI, NM_000581.2) or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site; Concrete gene order can be (referring to NCBI with the GPX1 gene, NM_000581.2) in the 2nd, 78,115, the encoding sequence of 156 and 202 halfcystine replaces with the resulting encoding gene of codon of Serine, it also can be gene (because the degeneracy of codon of other any coding GPX1 mutant protein, same aminoacid sequence can have multiple different encoding gene), but 3 of target gene ' end must contain whole base sequences of 3 of GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A, concrete sequence is referring to NCBI, NM_000581.2) or selenocysteine insertion sequence (SECIS); Contain the GPX1 mutant gene of specific restriction enzyme site and cells of mamma animals expression vector (such as pSecTag2A etc. with identical restriction endonuclease cutting two ends, Invitrogen company), the GPX1 mutant gene is assembled on the secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence by specific restriction enzyme site with dna ligase again; According to selenocysteine insertion sequence in the gene library in conjunction with albumen 2(SBP2, see NCBI, NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders are at biotech firm's its encoding gene of dna synthesizer synthetic, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site, with specific restriction enzyme site the SBP2 genome is installed in the born of the same parents on the type cells of mamma animals expression vector (such as pcDNA3.1/myc-His A etc., Invitrogen company); That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2), the screening of positive cell strain:

With the carrier that contains SBP2 and GPX1 mutant gene cotransfection cells of mamma animals under the mediation of transfection reagent, with the resistant gene on two carriers by antibiotic concentration from high to low step by step successive subtraction method screening have the positive cell clone of two kinds of resistances concurrently, with porous culture plate amplification culture positive colony step by step, finally obtain the positive cell strain that Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant again; Detect the expression amount of GPX1 mutant protein and screen the high cell strain of target protein expression amount;

3), the expression and purification of target protein:

In shaking flask, will express simultaneously the cell strain amplification culture of two kinds of foreign proteins, containing in the substratum of Sodium Selenite with cells of mamma animals expression GPX1 mutant, zymoprotein is secreted in the substratum with soluble form, with gsh (GSH) affinitive layer purification GPX1 mutant protein, namely get the zymoprotein sterling after the dialysis freeze-drying.

The expression amount of described detection GPX1 mutant protein can detect with the polyclonal antibody of anti-GPX1 and ELISA or GPX vitality test technology;

Described is to use pH7.5 with gsh GSH affinitive layer purification GPX1 mutant protein, and 50mmol/L Tris-Cl balance and wash-out foreign protein are with the buffer solution elution target protein that contains 10mmol/L GSH.

The 6th kind of method of the present invention is the substep carrier that contains the SBP2 gene and the same cells of mamma animals of carrier transfection that contains the GPX1 mutant gene.

1), the structure of expression vector:

Use the encoding gene of dna synthesizer synthetic GPX1 mutant in biotech firm according to the aminoacid sequence of GPX1 mutant of the present invention, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end is introduced whole base sequences of 3 of GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A in the terminator codon downstream, concrete sequence is referring to NCBI, NM_000581.2) or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site; Concrete gene order can be (referring to NCBI with the GPX1 gene, NM_000581.2) in the 2nd, 78,115, the encoding sequence of 156 and 202 halfcystine replaces with the resulting encoding gene of codon of Serine, it also can be gene (because the degeneracy of codon of other any coding GPX1 mutant protein, same aminoacid sequence can have multiple different encoding gene), but 3 of target gene ' end must contain whole base sequences of 3 of GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A, concrete sequence is referring to NCBI, NM_000581.2) or selenocysteine insertion sequence (SECIS); Contain the GPX1 mutant gene of specific restriction enzyme site and cells of mamma animals expression vector (such as pSecTag2A etc. with identical restriction endonuclease cutting two ends, Invitrogen company), the GPX1 mutant gene is assembled on the secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence by specific restriction enzyme site with dna ligase again; According to selenocysteine insertion sequence in the gene library in conjunction with albumen 2(SBP2, see NCBI, NM_024077.3) 512 amino acid whose gene orders of the 343-854 position of carboxyl terminal are at biotech firm's its encoding gene of dna synthesizer synthetic, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site, with specific restriction enzyme site the SBP2 genome is installed in the born of the same parents on the type cells of mamma animals expression vector (such as pcDNA3.1/myc-His A etc., Invitrogen company); That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2), the screening of positive cell strain:

First with the carrier transfection cells of mamma animals that contains the SBP2 gene, by the cell strain of the screening of the resistant gene on this carrier stably express SBP2.With the cell strain of the carrier transfection stably express SBP2 that contains the GPX1 mutant gene, express the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant with the screening of the resistant gene on two carriers Simultaneous Stabilization again; Detect the expression amount of GPX1 mutant protein and screen the high cell strain of target protein expression amount;

3), the expression and purification of target protein:

To express simultaneously the cell strain amplification culture of two kinds of foreign proteins in shaking flask, express the GPX1 mutant containing in the substratum of Sodium Selenite with cells of mamma animals, zymoprotein is secreted in the substratum with soluble form; With gsh affinitive layer purification GPX1 mutant protein, namely get the zymoprotein sterling after the dialysis freeze-drying.

In the screening of positive cell strain, the screening of described positive cell strain, also can first transfection GPX1 mutant gene, rear transfection SBP2 gene is expressed the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant again with the screening of the resistant gene on two carriers Simultaneous Stabilization.The expression amount of described detection GPX1 mutant protein can detect with the polyclonal antibody of anti-GPX1 and ELISA or GPX vitality test technology.

Described is to use pH7.5 with gsh affinitive layer purification GPX1 mutant, and 50mmol/L Tris-Cl balance and wash-out foreign protein are with the buffer solution elution target protein that contains 10mmol/L GSH.

The 7th kind of method: target gene and mammalian cell expression system with amplification prepare the GPX1 mutant protein

1), the structure of expression vector:

According to GPX1 gene order in the gene library (referring to NCBI, NM_000581.2) base sequence of the encoding gene and 3 of design primer amplification GPX1 ' end non-translational region, when the design primer, guarantee that 5 of target gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 of target gene ' end is introduced whole base sequences of 3 of GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A in the terminator codon downstream, concrete sequence is referring to NCBI, NM_000581.2) or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site, guarantee that the codon of the 2nd Cys replaces to the codon of Ser, other aminoacid sequence is constant; The target gene of specific restriction enzyme site and cells of mamma animals expression vector are arranged (such as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), the GPX1 gene is assembled on the secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region by specific restriction enzyme site with dna ligase again; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to the halfcystine that will sport Serine among the GPX1 and the amino acid whose gene order near it, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the GPX1 gene that is structured on the carrier for expression of eukaryon is become the codon of Serine; Determine suddenly change successfully, and without other unexpected transgenation generation the GPX1 mutant protein of the present invention of to encode of the gene after guaranteeing to suddenly change by dna sequencing.According to selenocysteine insertion sequence in the gene library in conjunction with albumen 2(SBP2, see NCBI, NM_024077.3) 512 of the 343-854 position of carboxyl terminal amino acid whose its encoding genes of gene order design primer amplification, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site; After cutting connection, enzyme with specific restriction enzyme site the SBP2 genome is installed in the born of the same parents on the type cells of mamma animals expression vector (such as pcDNA3.1/myc-His A etc., Invitrogen company) equally; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

The 8th kind of method of the present invention is the substep carrier that contains the SBP2 gene and the same cells of mamma animals of carrier transfection that contains the GPX1 mutant gene.

1), the structure of expression vector:

2), the screening of positive cell strain:

First with the carrier transfection cells of mamma animals that contains the SBP2 gene, by the cell strain of the screening of the resistant gene on this carrier stably express SBP2; With the cell strain of the carrier transfection stably express SBP2 that contains the GPX1 mutant gene, express the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant with the screening of the resistant gene on two carriers Simultaneous Stabilization again; Detect the expression amount of GPX1 mutant protein and screen the high cell strain of target protein expression amount;

3), the expression and purification of target protein:

The 9th kind of method of the present invention and first method are identical, just the encoding sequence of the 87th L-Ala is replaced with during the encoding gene of synthetic GPX1 mutant the codon of Serine, the 87th of the GPX1 mutant protein that obtains at last is Serine (SEQ ID No:2), rather than L-Ala, other aminoacid sequence and first method are identical.

