CN103756984B

CN103756984B - High vigor Selenoperoxidase GPX2 mutant and preparation method thereof

Info

Publication number: CN103756984B
Application number: CN201410054696.6A
Authority: CN
Inventors: 魏景艳; 郭笑; 宋健
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2014-02-18
Filing date: 2014-02-18
Publication date: 2015-10-14
Anticipated expiration: 2034-02-18
Also published as: CN103756984A

Abstract

High vigor Selenoperoxidase GPX2 mutant and preparation method thereof, belongs to biological technical field.The present invention with people GPX2 for template, rite-directed mutagenesis is fitted by computer mould, obtain a kind of novel high vigor Selenoperoxidase GPX2 mutant, it is made up of 203 amino acid, compared with people GPX2, it containing halfcystine, does not have higher GPX vigor and better stability, it is that cysteine mutation all in people GPX2 are become Serine, but the 40th remains seleno-cysteine (SeCys).It in mammalian cell strain or auxotroph prokaryotic expression system and SPP low temperature expression system thereof, directly expresses the method with the GPX2 mutant protein of enzymatic activity high with genetic engineering technique.Present method does not need chemically modified to prepare to have the novel artificial enzyme of high GPX vigor.The inventive method has broad application prospects in bio-pharmaceuticals.

Description

High vigor Selenoperoxidase GPX2 mutant and preparation method thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of high vigor Selenoperoxidase GPX2 mutant and preparation method thereof.

Background technology

Substrate containing 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) together form body anti-oxidative defense system.GPX plays an important role in this system, it with substrate GSH for reductive agent, hydrogen peroxide in decomposer and all kinds of hydroperoxide, thus can active oxygen (ROS) in purged body, prevent lipid peroxidation, treat the various diseases caused by active oxygen, as aging, ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataract, tumour etc.Different from other antioxidase, GPX is except removing except ROS, and lipid peroxide of can also degrading, prevents cellular superoxideization from damaging, and the function of the Cell protection of this uniqueness makes it in antioxidase system, occupy the position of particularly important.But, because the source of natural GPX is quite limited, poor stability, 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, about being only the thousandth of natural GPX.The GPX analogue enztme that macromolecular stand-in mainly contain abzyme (Chinese patent 94102481.4 and 96112628.0) and prepare for protein template with glutathione S-transferase (GST), its vigor is significantly higher than small molecule mimetics.Obviously, be that the macromole GPX analogue enztme of Template preparation have received better effect to have the albumen of gsh (GSH) combining site, therefore the GPX analogue enztme technology of preparing of these high vigor of exploitation preparation is with regard to the study hotspot of Cheng Liao academia, has broad application prospects for fields such as molecular biology, physiotechnology and medical science.The method that success has been developed so far has: the catalytic group seleno-cysteine (SeCys) 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, the preparation method of all kinds of selenium-containing abzyme disclosed in 200810050556.6 is exactly the catalytic group SeCys that applied chemistry sudden change (modification) method introduces GPX in antibody class template protein, there is following shortcoming: (1) is in preparation process, only chemical mutation (modification) this step will lose the zymoprotein of 20-40%, causes analogue enztme productive rate significantly to decline; (2) cycle of preparing analogue enztme is long, complex operation, and the time is long; (3) need to use phenylmethylsulfonyl fluoride, acetonitrile etc. in chemical mutation process, these materials are virose material; (4) lack targeting, for large protein molecular, chemical reaction different loci of being everlasting introduces multiple non-specific catalytic group, and therefore chemical mutation is introduced catalytic group and cannot be reached the such specificity of transgenation method.

Chinese patent 200810050556.6 also discloses the another kind of preparation method of people source strand selenium-containing abzyme, introduce catalytic group by auxotroph prokaryotic expression system (intestinal bacteria) and transgenation method, but it is only limitted to the preparation of people source strand selenium-containing abzyme, do not comprise the preparation method of Selenoperoxidase (GPX) mutant and other GPX analogue enztme.Have the glutathione S-transferase (GST) of GPX activity 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, it is only limitted to be that template protein prepares GPX analogue enztme with GST, does not comprise with natural GPX for Template preparation GPX mutant.In non-GPX class template albumen, introducing with auxotroph prokaryotic expression system catalytic group prepares analogue enztme method is catalytic group is incorporated into it that there is same substrate GSH combining site with GPX to plant in albumen and make it produce GPX vigor.

But do not possess the catalytic group of natural GPX self due to these template protein, be therefore difficult to find ideal and the position of catalytic group accurately; Simultaneously owing to not resembling catalytic triads desirable natural GPX in these template protein, therefore the catalytic efficiency of this kind of analogue enztme is not high yet.If with natural GPX for template gene engineering method prepares GPX mutant, these shortcomings can be overcome.Because the codon UGA of the catalytic group SeCys of the GPX that encodes is terminator codon, in common prokaryotic expression system, need in the open reading frame of GPX gene, next-door neighbour SeCys codon UGA downstream introduce neck ring structure UGA could be translated into SeCys instead of terminator codon, and the introducing of neck ring will inevitably cause the change of GPX space conformation in open reading frame, and then affect enzymic activity.Therefore common prokaryotic expression system is unsuitable for directly expressing the Selenonic protein with GPX activity.

Summary of the invention

The object of the present invention is to provide a kind of high vigor Selenoperoxidase GPX2 mutant and preparation method thereof.