The of the present invention ten kind of method and first method are identical, just the encoding sequence of second Serine is replaced with during the encoding gene of synthetic GPX1 mutant the codon of halfcystine, the second of the GPX1 mutant protein that obtains at last is seleno-cysteine (SEQ ID No:3), rather than Serine, other aminoacid sequence and first method are identical.

The 11 kind of method of the present invention and first method are identical, just owing to having used the pColdI(TAKARA company of being convenient to the target protein purifying) etc. secretor type protokaryon or carrier for expression of eukaryon and introduce Histidine purification tag on this carrier and other formed any one novel GPX1 mutant such as amino acid at the aminoterminal of target protein or carboxyl terminal.Such as introducing pColdI(TAKARA company at the aminoterminal of sequence SEQ ID No:1, SEQ ID No:2, SEQ ID No:3) formed sequence SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 behind the Histidine purification tag of prokaryotic expression carrier and the amino acid of factor Xa cleavage site.

The present invention has following characteristics:

⑴ the present invention uses simple gene to synthesize or the genetic engineering techniques such as amplification, transgenation and expression, and the preparation method is simple.

⑵ the GPX1 mutant protein vigor of the inventive method preparation is high, has surpassed an order of magnitude of natural GPX vigor, and Cell Biology Experiment has confirmed that the myocardial cell of Hydroperoxide injury is had stronger provide protection.

⑶ GPX1 mutant protein of the present invention has superpower stability, is difficult for inactivation, and this characteristic has remedied the defective of natural GPX.

⑷ the secreted expression carrier that the present invention uses, the external of colibacillary pericentral siphon chamber or cells of mamma animals can be expressed and be secreted into to the GPX1 mutant protein with soluble form, the targeting signal peptide of pilot protein secreting, expressing is excised automatically by thalline or cell, expressed albumen is solubility, activity with space conformation and Geng Gao of native protein, do not need renaturation, the productive rate that can avoid the renaturing inclusion bodies process to cause descends and inactivation, thereby with short production cycle.

⑸ the SPP low temperature expression system that the present invention uses can utilize MazF proteolytic enzyme specific recognition and cutting to contain the characteristic of the mRNA of ACA sequence, make thalline can only the composite coding gene in without the protein of ACA sequence, therefore new synthetic albumen mainly is foreign protein, thereby improve productive rate and Cys to the transformation efficiency of SeCys, therefore have the double dominant that vigor is high, productive rate is high.

⑹ the used eukaryotic expression of the present invention directly uses the selenocysteine insertion sequence (SECIS) of GPX self, do not need to introduce in addition SECIS and enter expression vector, thereby simple to operate and available conventional carrier for expression of eukaryon is implemented.

These advantages all are conducive to scale operation, for from now on practical application lays a solid foundation, have solved the problem that natural GPX source is not enough, character is unstable and vigor is low, have broad application prospects aspect bio-pharmaceuticals.

Figure of description

Fig. 1: on the SDS-PAGE(of the GPX1 mutant protein that purifying obtains) and under the Western blot() result: wherein M is molecular weight of albumen Marker, and the 1-11 swimming lane is the target protein of embodiment 1～11 preparation.

Embodiment

Embodiment 1: prepare genetically engineered people GPX1 mutant protein with synthetic gene associating SPP and auxotroph prokaryotic expression system

The sequence 1(SEQ ID No:1 according to the present invention) aminoacid sequence of described GPX1 mutant, biotech firm with the dna synthesizer synthetic can be in auxotrophic strain expressed sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, guarantee that 5 of GPX1 mutant gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, the encoding sequence TGA of No. 49 SeCys of GPX1 mutant gene replaces with the codon (TGC) of Cys, and full length gene does not contain the ACA sequence; Concrete target-gene sequence is (referring to NCBI with the GPX1 gene, NM_000581.2) in the 2nd, 78,115, the encoding sequence that the codon of 156 and 202 halfcystine replaces with Serine (is: AGT successively, AGC, AGC, AGT, AGT), the codon of the 49th seleno-cysteine replaces with the encoding sequence (TGC) of halfcystine, and with in the gene the 168th, 537,561 and No. 573 base C all replaces with the resulting GPX1 mutant gene that does not contain the ACA sequence of T, guarantee this gene energy code book invention sequence 1(SEQ ID No:1) described GPX1 mutant protein, and 5 of target gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, and 3 ' end contains terminator codon and Hind III restriction enzyme site; Behind restriction endonuclease Nde I and Hind III double digestion, be connected to pCold III(TAKARA, Cat.#3369 that same enzyme is cut) on the carrier.

Transform auxotroph e. coli bl21 (DE3) Cys with the carrier that the GPX1 mutant gene is housed (pCold III-GPX1 is prominent), be coated with the nutrient agar plate that contains Cys, the screening positive strain.Again with plasmid pMazF(TAKARA, the Cat.#3369 of express nucleic acid restriction endonuclease MazF) be transformed in this positive strain.Bacterium liquid is uniformly coated in the M9-CAA solid medium that contains 100 μ g/mL Amp, 25 μ g/mLKana and 25 μ g/mL screens positive transformant.With positive transformant be inoculated in 1.2L M9 defective express in the substratum (in the M9 substratum, adding each 100 μ g/ml of 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and 19 seed amino acids except Cys) to OD600 be 0.5.Bacterium liquid is placed-20 ℃ of refrigerator 5min, bacterium liquid is cooled off rapidly, afterwards bacterium liquid is placed 15 ℃ of relaying persistent oscillations to cultivate 45min.15 ℃ of centrifugal 5min of 5000rpm abandon supernatant.With the resuspended bacterial sediment of 0.9%NaCl, 15 ℃ of centrifugal 5min of 5000rpm abandon supernatant, repeat this step.Then use the resuspended thalline of productive culture base (in the M9 substratum, adding each the 50-400 μ g/ml of 19 seed amino acids except Cys), adding final concentration is that 1mmol/L IPTG and final concentration are 200 μ g/mL SeCys, 15 ℃ of shaking culture 16-72h, the GPX1 mutant protein is expressed in the pericentral siphon chamber of thalline with soluble form, and MazF can and cut off single stranded RNA particular sequence ACA by identification, suppresses the expression of host protein.Collect thalline (6000rpm, 10min) also with isopyknic bufferT(50mM Tris buffer, pH7.5 contains 1mM EDTA) washed twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), the ultrasonication thalline, 4 ℃, the centrifugal 30min of 12000rpm collects supernatant.By specification is processed GSH affinity post, uses damping fluid (50mmol/L Tris, pH7.5) balance, supernatant liquor is added on the GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein.With elutriant dialysis and freeze-drying, namely get people GPX1 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 1st swimming lane of Fig. 1.Its GPX vigor is 21786U/ μ mol, surpasses order of magnitude of natural GPX.

The SPP low temperature expression system that the present embodiment uses can utilize MazF proteolytic enzyme specific recognition and cutting to contain the characteristic of the mRNA of ACA sequence, make thalline can only the composite coding gene in without the protein of ACA sequence, therefore new synthetic albumen mainly is the external source recombinant protein, thereby improve productive rate and Cys to the transformation efficiency of SeCys, therefore have the high high yield double dominant of living.