Using gene engineering technique, cell culture technology, auxotroph prokaryotic expression technology and SPP(Single protein production, single protein production) system hypothermia expression technology prepares the GPX2 mutant with high GPX vigor, is in mammalian cell strain or auxotroph prokaryotic expression system and SPP low temperature expression system thereof, directly express the method with the GPX2 mutant protein of enzymatic activity high with genetic engineering technique.Present method does not need chemically modified to prepare to have the novel artificial enzyme of high GPX vigor.The inventive method has broad application prospects in bio-pharmaceuticals.

The present invention with people GPX2(see NCBI, NM_002083.3) be template, rite-directed mutagenesis is fitted by computer mould, obtain a kind of novel high vigor Selenoperoxidase GPX2 mutant, it is made up of 203 amino acid, compared with people GPX2, it is not containing halfcystine, there is higher GPX vigor and better stability, it is that cysteine mutation all in people GPX2 are become Serine, but the 40th remains seleno-cysteine (SeCys, one-letter abbreviations is U, is write as Xaa in sequence table).The aminoacid sequence (SEQ ID No:1) of described GPX2 mutant is as follows:

MAFIAKSFYDLSAISLDGEKVDFNTFRGRAVLIENVASLUGTTTRDFTQLNELQSRFPRRLVVLGFPSNQFGHQENSQNEEILNSLKYVRPGGGYQPTFTLVQKSEVNGQNEHPVFAYLKDKLPYPYDDPFSLMTDPKLIIWSPVRRSDVAWNFEKFLIGPEGEPFRRYSRTFPTINIEPDIKRLLKVAI

Further, the concrete aminoacid sequence of described GPX2 mutant also comprises, and any one or more L-Ala (Ala) in above-mentioned aminoacid sequence is sported any one novel GPX2 mutant that Serine (Ser) is formed.Such as 117 L-Ala (Ala) are sported Serine (Ser), the sequence formed is SEQ ID No:2(embodiment 9).

Further, the concrete aminoacid sequence of described GPX2 mutant also comprises, after any one or more of the 55th, 68,77,105 4 Serines in above-mentioned aminoacid sequence are replaced any one novel GPX2 mutant of being formed by L-Ala.After such as the 77th Serine is replaced the sequence SEQ ID No:3(embodiment 10 that formed by L-Ala).

Further, the concrete aminoacid sequence of described GPX2 mutant also comprises, owing to employing the pColdI(TAKARA company being convenient to target protein purifying) etc. secretor type protokaryon or carrier for expression of eukaryon and introduce any one novel GPX2 mutant that Histidine purification tag on this secretor type protokaryon or carrier for expression of eukaryon and other amino acid etc. formed at the aminoterminal of above-mentioned aminoacid sequence or carboxyl terminal.Such as introduce pColdI(TAKARA company at the aminoterminal of sequence SEQ ID No:1, SEQ ID No:2 and SEQ ID No:3) the sequence SEQ IDNo:4, the SEQ ID No:5 that are formed after the Histidine purification tag of prokaryotic expression carrier and the amino acid of factor Xa cleavage site and SEQ ID No:6(embodiment 11).

The method of preparation of the present invention high vigor Selenoperoxidase GPX2 mutant, specifically comprises following method:

Method 1:

First synthesizing target gene and being assembled into installs on secretor type prokaryotic expression carrier by GPX2 genome on secretor type prokaryotic expression carrier or first, target gene is obtained by transgenation (or amino acid replacement) method, again by auxotroph prokaryotic expression system or by auxotroph protokaryon and SPP low temperature Combined expression system, the catalytic group seleno-cysteine (SeCys) of GPX is incorporated into the substrate binding site of GPX2 mutant, thus the substrate binding site of existing GPX on this albumen, there are again catalytic group and the catalytic triads of GPX, result gives the activity of its high GPX, just create the Selenoperoxidase GPX2 mutant of high vigor.

Method 2:

Or first the gene of coding GPX2 mutant is assembled on secretor type cells of mamma animals expression vector together with selenocysteine insertion sequence, again by selenocysteine insertion sequence associated proteins 2(SBP2) be assembled on born of the same parents' inner mold cells of mamma animals expression vector, by two kinds of same cells of mamma animals strains of carrier cotransfection, then GPX2 mutant protein is prepared in the positive cell strain containing GPX2 mutant and SBP2 two kinds of genes while obtaining with screening, just create the genetically engineered Selenoperoxidase GPX2 mutant of high vigor in vitro, thus solve the problem of natural GPX limited source.

First method of the present invention is the gene first can expressing GPX2 mutant protein of the present invention at biotech firm's DNA synthesizer synthetic; guarantee not containing ACA sequence in gene, then combine preparation GPX2 mutant protein with single protein production systems and auxotroph prokaryotic expression system.

1), the structure of expression vector: according to the aminoacid sequence of GPX2 mutant of the present invention, the gene of GPX2 mutant protein can be expressed in auxotrophic strain at biotech firm's DNA synthesizer synthetic, guarantee that 5 ' end of target gene is containing initiator codon (ATG), 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 40 seleno-cysteines (SeCys) of target gene replaces with the codon (can be TGC etc.) of halfcystine (Cys), and full length gene is not containing ACA sequence, concrete gene order can be (see NCBI by GPX2 gene, NM_002083.3) in the 55th, 68, the encoding sequence of the halfcystine of 77 and 105 replaces with the codon of Serine, the codon of the 40th seleno-cysteine replaces with the codon of halfcystine, and according to the degeneracy of codon, under the prerequisite not changing aminoacid sequence, ACA sequences all in gene is all replaced with the encoding gene that non-ACA sequence obtains, also can be that other any one can express GPX2 mutant protein in auxotrophic strain, and the encoding gene of total length not containing ACA sequence is (due to the degeneracy of codon, multiple different encoding gene can be had) with amino acid sequence, contain GPX2 mutant gene and the secretor type prokaryotic expression carrier (as pCold series etc.) of specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type prokaryotic expression carrier with DNA ligase, described specific restriction enzyme site can be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in GPX2 mutant gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier,

2), the screening of positive transformant and the expression and purification of albumen:

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector containing GPX2 mutant gene built in step 1), be coated with the nutrient agar plate containing Cys, screening positive strain; Again by pMazF(TAKARA, the Cat#3367 containing express nucleic acid restriction endonuclease MazF) Plastid transformation positive strain, screens positive transformant with containing Double M9 solid medium; Positive transformant is spread cultivation after supporting, in the substratum containing SeCys, essential growth factor and nutrient substance, through IPTG low temperature 4-25 DEG C of abduction delivering, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX2 mutant containing SeCys at the combining site of substrate GSH, zymoprotein is expressed with soluble form and is secreted in the pericentral siphon chamber of thalline, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein; First cultivation screening high expression level strain in a small amount, then enlarged culturing and abduction delivering; Under collection, washing, ice bath, ultrasonication thalline, release zymoprotein, centrifugal by liquid cryogen, removes bacterial sediment, obtains supernatant liquor; With gsh (GSH) affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain GPX2 mutant protein sterling.Target protein is identified with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Westernblot).

The method introduces catalytic group by inducing auxotroph prokaryotic expression system under low temperature at the substrate binding site of GPX2 mutant, thus produces the GPX2 mutant of high vigor.

Described uses gsh (GSH) affinitive layer purification GPX2 mutant protein, is by pH7.5,50mmol/L Tris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

Described is centrifugal by liquid cryogen, can at 4 DEG C, the centrifugal 15-30min of 8000-12000g.

Second method of the present invention is the GPX2 gene that first increases, then with it for template, the gene expressing GPX2 mutant protein of the present invention is obtained by transgenation method, and ensureing that under the prerequisite that its aminoacid sequence is constant, the ACA sequence in gene is fallen in sudden change, obtain not containing the GPX2 mutant gene of ACA, then combine preparation GPX2 mutant protein with single protein production systems and auxotroph prokaryotic expression system.

1), the structure of expression vector:

Disclosed in gene library, the gene order of GPX2 is (see NCBI, NM_002083.3) primer is designed, increase its encoding gene, when designing primer, guarantee that 5 ' end of gene is containing initiator codon (ATG), 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends; After identical restriction endonuclease cut vector and target gene, then by specific restriction enzyme site, GPX2 genome is installed to (as pCold series etc.) on secretor type prokaryotic expression carrier with DNA ligase; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport 40 seleno-cysteines of halfcystine and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit, the sequence encoding mutant of the seleno-cysteine in the GPX2 gene be structured on prokaryotic expression carrier is become the codon of halfcystine; In like manner, guaranteeing under the prerequisite not introducing ACA sequence by the method for above-mentioned rite-directed mutagenesis, the sequence encoding mutant of other halfcystine in GPX2 gene is become the codon of Serine, again according to the degeneracy of codon, under the prerequisite not changing aminoacid sequence, ACA series jumps all in GPX2 gene are fallen; Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee that the gene after suddenling change can express GPX2 mutant protein of the present invention in auxotrophic strain; Described specific restriction enzyme site can be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

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

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector containing GPX2 mutant gene built in step 1), be coated with the nutrient agar plate containing halfcystine, screening positive strain, again the plasmid pMazF of express nucleic acid restriction endonuclease MazF is transformed positive strain, screen positive transformant with containing Double M9 solid medium, positive transformant is spread cultivation after supporting, containing seleno-cysteine, in the substratum of essential growth factor and nutrient substance, through isopropylthio-β-D-galactoside low temperature 4-25 DEG C of abduction delivering, auxotrophic strain-BL21 (DE3) Cys can synthesize seleno-cysteine with the codon of halfcystine, directly give expression to the restructuring GPX2 mutant containing seleno-cysteine at the combining site of substrate gsh, zymoprotein is expressed with soluble form and is secreted in the pericentral siphon chamber of thalline, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein, first cultivation screening high expression level strain in a small amount, then enlarged culturing and abduction delivering, under collection, washing, ice bath, ultrasonication thalline, release zymoprotein, centrifugal by liquid cryogen, removes bacterial sediment, obtains the supernatant liquor containing GPX2 mutant, with gsh (GSH) affinitive layer purification GPX, after dialysis freeze-drying, namely obtain zymoprotein sterling.

The method introduces catalytic group by inducing auxotroph prokaryotic expression system to combine the single protein production systems of SPP under low temperature at the substrate binding site of GPX2 mutant, thus produces the GPX2 mutant protein of high vigor.