Embodiment 2: prepare genetically engineered people GPX1 mutant protein with gene amplification and sudden change method and SPP associating auxotroph prokaryotic expression system

Extract in a small amount test kit (Sigma with mRNA, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, extract mRNA, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist down, by RT-PCR (RT-polymerase chain reaction) it is transcribed into cDNA.According to the gene of disclosed people GPX1 in the gene library (referring to NCBI, NM_000581.2) primers its encoding gene that increases, guarantee that 5 of gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, and the codon of the 2nd and No. 202 Cys being replaced to the codon of Ser, other aminoacid sequence is constant; Concrete 5 ' end primer is 5 '-GGAATTCCATATGAGTGCTGCTCGGC-3 ', and 3 ' end primer is 5 '-CCCAAGCTTCTAGGCACTGCTGGGC-3 '.Behind restriction endonuclease Nde I and Hind III double digestion, be connected to pCold III(TAKARA, the Cat.#3369 that same enzyme is cut with dna ligase) on the carrier.Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to the amino acid whose gene order near No. 49 SeCys, the long 25-50bp of primer, centered by the codon (TGA) of No. 49 SeCys, the sequence of sense primer is 5 '-GAATGTGGCGTCCCTCTG CGGCACCACGGTCCGGGAC-3 '.Primer and Fast Fixed-point sudden change test kit (Invitrogen company with these two complete complementaries, press the operation of test kit specification sheets), the encoding sequence TGA that is structured in No. 49 SeCys of the GPX1 gene on the prokaryotic expression carrier pCold III is mutated into the codon (TGC) of Cys, determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation.The codon mutation of other all Cys in the people GPX1 gene is become the codon of Ser with same method; Corresponding sense primer is respectively: C78S5 '-GTGCTCGGCTTCCCG AGCAACCAGTTTGGGC-3 ', C115S5 '-CATGCTCTTCGAGAAG AGCGAGGTGAACGGTGC-3 ', C156S5 '-CACCTGGTCTCCGGTG AGTCGCAACGATGTTGC-3 '.All ACA sequences in the gene are fallen in sudden change under keeping the constant prerequisite of GPX1 mutant aminoacid sequence at last, be about to the 168th, 537,561 and No. 573 base C and all sport T, share 3 primers, its just sequence is respectively 5 '-CACGGTCCGGGACTATACCCAGATGAACGAG-3 ', 5 '-CCCCTACGCAGGTATAGCCGCCGCTTCC-3 ', 5 '-CGCTTCCAGACCATTGATATCGAGCCTGATATCGAAGCCCTGCTGTC-3 '; Determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the gene energy code book invention sequence 1(Seq1 after guaranteeing to suddenly change) described GPX1 mutant protein.

Transform auxotroph e. coli bl21 (DE3) Cys with the carrier that the GPX1 mutant gene is housed (pCold III-GPX1 is prominent), be coated with the nutrient agar plate that contains Cys, the screening positive strain.Again with plasmid pMazF(TAKARA, the Cat.#3369 of express nucleic acid restriction endonuclease MazF) be transformed in this positive strain.Bacterium liquid is uniformly coated in the M9-CAA solid medium that contains 100 μ g/mL Amp, 25 μ g/mLKana and 25 μ g/mL screens positive transformant.With positive transformant be inoculated in 1.2L M9 defective express in the substratum (in the M9 substratum, adding each 100 μ g/ml of 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and 19 seed amino acids except Cys) to OD600 be 0.5.Bacterium liquid is placed-20 ℃ of refrigerator 5min, bacterium liquid is cooled off rapidly, afterwards bacterium liquid is placed 15 ℃ of relaying persistent oscillations to cultivate 45min.15 ℃ of centrifugal 5min of 5000rpm abandon supernatant.The resuspended bacterial sediment of 0.9%NaCl, 15 ℃ of centrifugal 5min of 5000rpm abandon supernatant, repeat this step.Then use the resuspended thalline of productive culture base (in the M9 substratum, adding each the 50-400 μ g/ml of 19 seed amino acids except Cys), adding final concentration is that 1mmol/LIPTG and final concentration are 200 μ g/mL SeCys, 15 ℃ of shaking culture 16-72h, the GPX1 mutant protein is expressed in the pericentral siphon chamber of thalline with soluble form, and MazF can and cut off single stranded RNA particular sequence ACA by identification, suppresses the expression of host protein.Collect thalline (6000rpm, 10min) also with isopyknic bufferT(50mM Tris buffer, pH7.5 contains 1mM EDTA) washed twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), the ultrasonication thalline, 4 ℃, the centrifugal 30min of 12000rpm collects supernatant.By specification is processed GSH affinity post, uses damping fluid (50mmol/L Tris, pH7.5) balance, supernatant liquor is added on the GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein.With elutriant dialysis and freeze-drying, namely get people GPX1 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 2nd swimming lane of Fig. 1.Its GPX vigor is 21560U/ μ mol, surpasses order of magnitude of natural GPX.

Embodiment 3: prepare genetically engineered people GPX1 mutant protein with synthetic gene and auxotroph prokaryotic expression system

The sequence 1(SEQ ID No:1 according to the present invention) aminoacid sequence of described GPX1 mutant, biotech firm with the dna synthesizer synthetic can be in auxotrophic strain expressed sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, guarantee that 5 of GPX1 mutant gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, and the encoding sequence TGA of No. 49 SeCys of GPX1 mutant gene replaces with the codon (TGC) of Cys; Concrete target-gene sequence is (referring to NCBI with the GPX1 gene, NM_000581.2) in the 2nd, 78,115, the encoding sequence that the codon of 156 and 202 halfcystine replaces with Serine (is: AGT successively, AGC, AGC, AGT, AGT), the codon of the 49th seleno-cysteine replaces with that the encoding sequence (TGC) of halfcystine is resulting can code book invention sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, and 5 of target gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, and 3 ' end contains terminator codon and Hind III restriction enzyme site; Behind restriction endonuclease Nde I and HindIII double digestion, be connected to pCold III(TAKARA, Cat.#3369 that same enzyme is cut) on the carrier.

Transform auxotroph e. coli bl21 (DE3) Cys with the carrier that the GPX1 mutant gene is housed (pCold III-GPX1 is prominent), be coated with the nutrient agar plate that contains Cys, the screening positive transformant.With positive transformant be inoculated in 1.2L M9 defective express in the substratum (in the M9 substratum, adding each 100 μ g/ml of 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and 19 seed amino acids except Cys) to OD600 be 0.1, be cultured to OD600 and be about 0.3-1, the IPTG that adds final concentration 0.1-1mM, the paraxin of adding final concentration 10 μ g/ml behind the 10min.Adding paraxin medium centrifugal after five minutes, centrifugal 5 minutes of low temperature 6000rpm, wash twice with the physiological salt solution of ice bath, each 1.2L that uses, then use productive culture base (in the M9 substratum add except Cys each 50-400 μ g/ml of 19 seed amino acids) resuspended, and replenish Rifampin and the 600 μ M SeCys of final concentration 400 μ g in the substratum.Continue to cultivate 2-12h, GPX1 is expressed in the pericentral siphon chamber of thalline with soluble form.Collect thalline (6000rpm, 10min) also with isopyknic bufferT(50mM Tris buffer, pH7.5 contains 1mM EDTA) washed twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), the ultrasonication thalline, 4 ℃, the centrifugal 30min of 12000rpm collects supernatant.By specification is processed GSH affinity post, uses damping fluid (50mmol/L Tris, pH7.5) balance, supernatant liquor is added on the GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein.With elutriant dialysis and freeze-drying, namely get people GPX1 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 3rd swimming lane of Fig. 1.Its GPX vigor is 5320U/ μ mol, reaches the order of magnitude level of natural GPX.

Auxotroph e. coli bl21 (DE3) Cys derives from exercise question and is the article of " Structure of the Cathelicidin Motif of Protegrin-3Precursor:Structural Insights into the Activation Mechanism of an Antimicrobial Protein ", be published in Structure the 10th volume in 2002,1370 pages of 1363 –.Obtaining of this bacterial classification can be granted by author or August professor Bock.