Described uses gsh (GSH) affinitive layer purification GPX2 mutant, is by pH7.5,50mmol/L Tris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

The third method of the present invention first can express GPX2 mutant protein gene of the present invention at biotech firm's DNA synthesizer synthetic, then prepares GPX2 mutant protein with auxotroph prokaryotic expression system.

1), the structure of expression vector:

According to the aminoacid sequence of GPX2 mutant of the present invention, the gene of GPX2 mutant protein can be expressed in auxotrophic strain at biotech firm's DNA synthesizer synthetic, guarantee that 5 ' end of target gene is containing initiator codon (ATG), 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 40 seleno-cysteines (SeCys) of target gene replaces with the codon (can be TGC etc.) of halfcystine (Cys); Concrete gene order can be (see NCBI by GPX2 gene, NM_002083.3) in, the encoding sequence of halfcystine of the 55th, 68,77 and 105 replaces with the codon of Serine, the encoding gene that the codon that the codon of the 40th seleno-cysteine replaces with halfcystine obtains, also can be the encoding gene (due to the degeneracy of codon, multiple different encoding gene can be had with amino acid sequence) that other any one can express GPX2 mutant protein in auxotrophic strain; Contain GPX2 mutant gene and the secretor type prokaryotic expression carrier (as pCold series etc.) of specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type prokaryotic expression carrier with DNA ligase; Described specific restriction enzyme site can be in the multiple clone site of expression vector containing and any one restriction enzyme site non-existent in GPX2 mutant gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector containing GPX2 mutant gene built in step 1), be coated with the nutrient agar plate containing Cys, screening positive transformant; Positive transformant is spread cultivation after supporting, in the substratum containing SeCys, essential growth factor and nutrient substance, induce through isopropylthio-β-D-galactoside (IPTG), auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, finally directly give expression to the GPX2 mutant protein containing SeCys at the combining site of substrate GSH, zymoprotein is expressed with soluble form and is secreted in the pericentral siphon chamber of thalline; First cultivation screening high expression level strain in a small amount, then enlarged culturing and abduction delivering; Under collection, washing, ice bath, ultrasonication thalline, release zymoprotein, centrifugal by liquid cryogen, removes bacterial sediment, obtains supernatant liquor; With gsh (GSH) affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain GPX2 mutant protein sterling.

The method introduces catalytic group by transgenation and auxotroph prokaryotic expression system at the substrate binding site of GPX2 mutant, thus produces the GPX2 mutant protein of high vigor.

4th kind of method of the present invention is the GPX2 gene that first increases, then with it for template, obtain by transgenation method and can express the gene of GPX2 mutant protein of the present invention, then prepare GPX2 mutant protein with auxotroph prokaryotic expression system.

1), the structure of expression vector:

Disclosed in gene library, the gene order of GPX2 is (see NCBI, NM_002083.3) primer is designed, increase its encoding gene, when designing primer, guarantee that 5 ' end of gene is containing initiator codon (ATG), 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends; After identical restriction endonuclease cut vector and target gene, then by specific restriction enzyme site, GPX2 genome is installed to (as pCold series etc.) on secretor type prokaryotic expression carrier with DNA ligase; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport 40 SeCys of halfcystine (Cys) and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer; With rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit (Invitrogen company, by the operation of test kit specification sheets), the sequence encoding mutant of the SeCys in the GPX2 gene be structured on prokaryotic expression carrier is become the codon (such as TGC) of Cys; In like manner, by the method for above-mentioned rite-directed mutagenesis, the sequence encoding mutant of other halfcystine in GPX2 gene is become the codon of Serine; Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee that the gene after suddenling change can express GPX2 mutant protein of the present invention in auxotrophic strain; Described specific restriction enzyme site can be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

Lung biopsy: prepare GPX2 mutant protein with the target gene synthesized and mammalian cell expression system

1), the structure of expression vector:

According to the aminoacid sequence of GPX2 mutant of the present invention, encoding gene in biotech firm with DNA synthesizer synthetic GPX2 mutant, guarantee that 5 ' end of gene is containing initiator codon (ATG) and specific restriction enzyme site, 3 ' holds whole base sequences of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A, concrete sequence is see NCBI, NM_002083.3) or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site, concrete gene order can be (see NCBI by GPX2 gene, NM_002083.3) in the 55th, 68, the encoding gene that the codon that the encoding sequence of the halfcystine of 77 and 105 replaces with Serine obtains, also can be that the gene of other any one coding GPX2 mutant protein is (due to the degeneracy of codon, multiple different encoding gene can be had) with amino acid sequence, but 3 ' of target gene end must hold whole base sequences of non-translational region (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A containing 3 ' of GPX2 gene, concrete sequence is see NCBI, or selenocysteine insertion sequence (SECIS) NM_002083.3), with identical restriction endonuclease cutting two ends containing the GPX2 mutant gene of specific restriction enzyme site and cells of mamma animals expression vector (as pSecTag2A etc., Invitrogen company), then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence with DNA ligase, according to selenocysteine insertion sequence associated proteins 2(SBP2 in gene library, see NCBI, NM_024077.3) 512 amino acid whose gene orders of carboxyl terminal 343-854 position are at biotech firm's its encoding gene of DNA synthesizer synthetic, guarantee that 5 ' end of gene is containing initiator codon (ATG) and specific restriction enzyme site, 3 ' end is containing terminator codon and specific restriction enzyme site, with specific restriction enzyme site, SBP2 genome is installed on born of the same parents' inner mold cells of mamma animals expression vector (as pcDNA3.1/myc-His A etc., Invitrogen company), described specific restriction enzyme site can be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier,

2), the screening of positive cell strain:

With the cotransfection cells of mamma animals under the mediation of transfection reagent of the carrier containing SBP2 and GPX2 mutant gene, 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, again with porous culture plate amplification culture positive colony step by step, the final positive cell strain obtaining Simultaneous Stabilization expression SBP2 and GPX2 mutant two kinds of foreign proteins; Detect the expression amount of GPX2 mutant protein and screen the high cell strain of target protein expression amount;

3), the expression and purification of target protein:

The cell strain amplification culture of two kinds of foreign proteins will be expressed in shaking flask simultaneously, GPX2 mutant is being expressed with cells of mamma animals containing in the substratum of Sodium Selenite, zymoprotein is secreted in substratum with soluble form, with gsh (GSH) affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain zymoprotein sterling.

The expression amount of described detection GPX2 mutant protein, can detect by the polyclonal antibody of anti-GPX2 and ELISA or GPX vitality test technology;

Described uses gsh GSH affinitive layer purification GPX2 mutant protein, is by pH7.5,50mmol/L Tris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

6th kind of method of the present invention is the carrier of substep containing SBP2 gene and the same cells of mamma animals of carrier transfection containing GPX2 mutant gene.

1), the structure of expression vector:

According to the encoding gene of aminoacid sequence in biotech firm with DNA synthesizer synthetic GPX2 mutant of GPX2 mutant of the present invention, guarantee that 5 ' end of gene is containing initiator codon (ATG) and specific restriction enzyme site, 3 ' holds whole base sequences of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A, concrete sequence is see NCBI, NM_002083.3) or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site, concrete gene order can be (see NCBI by GPX2 gene, NM_002083.3) in the 55th, 68, the encoding gene that the codon that the encoding sequence of the halfcystine of 77 and 105 replaces with Serine obtains, also can be that the gene of other any one coding GPX2 mutant protein is (due to the degeneracy of codon, multiple different encoding gene can be had) with amino acid sequence, but 3 ' of target gene end must hold whole base sequences of non-translational region (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A containing 3 ' of GPX2 gene, concrete sequence is see NCBI, or selenocysteine insertion sequence (SECIS) NM_002083.3), with identical restriction endonuclease cutting two ends containing the GPX2 mutant gene of specific restriction enzyme site and cells of mamma animals expression vector (as pSecTag2A etc., Invitrogen company), then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence with DNA ligase, according to selenocysteine insertion sequence associated proteins 2(SBP2 in gene library, 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 ' end of gene is containing initiator codon (ATG) and specific restriction enzyme site, 3 ' end is containing terminator codon and specific restriction enzyme site, with specific restriction enzyme site, SBP2 genome is installed on born of the same parents' inner mold cells of mamma animals expression vector (as pcDNA3.1/myc-His A etc., Invitrogen company), described specific restriction enzyme site can be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier,

2), the screening of positive cell strain:

First with the carrier transfection cells of mamma animals containing SBP2 gene, by the cell strain of the resistant gene screening stably express SBP2 on this carrier.Again with the cell strain of the carrier transfection stably express SBP2 containing GPX2 mutant gene, express the cell strain of SBP2 and GPX2 mutant two kinds of foreign proteins with the resistant gene screening Simultaneous Stabilization on two carriers; Detect the expression amount of GPX2 mutant protein and screen the high cell strain of target protein expression amount;

3), the expression and purification of target protein:

To express the cell strain amplification culture of two kinds of foreign proteins in shaking flask, expressing GPX2 mutant containing in the substratum of Sodium Selenite with cells of mamma animals, zymoprotein is secreted in substratum with soluble form simultaneously; With gsh affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain zymoprotein sterling.

In the screening of positive cell strain, the screening of described positive cell strain, also can first transfection GPX2 mutant gene, rear transfection SBP2 gene, then the cell strain of expressing SBP2 and GPX2 mutant two kinds of foreign proteins with the resistant gene screening Simultaneous Stabilization on two carriers.The expression amount of described detection GPX2 mutant protein, can detect by the polyclonal antibody of anti-GPX2 and ELISA or GPX vitality test technology.

Described uses gsh affinitive layer purification GPX2 mutant, is by pH7.5,50mmol/L Tris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

7th kind of method: prepare GPX2 mutant protein with the target gene increased and mammalian cell expression system

1), the structure of expression vector:

According to GPX2 gene order in gene library (see NCBI, NM_002083.3) encoding gene of primer amplification GPX2 and the base sequence of 3 ' end non-translational region thereof is designed, when designing primer, guarantee that 5 ' end of target gene is containing initiator codon (ATG) and specific restriction enzyme site, 3 ' of target gene holds whole base sequences of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A, concrete sequence is see NCBI, or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site NM_002083.3), 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 is had (as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), then by specific restriction enzyme site, GPX2 gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region with DNA ligase, keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit, the sequence encoding mutant of the whole halfcystines in the GPX2 gene be structured on carrier for expression of eukaryon is become the codon of Serine, determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene encodes GPX2 mutant protein of the present invention after suddenling change.According to selenocysteine insertion sequence associated proteins 2(SBP2 in gene library, see NCBI, NM_024077.3) 512 amino acid whose its encoding genes of gene order design primer amplification of the 343-854 position of carboxyl terminal, guarantee that 5 ' end of gene is containing initiator codon (ATG) and specific restriction enzyme site, 3 ' end is containing terminator codon and specific restriction enzyme site; SBP2 genome installs on born of the same parents' inner mold cells of mamma animals expression vector (as pcDNA3.1/myc-His A etc., Invitrogen company) with specific restriction enzyme site by same cutting after connection through enzyme; Described specific restriction enzyme site can be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

8th kind of method of the present invention is the carrier of substep containing SBP2 gene and the same cells of mamma animals of carrier transfection containing GPX2 mutant gene.