Embodiment 4: prepare genetically engineered people GPX1 mutant protein with gene amplification and sudden change method and auxotroph prokaryotic expression system

Extract in a small amount test kit (Sigma with mRNA, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, extract mRNA, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist down, by RT-PCR (RT-polymerase chain reaction) it is transcribed into cDNA.According to the gene of disclosed people GPX1 in the gene library (referring to NCBI, NM_000581.2) primers its encoding gene that increases, guarantee that 5 of gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, and the codon of the 2nd and No. 202 Cys being replaced to the codon of Ser, other aminoacid sequence is constant; Concrete 5 ' end primer is 5 '-GGAATTCCATATGAGTGCTGCTCGGC-3 ', and 3 ' end primer is 5 '-CCCAAGCTTCTAGGCACTGCTGGGC-3 '.Behind restriction endonuclease Nde I and Hind III double digestion, be connected to pCold III(TAKARA, the Cat.#3369 that same enzyme is cut with dna ligase) on the carrier.Keeping under the constant prerequisite of other aminoacid sequence, designing two rite-directed mutagenesis primers of complete isometric complementation according to the amino acid whose gene order near No. 49 SeCys, the long 25-50bp of primer is centered by the codon (TGA) of No. 49 SeCys.Primer and Fast Fixed-point sudden change test kit (Invitrogen company with these two complete complementaries, press the operation of test kit specification sheets), the encoding sequence TGA that is structured in No. 49 SeCys of the GPX1 gene on the prokaryotic expression carrier pCold III is mutated into the codon (TGC) of Cys, determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the sequence encoding mutant of other all Cys in the people GPX1 gene is become the codon of Ser with same method, be the codon (TCC) of Ser with the sequence encoding mutant of 87 Ala, corresponding sense primer is respectively: C78S5 '-GTGCTCGGCTTCCCG AGCAACCAGTTTGGGC-3 ', C115S5 '-CATGCTCTTCGAGAAG AGCGAGGTGAACGGTGC-3 ', C156S5 '-CACCTGGTCTCCGGTG AGTCGCAACGATGTTGC-3 ' and A87S5 '-GTTTGGGCATCAGGAGAAC TCCAAGAACGAAGAGATTC-3 '.Determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the gene energy code book invention sequence 2(SEQ ID No:2 after guaranteeing to suddenly change) described GPX1 mutant protein.

Transform auxotroph e. coli bl21 (DE3) Cys with the carrier that the GPX1 mutant gene is housed (pCold III-GPX1 is prominent), be coated with the nutrient agar plate that contains Cys, the screening positive transformant.With positive transformant be inoculated in 1.2L M9 defective express in the substratum (in the M9 substratum, adding each 100 μ g/ml of 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and 19 seed amino acids except Cys) to OD600 be 0.1, be cultured to OD600 and be about 0.3-1, the IPTG that adds final concentration 0.1-1mM, the paraxin of adding final concentration 10 μ g/ml behind the 10min.Adding paraxin medium centrifugal after five minutes, centrifugal 5 minutes of low temperature 6000rpm, wash twice with the physiological salt solution of ice bath, each 1.2L that uses, then use productive culture base (in the M9 substratum add except Cys each 50-400 μ g/ml of 19 seed amino acids) resuspended, and replenish Rifampin and the 600 μ M SeCys of final concentration 400 μ g in the substratum.Continue to cultivate 2-12h, GPX1 is expressed in the pericentral siphon chamber of thalline with soluble form.Collect thalline (6000rpm, 10min) also with isopyknic bufferT(50mM Tris buffer, pH7.5 contains 1mM EDTA) washed twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), the ultrasonication thalline, 4 ℃, the centrifugal 30min of 12000rpm collects supernatant.By specification is processed GSH affinity post, uses damping fluid (50mmol/L Tris, pH7.5) balance, supernatant liquor is added on the GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein.With elutriant dialysis and freeze-drying, namely get people GPX1 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 4th swimming lane of Fig. 1.Its GPX vigor is 5130U/ μ mol, reaches the order of magnitude level of natural GPX.The GPX1 mutant of the aminoacid sequence of (present method also can be used for preparing 87 Ala do not sport Serine namely have sequence 1(SEQ ID No:1), concrete grammar is the codon of Ser this step except saving sequence encoding mutant with 87 Ala, and other and this law are identical.）

Embodiment 5: prepare people GPX1 mutant protein with synthetic gene and the one step transfection of mammalian cell expression system

1. the structure of people GPX1 mutant protein expression vector: the sequence 1(SEQ ID No:1 according to the present invention) aminoacid sequence of described GPX1 mutant in biological reagent company with dna synthesizer synthetic GPX1 mutant gene, guarantee that 5 of gene ' end contains initiator codon (ATG) and Hind III restriction enzyme site, 3 ' end at whole base sequences of 3 of people GPX1 mutant gene terminator codon downstream GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A, concrete sequence is referring to NCBI, NM_000581.2), insert the BamHI restriction enzyme site and after the previous base of the poly oligonucleotide A of GPX1 gene; Concrete target-gene sequence is (referring to NCBI with the GPX1 gene, NM_000581.2) in the 2nd, 78,115, the encoding sequence that the codon of 156 and 202 halfcystine replaces with Serine (is: AGT successively, AGC, AGC, AGT, AGT) the resulting energy code book invention sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, and 3 of target gene ' end contains whole base sequences of 3 of GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A, concrete sequence is referring to NCBI, NM_000581.2), insert the BamHI restriction enzyme site and after the previous base of the poly oligonucleotide A of GPX1 gene.Cut latter linked method with first enzyme, by Hind III/BamHI restriction enzyme site the GPX1 mutant gene is connected on the cells of mamma animals expression vector pSecTag2A (Invitrogen company) that same enzyme cuts together with selenocysteine insertion sequence; In like manner according to human selenocysteine insertion sequence in the gene library in conjunction with albumen 2(SBP2; NCBI; NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders are at biotech firm's its encoding gene of dna synthesizer synthetic; guarantee that 5 of gene ' end contains initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end contains terminator codon and EcoRI restriction enzyme site.Cut latter linked method with first enzyme, by the XbaI/EcoRI restriction enzyme site carboxyl terminal (343-854aa) genome of SBP2 is installed in the born of the same parents that same enzyme cuts on the type cells of mamma animals expression vector pcDNA3.1/myc-His A (Invitrogen company).

2. the screening of people GPX1 mutant gene positive cell strain: when Growth of Cells to 1 * 10 ⁶During the density of/ml, with the expression vector that contains SBP2 and GPX1 mutant gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower cotransfection 293F cell (Invitrogen of mediation, R79007), cell changed in 72 hours after the transfection and begin adherent culture in the culture dish, the common 2-50 of adherent culture 48() hour change the nutrient solution that contains G418 and Zeocin after, it is the positive cell clone that Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant that screening has two kinds of resistances, G418 and Zeocin concentration are progressively successively decreased from 800-200 μ g/ml and 400-100 μ g/ml respectively during this time, subtract respectively 200 μ g/ml and 100 μ g/ml at every turn, each concentration was used 5 days, changed nutrient solution in 2-3 days one time, screening and culturing after 20 days with positive cell clone with 96,48,24,12 and 6 well culture plates are amplification culture step by step.Detect the expression amount of GPX1 mutant protein with anti-GPX1 polyclonal antibody and elisa technique, select the high cell strain of target protein expression amount and be used for spreading cultivation foster and frozen.

3. the expression and purification of people GPX1 mutant protein: the 293F that contains 1-10 μ M Sodium Selenite at large capacity expresses to spread cultivation in the substratum and supports the cell strain that screening obtains in 2 Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant.Under the common participation of other protein factor, the GPX1 mutant protein is expressed with soluble form and is secreted in substratum in SBP2, SECIS and 293 cells, nutrient solution of collection in per 48 hours.Nutrient solution with GSH affinity chromatography column purification GPX1 mutant protein, is collected the elutriant that contains GPX4 behind concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, small-molecule substance is removed in dialysis, and freeze-drying namely gets people GPX1 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 5th swimming lane of Fig. 1.Its GPX vigor is 22689U/ μ mol, surpasses order of magnitude of natural GPX.

Embodiment 6: prepare people GPX1 mutant protein with synthetic gene and the transfection of mammalian cell expression system substep

2. the screening of people GPX1 mutant gene positive cell strain: when Growth of Cells to 1 * 10 ⁶During the density of/ml, with the expression vector that contains the SBP2 gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower transfection 293F cell (Invitrogen of mediation, R79007), cell changed in 72 hours after the transfection and begin adherent culture in the culture dish, the common 2-50 of adherent culture 48() hour change the nutrient solution that contains G418 after, screening has the positive cell clone that the G418 resistance is stably express SBP2, G418 concentration progressively is decremented to 200 μ g/ml from 800 μ g/ml during this time, subtract 200 μ g/ml at every turn, each concentration was used 5 days, changed nutrient solution in 2-3 days one time, screening and culturing after 20 days with positive cell clone with 96,48,24,12 and 6 well culture plates are amplification culture step by step.Again with containing the expression vector of GPX1 mutant gene at lipofectamine2000 transfection reagent (Invitrogen, the by specification use) the 293F cell (Invitrogen of the lower transfection stably express SBP2 of mediation, R79007), cell changed in 72 hours after the transfection and begin adherent culture in the culture dish, the common 2-50 of adherent culture 48() hour changing the nutrient solution screening that contains G418 and Zeocin after, to have two kinds of resistances be the positive cell clone that Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant, G418 concentration maintains 200 μ g/ml during this time, Zeocin concentration progressively is decremented to 100 μ g/ml from 400 μ g/ml, subtract 100 μ g/ml at every turn, each concentration was used 5 days, changed nutrient solution in 2-3 days one time, screening and culturing will have the positive cell clone of two kinds of resistances with 96 after 20 days, 48,24,12 and 6 well culture plates are amplification culture step by step.With the polyclonal antibody of anti-GPX1 and the expression amount of elisa technique detection GPX1 mutant protein, select the high cell strain of target protein expression amount and be used for spreading cultivation foster and frozen.

Aforesaid method also can first transfection GPX1 mutant gene, rear transfection SBP2 gene.

3. the expression and purification of people GPX1 mutant: the 293F that contains 1-10 μ M Sodium Selenite at large capacity expresses to spread cultivation in the substratum and supports the cell strain that screening obtains in 2 Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant.Under the common participation of other protein factor, the GPX1 mutant is expressed with soluble form and is secreted in substratum in SBP2, SECIS and 293F cell, nutrient solution of collection in per 48 hours.Nutrient solution with GSH affinity chromatography column purification GPX1 mutant, is collected the elutriant that contains the GPX1 mutant behind concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, small-molecule substance is removed in dialysis, and freeze-drying namely gets people GPX1 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 6th swimming lane of Fig. 1.Its GPX vigor is 21968U/ μ mol, surpasses order of magnitude of natural GPX.

Embodiment 7: one step transfection prepares people GPX1 mutant protein with the target gene of amplification with the mammalian cell expression system

1. the structure of people GPX1 mutant protein expression vector: extract in a small amount test kit (Sigma with mRNA, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, extract mRNA, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist down, by RT-PCR (RT-polymerase chain reaction) it is transcribed into cDNA.According to the gene order of disclosed people GPX1 in the gene library (referring to NCBI, NM_000581.2) base sequence of the encoding gene and 3 of design primer amplification GPX1 ' end non-translational region, when the design primer, guarantee that 5 of target gene ' end contains initiator codon (ATG) and Hind III restriction enzyme site, the codon of the 2nd Cys replaces with the encoding sequence of Ser, other aminoacid sequence is constant, 3 ' end is introduced whole base sequences of 3 of GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A in the terminator codon downstream, concrete sequence is referring to NCBI, NM_000581.2) and Hind III restriction enzyme site; The target gene of specific restriction enzyme site and cells of mamma animals expression vector are arranged (such as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), the GPX1 gene is assembled on the secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region by specific restriction enzyme site with dna ligase again; Concrete 5 ' end primer is 5 '-CCCAAGCTTCATATGAGTGCTGCTCGGC-3 ', and 3 ' end primer is 5 '-CGGGATCCAGT GGGGAAACTCG-3 '.Cut latter linked method with first enzyme, by Hind III/BamHI restriction enzyme site the GPX1 gene is connected on the cells of mamma animals expression vector pSecTag2A (Invitrogen company) that same enzyme cuts together with selenocysteine insertion sequence; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to the halfcystine that will sport Serine among the GPX1 and the amino acid whose gene order near it, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the people GPX1 gene that is structured on the prokaryotic expression carrier is become the codon of Serine; Concrete sense primer is respectively: C78S5 '-GTGCTCGGCTTCCCG AGCAACCAGTTTGGGC-3 ', C115S5 '-CATGCTCTTCGAGAAG AGCGAGGTGAACGGTGC-3 ', C156S5 '-CACCTGGTCTCCGGTG AGTCGCAACGATGTTGC-3 ' and C202S5 '-GTCTCAAGGGCCCAGCTCTGCCTAGGGCGCCCCTC-3 '; Determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the gene energy code book invention sequence 1(SEQ ID No:1 after guaranteeing to suddenly change) described GPX1 mutant protein.According to human selenocysteine insertion sequence in the gene library in conjunction with albumen 2(SBP2, NCBI, NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders, its encoding gene of design primer amplification, guarantee that 5 of gene ' end contains initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end contains terminator codon and EcoRI restriction enzyme site; Concrete 5 ' end primer is 5 '-GCTCTAGAATGGAAGCTTTATCTTC-3 ', and 3 ' end primer is 5 '-CGGAATTCTAAATTCAAATTC-3 '.Cut latter linked method with first enzyme, by the XbaI/EcoRI restriction enzyme site carboxyl terminal (343-854aa) genome of SBP2 is installed in the born of the same parents that same enzyme cuts on the type cells of mamma animals expression vector pcDNA3.1/myc-His A (Invitrogen company).

2. the screening of people GPX1 mutant gene positive cell strain: when the density of Growth of Cells to 1 * 106/ml, with the expression vector that contains SBP2 and GPX1 mutant gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower cotransfection 293F cell (Invitrogen of mediation, R79007), cell changed in 72 hours after the transfection and begin adherent culture in the culture dish, the common 2-50 of adherent culture 48() hour change the nutrient solution that contains G418 and Zeocin after, it is the positive cell clone that Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant that screening has two kinds of resistances, G418 and Zeocin concentration are progressively successively decreased from 800-200 μ g/ml and 400-100 μ g/ml respectively during this time, subtract respectively 200 μ g/ml and 100 μ g/ml at every turn, each concentration was used 5 days, changed nutrient solution in 2-3 days one time, screening and culturing after 20 days with positive cell clone with 96,48,24,12 and 6 well culture plates are amplification culture step by step.Detect the expression amount of GPX1 mutant protein with anti-GPX1 polyclonal antibody and elisa technique, select the high cell strain of target protein expression amount and be used for spreading cultivation foster and frozen.

3. the expression and purification of people GPX1 mutant protein: the 293F that contains 1-10 μ M Sodium Selenite at large capacity expresses to spread cultivation in the substratum and supports the cell strain that screening obtains in 2 Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant.Under the common participation of other protein factor, the GPX1 mutant protein is expressed with soluble form and is secreted in substratum in SBP2, SECIS and 293 cells, nutrient solution of collection in per 48 hours.Nutrient solution with GSH affinity chromatography column purification GPX1 mutant protein, is collected the elutriant that contains GPX4 behind concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, small-molecule substance is removed in dialysis, and freeze-drying namely gets people GPX1 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 7th swimming lane of Fig. 1.Its GPX vigor is 22689U/ μ mol, surpasses order of magnitude of natural GPX.