1), the structure of expression vector:

According to GPX2 gene order in gene library (see NCBI, NM_002083.3) encoding gene of primer amplification GPX2 and the base sequence of 3 ' end non-translational region thereof is designed, when designing primer, guarantee that 5 ' end of target gene is containing initiator codon (ATG) and specific restriction enzyme site, 3 ' of target gene holds whole base sequences of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A, concrete sequence is see NCBI, or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site NM_002083.3), 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 is had (as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), then by specific restriction enzyme site, GPX2 gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region with DNA ligase, keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit, the sequence encoding mutant of the whole halfcystines in the GPX2 gene be structured on carrier for expression of eukaryon is become the codon of Serine, determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee that the gene after suddenling change can be encoded GPX2 mutant protein of the present invention.According to selenocysteine insertion sequence associated proteins 2(SBP2 in gene library, see NCBI, NM_024077.3) 512 amino acid whose its encoding genes of gene order design primer amplification of the 343-854 position of carboxyl terminal, guarantee that 5 ' end of gene is containing initiator codon (ATG) and specific restriction enzyme site, 3 ' end is containing terminator codon and specific restriction enzyme site; SBP2 genome installs on born of the same parents' inner mold cells of mamma animals expression vector (as pcDNA3.1/myc-His A etc., Invitrogen company) with specific restriction enzyme site by same cutting after connection through enzyme; Described specific restriction enzyme site can be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

2), the screening of positive cell strain:

First with the carrier transfection cells of mamma animals containing SBP2 gene, by the cell strain of the resistant gene screening stably express SBP2 on this carrier; Again with the cell strain of the carrier transfection stably express SBP2 containing GPX2 mutant gene, express the cell strain of SBP2 and GPX2 mutant two kinds of foreign proteins with the resistant gene screening Simultaneous Stabilization on two carriers; Detect the expression amount of GPX2 mutant protein and screen the high cell strain of target protein expression amount;

3), the expression and purification of target protein:

9th kind of method of the present invention is identical with first method, when being the encoding gene of synthesis GPX2 mutant, the encoding sequence of the 117th L-Ala is replaced with the codon of Serine, the 117th of the GPX2 mutant protein finally obtained is Serine (SEQ ID No:2), instead of L-Ala, other step is identical with first method.

Of the present invention ten kind of method is identical with first method, when being the encoding gene of synthesis GPX2 mutant, the encoding sequence of the 77th Serine is replaced with the codon of L-Ala, the 77th of the GPX2 mutant protein finally obtained is L-Ala (SEQ ID No:3), instead of Serine, other step is identical with first method.

11 kind of method of the present invention is identical with first method, just owing to employing the pColdI(TAKARA company being convenient to target protein purifying) etc. secretor type protokaryon or carrier for expression of eukaryon and introduce any one novel GPX2 mutant that Histidine purification tag on this carrier and other amino acid etc. formed at the aminoterminal of target protein or carboxyl terminal.Such as introduce pColdI(TAKARA company at the aminoterminal of sequence SEQ ID No:1, SEQ ID No:2, SEQ ID No:3) the sequence SEQ ID No:4, the SEQ ID No:5 that are formed after the Histidine purification tag of prokaryotic expression carrier and the amino acid of factor Xa cleavage site, SEQ ID No:6.The present invention has following characteristics:

(1) the present invention uses the genetic engineering technique such as simple gene chemical synthesis or amplification, transgenation and expression, and preparation method is simple.

(2) the GPX2 mutant protein vigor prepared of the inventive method is high, and reached the same order of magnitude of natural GPX vigor, Cell Biology Experiment has confirmed there is stronger provide protection to the myocardial cell of Hydroperoxide injury.

(3) GPX2 mutant protein of the present invention has superpower stability, not easy in inactivation, this feature supplements defect of natural GPX.

(4) the secreted expression carrier of the present invention's use, GPX2 mutant protein can be expressed with soluble form and be secreted into the external of colibacillary pericentral siphon chamber or cells of mamma animals, the targeting signal peptide of pilot protein secreting, expressing is excised automatically by thalline or cell, expressed albumen is solubility, there is the space conformation of native protein and the activity of Geng Gao, do not need renaturation, the productive rate that renaturing inclusion bodies process can be avoided to cause declines and inactivation, thus with short production cycle.

(5) the SPP low temperature expression system that the present invention uses can utilize the identification of MazF protease specificity and the characteristic of the mRNA of cutting containing ACA sequence, make thalline can only without the protein of ACA sequence in composite coding gene, therefore the albumen mainly foreign protein of new synthesis, thus raising productive rate and Cys are to the transformation efficiency of SeCys, therefore have the double dominant that vigor is high, productive rate is high.

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

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

Accompanying drawing explanation

Fig. 1: on the SDS-PAGE(of the GPX2 mutant protein that purifying obtains) and Western blot(under) result: wherein M to be molecular weight of albumen Marker, 1-11 swimming lane be target protein prepared by embodiment 1 ~ 11.

Embodiment

Embodiment 1: prepare genetically engineered people GPX2 mutant protein with the gene association SPP synthesized and auxotroph prokaryotic expression system

According to sequence 1(SEQ ID No:1 of the present invention) described in the aminoacid sequence of GPX2 mutant, biotech firm with DNA synthesizer synthetic can in auxotrophic strain expressed sequence 1(SEQ ID No:1) described in the gene of GPX2 mutant protein, guarantee that 5 ' end of GPX2 mutant gene is containing initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end is containing terminator codon and Hind III digestion site, the encoding sequence TGA of No. 40 SeCys of GPX2 mutant gene replaces with the codon (TGC) of Cys, and full length gene is not containing ACA sequence, concrete target-gene sequence is (see NCBI by GPX2 gene, NM_002083.3) in the 55th, 68, the codon of the halfcystine of 77 and 105 replaces with the encoding sequence of Serine (successively: AGC, AGC, AGT, AGT), the codon of the 40th seleno-cysteine replaces with the encoding sequence (TGC) of halfcystine, again according to the degeneracy of codon, under the prerequisite not changing aminoacid sequence, ACA series jumps all in GPX2 gene are fallen, obtain the GPX2 mutant gene without ACA sequence, guarantee this gene encodes sequence 1(SEQ ID of the present invention No:1) described in GPX2 mutant protein, and 5 ' of target gene end is containing initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end is containing terminator codon and Hind III digestion site, after restriction endonuclease Nde I and Hind III double digestion, be connected to pCold III(TAKARA, Cat.#3369 that same enzyme is cut) on carrier.

Auxotrophic E. coli BL21(DE3 is transformed with the carrier (pCold III-GPX2 dashes forward) that GPX2 mutant gene is housed) Cys, be coated with the nutrient agar plate containing Cys, screening positive strain.Again by 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 containing 100 μ g/mL Amp, 25 μ g/mLKana and 25 μ g/mL and screens positive transformant.Positive transformant is inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.5.Bacterium liquid is placed in-20 DEG C of refrigerator 5min, bacterium liquid is cooled rapidly, afterwards bacterium liquid is placed in 15 DEG C of relaying persistent oscillations and cultivates 45min.15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant.With the resuspended bacterial sediment of 0.9%NaCl, 15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant, repeat this step.Then productive culture base (adding the 19 seed amino acid each 50-400 μ g/ml except Cys in M9 substratum) resuspended thalline is used, to add final concentration be 1mmol/L IPTG and final concentration is 200 μ g/mL SeCys, 15 DEG C of shaking culture 16-72h, GPX2 mutant protein is expressed in the pericentral siphon chamber of thalline with soluble form, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein.Collect thalline (6000rpm, 10min) with isopyknic bufferT(50mM Tris buffer, pH7.5, containing 1mM EDTA) wash twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collects supernatant.By specification process GSH affinity post, application damping fluid (50mmol/L Tris, pH7.5) balance, adds on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein by supernatant liquor.By elutriant dialysis also freeze-drying, obtain people GPX2 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and is combined with anti-GPX2 antibodies specific, proves to have successfully been obtained target protein, sees the 1st swimming lane of Fig. 1.Its GPX vigor is 2636U/ μm of ol, reaches the same order of magnitude of natural GPX.

The SPP low temperature expression system that the present embodiment uses can utilize the identification of MazF protease specificity and the characteristic of the mRNA of cutting containing ACA sequence, make thalline can only without the protein of ACA sequence in composite coding gene, therefore the albumen mainly foreign recombinant proteins of new synthesis, thus raising productive rate and Cys are to the transformation efficiency of SeCys, therefore have high high yield double dominant of living.

Embodiment 2: combine auxotroph prokaryotic expression system with gene amplification and sudden change method and SPP and prepare genetically engineered people GPX2 mutant protein