Embodiment 8: transfection prepares people GPX1 mutant protein with the target gene of amplification with mammalian cell expression system substep

1. the structure of people GPX1 mutant protein expression vector: extract in a small amount test kit (Sigma with mRNA, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, extract mRNA, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist down, by RT-PCR (RT-polymerase chain reaction) it is transcribed into cDNA.According to the gene order of disclosed people GPX1 in the gene library (referring to NCBI, NM_000581.2) base sequence of the encoding gene and 3 of design primer amplification GPX1 ' end non-translational region, when the design primer, guarantee that 5 of target gene ' end contains initiator codon (ATG) and Hind III restriction enzyme site, the codon of the 2nd Cys replaces with the encoding sequence of Ser, other aminoacid sequence is constant, 3 ' end is introduced whole base sequences of 3 of GPX1 gene ' end non-translational region (from first base after the terminator codon of GPX1 gene to the previous base of poly oligonucleotide A in the terminator codon downstream, concrete sequence is referring to NCBI, NM_000581.2) and Hind III restriction enzyme site; The target gene of specific restriction enzyme site and cells of mamma animals expression vector are arranged (such as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), the GPX1 gene is assembled on the secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region by specific restriction enzyme site with dna ligase again; Concrete 5 ' end primer is 5 '-CCCAAGCTTCATATGAGTGCTGCTCGGC-3 ', and 3 ' end primer is 5 '-CG GGA TCCAGT GGGGAAACTCG-3 '.Cut latter linked method with first enzyme, by Hind III/BamHI restriction enzyme site the GPX1 gene is connected on the cells of mamma animals expression vector pSecTag2A (Invitrogen company) that same enzyme cuts together with selenocysteine insertion sequence; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to the halfcystine that will sport Serine among the GPX1 and the amino acid whose gene order near it, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the people GPX1 gene that is structured on the prokaryotic expression carrier is become the codon of Serine; Concrete sense primer is respectively: C78S5 '-GTGCTCGGCTTCCCG AGCAACCAGTTTGGGC-3 ', C115S5 '-CATGCTCTTCGAGAAG AGCGAGGTGAACGGTGC-3 ', C156S5 '-CACCTGGTCTCCGGTG AGTCGCAACGATGTTGC-3 ' and C202S5 '-GTCTCAAGGGCCCAGCTCTGCCTAGGGCGCCCCTC-3 '; Determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the gene energy code book invention sequence 1(SEQ ID No:1 after guaranteeing to suddenly change) described GPX1 mutant protein.According to human selenocysteine insertion sequence in the gene library in conjunction with albumen 2(SBP2, NCBI, NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders, its encoding gene of design primer amplification, guarantee that 5 of gene ' end contains initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end contains terminator codon and EcoRI restriction enzyme site; Concrete 5 ' end primer is 5 '-GCTCTAGAATGGAAGCTTTATCTTC-3 ', and 3 ' end primer is 5 '-CGGAATTCTAAATTCAAATTC-3 '.Cut latter linked method with first enzyme, by the XbaI/EcoRI restriction enzyme site carboxyl terminal (343-854aa) genome of SBP2 is installed in the born of the same parents that same enzyme cuts on the type cells of mamma animals expression vector pcDNA3.1/myc-His A (Invitrogen company).

3. the expression and purification of people GPX1 mutant: the 293F that contains 1-10 μ M Sodium Selenite at large capacity expresses to spread cultivation in the substratum and supports the cell strain that screening obtains in 2 Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant.Under the common participation of other protein factor, the GPX1 mutant is expressed with soluble form and is secreted in substratum in SBP2, SECIS and 293F cell, nutrient solution of collection in per 48 hours.Nutrient solution with GSH affinity chromatography column purification GPX1 mutant, is collected the elutriant that contains the GPX1 mutant behind concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, small-molecule substance is removed in dialysis, and freeze-drying namely gets people GPX1 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 8th swimming lane of Fig. 1.Its GPX vigor is 20968U/ μ mol, surpasses order of magnitude of natural GPX.

Embodiment 9:

Identical with embodiment 1 preparation method, just the encoding sequence of the 87th L-Ala is replaced with during the encoding gene of synthetic GPX1 mutant the codon (TCC) of Serine, the 87th of the GPX1 mutant protein that obtains at last is Serine, rather than L-Ala, other aminoacid sequence and first method are identical, i.e. sequence 2(SEQ ID No:2 of the present invention) described GPX1 mutant protein.This mutant molecule amount only has small variation, and is as broad as long on SDS-PAGE and Western Blot result, sees the 9th swimming lane of Fig. 1.But its GPX vigor is 23619U/ μ mol, is higher than embodiment 1, surpasses order of magnitude of natural GPX.

Embodiment 10:

Identical with embodiment 1 preparation method, just the encoding sequence of the 2nd Serine is replaced with during the encoding gene of synthetic GPX1 mutant the codon of halfcystine, the second of the GPX1 mutant protein that obtains at last is seleno-cysteine, rather than Serine, other aminoacid sequence and first method are identical, i.e. sequence 3(SEQ ID No:3 of the present invention) described GPX1 mutant protein.This mutant molecule amount only has small variation, and is as broad as long on SDS-PAGE and Western Blot result, sees the 10th swimming lane of Fig. 1.But its GPX vigor is 15358U/ μ mol, is lower than embodiment 1, but still surpasses order of magnitude of natural GPX.

Embodiment 11:

Except the expression vector pCold III(TAKARA that embodiment 1 is used, Cat.#3369) change pColdI(TAKARA with the Histidine purification tag into, Cat.#3367), all the other and embodiment 1 are identical, namely obtain the most at last sequence 4(SEQ ID No:4 of the present invention) described GPX1 mutant protein.This mutant has been introduced 16 exogenous amino acids that carrier comprises 6 Histidines at aminoterminal, therefore molecular weight increases to some extent, can see on SDS-PAGE and Western Blot result, see the 11st swimming lane of Fig. 1, its advantage is also can use the affine series of strata purification of target of nickel albumen.Its GPX vigor is 20532U/ μ mol, a little less than embodiment 1, proves purification tag to the not obviously impact of vigor of albumen, and vigor surpasses order of magnitude of natural GPX.

Claims

1. one kind high vigor Selenoperoxidase GPX1 mutant, its aminoacid sequence is shown in SEQ ID No:1.

2. a kind of high vigor Selenoperoxidase GPX1 mutant as claimed in claim 1 is characterized in that: any one or a plurality of L-Ala Ala in the aminoacid sequence shown in the SEQ ID No:1 are sported the formed Selenoperoxidase GPX1 of Serine Ser mutant.

3. a kind of high vigor Selenoperoxidase GPX1 mutant as claimed in claim 2, it is characterized in that: the aminoacid sequence of formed mutant is shown in SEQ ID No:2.

4. a kind of high vigor Selenoperoxidase GPX1 mutant as claimed in claim 1 is characterized in that: one or more in the aminoacid sequence shown in the SEQ ID No:1 in 5 Serines of the 2nd, 78,115,156 and 202 are replaced formed Selenoperoxidase GPX1 mutant by seleno-cysteine.

5. a kind of high vigor Selenoperoxidase GPX1 mutant as claimed in claim 4, it is characterized in that: the aminoacid sequence of formed mutant is shown in SEQ ID No:3.

6. one kind high vigor Selenoperoxidase GPX1 mutant, its aminoacid sequence is shown in SEQ ID No:4, SEQ ID No:5 or SEQ ID No:6.

7. the preparation method of high vigor Selenoperoxidase GPX1 mutant claimed in claim 1, its step is as follows:

1), the structure of expression vector:

According to aminoacid sequence SEQ ID No:1, can in auxotrophic strain, express the gene of GPX1 mutant protein with the dna synthesizer synthetic, guarantee that 5 of target gene ' end contains initiator codon ATG, 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 49 seleno-cysteine SeCys of target gene replaces with the codon of halfcystine Cys, and full length gene does not contain the ACA sequence; Then contain GPX1 mutant gene and the secretor type prokaryotic expression carrier of specific restriction enzyme site with identical restriction endonuclease cutting two ends, the GPX1 mutant gene is assembled on the secretor type prokaryotic expression carrier by specific restriction enzyme site with dna ligase again; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the GPX1 mutant gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains the GPX1 mutant gene that makes up in the step 1), be coated with the nutrient agar plate that contains Cys, the screening positive strain; The pMazF Plasmid Transformation positive strain that will contain again express nucleic acid restriction endonuclease MazF is with the M9 solid medium screening positive transformant that contains two resistances; With positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, through 4-25 ℃ of abduction delivering of IPTG low temperature, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX1 mutant that contains SeCys at the combining site of substrate GSH, zymoprotein is expressed and is secreted in the pericentral siphon chamber of thalline with soluble form, and MazF can and cut off single stranded RNA particular sequence ACA by identification, suppresses the expression of host protein; Cultivate in a small amount first the strain of screening high expression level, again enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, the ice bath with the liquid low-temperature centrifugation, are removed bacterial sediment, are obtained supernatant liquor; With gsh GSH affinitive layer purification GPX1 mutant protein, namely get GPX1 mutant protein sterling after the dialysis freeze-drying, identify target protein with denaturing polyacrylamide gel electrophoresis SDS-PAGE and Western blot Western blot;

Or,

1), the structure of expression vector:

Gene order design primer according to GPX1, its encoding gene increases, when the design primer, guarantee that 5 of gene ' end contains initiator codon ATG, 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantees that the codon of the 2nd and 202 Cys replaces to the codon of Ser, and other aminoacid sequence is constant; After identical restriction endonuclease cut vector and target gene, the GPX1 genome is installed on the secretor type prokaryotic expression carrier by specific restriction enzyme site with dna ligase again; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to 49 seleno-cysteines that will sport halfcystine among the GPX1 and the amino acid whose gene order near it, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the seleno-cysteine in the GPX1 gene that is structured on the prokaryotic expression carrier is become the codon of halfcystine; In like manner, guaranteeing not introduce under the prerequisite of ACA sequence, the sequence encoding mutant of other halfcystine in the GPX1 gene is become the codon of Serine with the method for above-mentioned rite-directed mutagenesis; According to the degeneracy of codon, under the prerequisite that does not change aminoacid sequence, all ACA series jumps in the GPX1 gene are fallen again; Determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the gene after guaranteeing to suddenly change can be expressed GPX1 mutant protein of the present invention in auxotrophic strain; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains the GPX1 mutant gene that makes up in the step 1), be coated with the nutrient agar plate that contains halfcystine, the screening positive strain; Plasmid pMazF with express nucleic acid restriction endonuclease MazF transforms positive strain again, with the M9 solid medium screening positive transformant that contains two resistances; With positive transformant spread cultivation support after, containing seleno-cysteine, in the substratum of essential growth factor and nutrient substance, through 4-25 ℃ of abduction delivering of isopropylthio-β-D-galactoside low temperature, auxotrophic strain-BL21 (DE3) Cys can synthesize seleno-cysteine with the codon of halfcystine, directly give expression to the restructuring GPX1 mutant that contains seleno-cysteine at the combining site of substrate gsh, zymoprotein is expressed and is secreted in the pericentral siphon chamber of thalline with soluble form, and MazF can and cut off single stranded RNA particular sequence ACA by identification, suppresses the expression of host protein; Cultivate in a small amount first the strain of screening high expression level, again enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, the ice bath, with the liquid low-temperature centrifugation, removal bacterial sediment, acquisition contain the supernatant liquor of GPX1 mutant; With gsh GSH affinitive layer purification GPX, namely get the zymoprotein sterling after the dialysis freeze-drying;

Or,

1), the structure of expression vector:

According to aminoacid sequence SEQ ID No:1, can in auxotrophic strain, express the gene of GPX1 mutant protein with the dna synthesizer synthetic, guarantee that 5 of target gene ' end contains initiator codon ATG, 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantees that the encoding sequence of 49 seleno-cysteine SeCys of target gene replaces with the codon of halfcystine Cys; Contain GPX1 mutant gene and the secretor type prokaryotic expression carrier of specific restriction enzyme site with identical restriction endonuclease cutting two ends, the GPX1 mutant gene is assembled on the secretor type prokaryotic expression carrier by specific restriction enzyme site with dna ligase again; Containing in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the GPX1 mutant gene, is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains the GPX1 mutant gene that makes up in the step 1), be coated with the nutrient agar plate that contains Cys, the screening positive transformant; With positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, IPTG induces through isopropylthio-β-D-galactoside, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, the last GPX1 mutant protein that contains SeCys at the combining site of substrate GSH that directly gives expression to, zymoprotein is expressed and is secreted in the pericentral siphon chamber of thalline with soluble form; Cultivate in a small amount first the strain of screening high expression level, again enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, the ice bath with the liquid low-temperature centrifugation, are removed bacterial sediment, are obtained supernatant liquor; With gsh GSH affinitive layer purification GPX1 mutant protein, namely get GPX1 mutant protein sterling after the dialysis freeze-drying;

Or,

1), the structure of expression vector:

Gene order design primer according to GPX1, its encoding gene increases, when the design primer, guarantee that 5 of gene ' end contains initiator codon ATG, 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantees that the codon of the 2nd and 202 Cys replaces to the codon of Ser, and other aminoacid sequence is constant; After identical restriction endonuclease cut vector and target gene, the GPX1 genome is installed on the secretor type prokaryotic expression carrier by specific restriction enzyme site with dna ligase again; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to 49 SeCys that will sport halfcystine Cys among the GPX1 and the amino acid whose gene order near it, centered by the codon of mutating acid, the long 25-50bp of primer; With rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the SeCys in the GPX1 gene that is structured on the prokaryotic expression carrier is become the codon of Cys; In like manner, with the method for above-mentioned rite-directed mutagenesis the sequence encoding mutant of other halfcystine in the GPX1 gene is become the codon of Serine; Determine to suddenly change successfully by dna sequencing, and occur without other unexpected transgenation, the gene after guaranteeing to suddenly change can be expressed GPX1 mutant protein of the present invention in auxotrophic strain; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

Or,

1), the structure of expression vector:

According to aminoacid sequence SEQ ID No:1, gene with dna synthesizer synthetic GPX1 mutant protein, guarantee that 5 of gene ' end contains initiator codon ATG and specific restriction enzyme site, 3 ' end is introduced whole base sequences or selenocysteine insertion sequence SECIS and the specific restriction enzyme site of 3 of GPX1 gene ' end non-translational region in the terminator codon downstream; The GPX1 mutant gene and the cells of mamma animals expression vector that contain specific restriction enzyme site with identical restriction endonuclease cutting two ends are assembled into whole base sequences or the selenocysteine insertion sequence of GPX1 mutant gene together with its 3 ' end non-translational region on the secretor type cells of mamma animals expression vector by specific restriction enzyme site with dna ligase again; According to 512 the amino acid whose gene orders of selenocysteine insertion sequence in conjunction with Protein S BP2 carboxyl terminal 343-854 position, with its encoding gene of dna synthesizer synthetic, guarantee that 5 of gene ' end contains initiator codon ATG and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site, with specific restriction enzyme site the SBP2 genome is installed in the born of the same parents on the type cells of mamma animals expression vector; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

In shaking flask, will express simultaneously the cell strain amplification culture of two kinds of foreign proteins, containing in the substratum of Sodium Selenite with cells of mamma animals expression GPX1 mutant, zymoprotein is secreted in the substratum with soluble form, with gsh GSH affinitive layer purification GPX1 mutant protein, namely get the zymoprotein sterling after the dialysis freeze-drying;

Or,

1), the structure of expression vector:

According to the aminoacid sequence SEQ ID NO:1 of GPX1 mutant with the encoding gene of dna synthesizer synthetic GPX1 mutant, guarantee that 5 of gene ' end contains initiator codon ATG and specific restriction enzyme site, 3 ' end is introduced whole base sequences or selenocysteine insertion sequence SECIS and the specific restriction enzyme site of 3 of GPX1 gene ' end non-translational region in the terminator codon downstream; Contain GPX1 mutant gene and the cells of mamma animals expression vector of specific restriction enzyme site with identical restriction endonuclease cutting two ends, the GPX1 mutant gene is assembled on the secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence by specific restriction enzyme site with dna ligase again; According to 512 the amino acid whose gene orders of selenocysteine insertion sequence in conjunction with Protein S BP2 carboxyl terminal 343-854 position, with its encoding gene of dna synthesizer synthetic, guarantee that 5 of gene ' end contains initiator codon ATG and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site, with specific restriction enzyme site the SBP2 genome is installed in the born of the same parents on the type cells of mamma animals expression vector; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

To express simultaneously the cell strain amplification culture of two kinds of foreign proteins in shaking flask, express the GPX1 mutant containing in the substratum of Sodium Selenite with cells of mamma animals, zymoprotein is secreted in the substratum with soluble form; With gsh affinitive layer purification GPX1 mutant protein, namely get the zymoprotein sterling after the dialysis freeze-drying;

Or,

1), the structure of expression vector:

According to the GPX1 gene order, the base sequence of the encoding gene and 3 of design primer amplification GPX1 ' end non-translational region, when the design primer, guarantee that 5 of target gene ' end contains initiator codon ATG and specific restriction enzyme site, 3 of target gene ' end is introduced whole base sequences or selenocysteine insertion sequence SECIS and the specific restriction enzyme site of 3 of GPX1 gene ' end non-translational region in the terminator codon downstream, guarantee that the codon of the 2nd Cys replaces to the codon of Ser, other aminoacid sequence is constant; Target gene and cells of mamma animals expression vector that specific restriction enzyme site is arranged with identical restriction endonuclease cutting two ends are assembled into the GPX1 gene on the secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region by specific restriction enzyme site with dna ligase again; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to the halfcystine that will sport Serine among the GPX1 and the amino acid whose gene order near it, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the GPX1 gene that is structured on the carrier for expression of eukaryon is become the codon of Serine; Determine suddenly change successfully, and without other unexpected transgenation generation the GPX1 mutant protein of the present invention of to encode of the gene after guaranteeing to suddenly change by dna sequencing; According to 512 the amino acid whose gene orders of selenocysteine insertion sequence in conjunction with Protein S BP2 carboxyl terminal 343-854 position, its encoding gene of design primer amplification, guarantee that 5 of gene ' end contains initiator codon ATG and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site; After cutting connection, enzyme with specific restriction enzyme site the SBP2 genome is installed in the born of the same parents on the type cells of mamma animals expression vector equally; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

Or,

1), the structure of expression vector:

Base sequence according to the encoding gene and 3 of GPX1 gene order design primer amplification GPX1 ' end non-translational region, when the design primer, guarantee that 5 of target gene ' end contains initiator codon ATG and specific restriction enzyme site, 3 of target gene ' end is introduced whole base sequences or selenocysteine insertion sequence SECIS and the specific restriction enzyme site of 3 of GPX1 gene ' end non-translational region in the terminator codon downstream, guarantee that the codon of the 2nd Cys replaces to the codon of Ser, other aminoacid sequence is constant; Target gene and cells of mamma animals expression vector that specific restriction enzyme site is arranged with identical restriction endonuclease cutting two ends are assembled into the GPX1 gene on the secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region by specific restriction enzyme site with dna ligase again; Keeping under the constant prerequisite of other aminoacid sequence, design two rite-directed mutagenesis primers of complete isometric complementation according to the halfcystine that will sport Serine among the GPX1 and the amino acid whose gene order near it, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the GPX1 gene that is structured on the carrier for expression of eukaryon is become the codon of Serine; Determine suddenly change successfully, and without other unexpected transgenation generation the GPX1 mutant protein of the present invention of to encode of the gene after guaranteeing to suddenly change by dna sequencing; According to 512 the amino acid whose gene orders of selenocysteine insertion sequence in conjunction with Protein S BP2 carboxyl terminal 343-854 position, its encoding gene of design primer amplification, guarantee that 5 of gene ' end contains initiator codon ATG and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site; After cutting connection, enzyme with specific restriction enzyme site the SBP2 genome is installed in the born of the same parents on the type cells of mamma animals expression vector equally; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the target gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

8. the preparation method of high vigor Selenoperoxidase GPX1 mutant claimed in claim 3, its step is as follows:

1), the structure of expression vector:

According to aminoacid sequence SEQ ID No:1, can in auxotrophic strain, express the gene of GPX1 mutant protein with the dna synthesizer synthetic, guarantee that 5 of target gene ' end contains initiator codon ATG, 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 49 seleno-cysteine SeCys of target gene replaces with the codon of halfcystine Cys, the encoding sequence of the 87th L-Ala replaces with the codon of Serine, and full length gene does not contain the ACA sequence; Then contain GPX1 mutant gene and the secretor type prokaryotic expression carrier of specific restriction enzyme site with identical restriction endonuclease cutting two ends, the GPX1 mutant gene is assembled on the secretor type prokaryotic expression carrier by specific restriction enzyme site with dna ligase again; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the GPX1 mutant gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains the GPX1 mutant gene that makes up in the step 1), be coated with the nutrient agar plate that contains Cys, the screening positive strain; The pMazF Plasmid Transformation positive strain that will contain again express nucleic acid restriction endonuclease MazF is with the M9 solid medium screening positive transformant that contains two resistances; With positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, through 4-25 ℃ of abduction delivering of IPTG low temperature, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX1 mutant that contains SeCys at the combining site of substrate GSH, zymoprotein is expressed and is secreted in the pericentral siphon chamber of thalline with soluble form, and MazF can and cut off single stranded RNA particular sequence ACA by identification, suppresses the expression of host protein; Cultivate in a small amount first the strain of screening high expression level, again enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, the ice bath with the liquid low-temperature centrifugation, are removed bacterial sediment, are obtained supernatant liquor; With gsh GSH affinitive layer purification GPX1 mutant protein, namely get GPX1 mutant protein sterling after the dialysis freeze-drying, identify target protein with denaturing polyacrylamide gel electrophoresis SDS-PAGE and Western blot Western blot.

9. the preparation method of high vigor Selenoperoxidase GPX1 mutant claimed in claim 5, its step is as follows:

1), the structure of expression vector:

According to aminoacid sequence SEQ ID No:1, can in auxotrophic strain, express the gene of GPX1 mutant protein with the dna synthesizer synthetic, guarantee that 5 of target gene ' end contains initiator codon ATG, 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 49 seleno-cysteine SeCys of target gene replaces with the codon of halfcystine Cys, the encoding sequence of second Serine replaces with the codon of halfcystine, and full length gene does not contain the ACA sequence; Then contain GPX1 mutant gene and the secretor type prokaryotic expression carrier of specific restriction enzyme site with identical restriction endonuclease cutting two ends, the GPX1 mutant gene is assembled on the secretor type prokaryotic expression carrier by specific restriction enzyme site with dna ligase again; That contain in the multiple clone site that described specific restriction enzyme site can be expression vector and non-existent any one restriction enzyme site in the GPX1 mutant gene is the dna sequence dna of intrinsic based composition by restriction endonuclease identification on the carrier;

10. the preparation method of high vigor Selenoperoxidase GPX1 mutant claimed in claim 6, its step is as follows: introduce formed sequence SEQ ID No:4, SEQ ID No:5 or SEQ ID No:6 behind the amino acid of the Histidine purification tag of pColdI prokaryotic expression carrier and factor Xa cleavage site at the aminoterminal of sequence SEQ ID No:1, SEQ ID No:2 or SEQ ID No:3.