With mRNA Mini Kit (Sigma, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, mRNA is extracted, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist under, be transcribed into cDNA by RT-PCR (RT-polymerase chain reaction).Disclosed in gene library, the gene of people GPX2 is (see NCBI, NM_002083.3) primers increases its encoding gene, guarantee that 5 ' end of gene is containing initiator codon (ATG) and NdeI restriction enzyme site, 3 ' end is containing terminator codon and Hind III digestion site; 5 ' concrete end primer is 5 '-GGAATTC CATATGGCTTTCATTGCCAAGTCCT-3 ', and 3 ' end primer is 5 '-CGG AAGCTTCTATATGGCAACTTTAAGGAGG-3 '.After restriction endonuclease Nde I and Hind III double digestion, be connected to DNA ligase pCold III(TAKARA, the Cat.#3369 that same enzyme cuts) on carrier.Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers of the complete isometric complementation of amino acid whose gene order design near No. 49 SeCys, the long 25-50bp of primer, centered by the codon of No. 40 SeCys (TGA), the sequence of sense primer is 5 '-GAATGTGGCTTCGCTCTGCGGCACGACCACCCGGGAC-3 '.With primer and Fast Fixed-point mutagenesis kit (the Invitrogen company of these two complete complementaries, by the operation of test kit specification sheets), the encoding sequence TGA of the GPX2 gene be structured on prokaryotic expression carrier pCold III No. 40 SeCys is mutated into the codon (TGC) of Cys, determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation.With same method, the codon mutation of other all Cys in people GPX2 gene is become the codon of Ser; Corresponding sense primer is respectively: C55S5 '-GCTCAACGAGCTGCAAT ccCGCTTTCCCAGGCGCCTG-3 ', C68S5 '-GTGGTCCTTGGCTTCCCTT ccAACCAATTTGGCCATCAGGAG-3 ', C77S 5 '-CAGGAGAACT ctCAGAATGAGGAGATCCTGAA taGTCTCAAGTATG-3 ', C105S5 '-CTTCACCCTTGTCCAAAAAT ctGAGGTGAATGGGCAGAAC-3 '.Finally sudden change under the prerequisite that GPX2 variant amino acid sequence is constant is being kept to fall all ACA sequences in gene, all T is sported by the 363rd, 507,531 and No. 543 base C, share 3 primers, its Sense sequences is 5 '-GTCTTCGCCTACCTGAAGGA respectively taAGCTCCCCTACCCTTATGATG-3 ', 5 '-GGAGAGCCCTTCCGACGCTA taGCCGCACCTTCCCAACC-3 ', 5 '-CCTTCCCAACCATCAATATTGAGCCTGA taTCAAGCGCCTCC-3 '; Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene encodes sequence 1(Seq1 of the present invention after suddenling change) described in GPX2 mutant protein.

Auxotrophic E. coli BL21(DE3 is transformed with the carrier (pCold III-GPX2 dashes forward) that GPX2 mutant gene is housed) Cys, be coated with the nutrient agar plate containing Cys, screening positive strain.Again by 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 containing 100 μ g/mL Amp, 25 μ g/mLKana and 25 μ g/mL and screens positive transformant.Positive transformant is inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.5.Bacterium liquid is placed in-20 DEG C of refrigerator 5min, bacterium liquid is cooled rapidly, afterwards bacterium liquid is placed in 15 DEG C of relaying persistent oscillations and cultivates 45min.15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant.The resuspended bacterial sediment of 0.9%NaCl, 15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant, repeat this step.Then productive culture base (adding the 19 seed amino acid each 50-400 μ g/ml except Cys in M9 substratum) resuspended thalline is used, to add final concentration be 1mmol/LIPTG and final concentration is 200 μ g/mL SeCys, 15 DEG C of shaking culture 16-72h, GPX2 mutant protein is expressed in the pericentral siphon chamber of thalline with soluble form, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein.Collect thalline (6000rpm, 10min) with isopyknic bufferT(50mM Tris buffer, pH7.5, containing 1mM EDTA) wash twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collects supernatant.By specification process GSH affinity post, application damping fluid (50mmol/LTris, pH7.5) balance, adds on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein by supernatant liquor.By elutriant dialysis also freeze-drying, obtain people GPX2 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and is combined with anti-GPX2 antibodies specific, proves to have successfully been obtained target protein, sees the 2nd swimming lane of Fig. 1.Its GPX vigor is 2360U/ μm of ol, reaches the same order of magnitude of natural GPX.

Embodiment 3: prepare genetically engineered people GPX2 mutant protein with the gene synthesized and auxotroph prokaryotic expression system

According to sequence 1(SEQ ID No:1 of the present invention) described in the aminoacid sequence of GPX2 mutant, biotech firm with DNA synthesizer synthetic can in auxotrophic strain expressed sequence 1(SEQ ID No:1) described in the gene of GPX2 mutant protein, guarantee that 5 ' end of GPX2 mutant gene is containing initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end is containing terminator codon and Hind III digestion site, and the encoding sequence TGA of No. 49 SeCys of GPX2 mutant gene replaces with the codon (TGC) of Cys, concrete target-gene sequence is (see NCBI by GPX2 gene, NM_002083.3) in the 55th, 68, the codon of the halfcystine of 77 and 105 replaces with the encoding sequence of Serine (successively: AGT, AGC, AGC, AGT, AGT), what the codon of the 49th seleno-cysteine replaced with that the encoding sequence (TGC) of halfcystine obtains can code book invention sequence 1(SEQ ID No:1) described in the gene of GPX2 mutant protein, and 5 ' of target gene end is containing initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end is containing terminator codon and Hind III digestion site, after restriction endonuclease Nde I and HindIII double digestion, be connected to pCold III(TAKARA, Cat.#3369 that same enzyme is cut) on carrier.

Auxotrophic E. coli BL21(DE3 is transformed with the carrier (pCold III-GPX2 dashes forward) that GPX2 mutant gene is housed) Cys, be coated with the nutrient agar plate containing Cys, screening positive transformant.Positive transformant is inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.1, be cultured to OD600 and be about 0.3-1, add the IPTG of final concentration 0.1-1mM, after 10min, add the paraxin of final concentration 10 μ g/ml.Add paraxin medium centrifugal after five minutes, centrifugal 5 minutes of low temperature 6000rpm, twice is washed with the physiological salt solution of ice bath, each 1.2L, then use productive culture base (adding the 19 seed amino acid each 50-400 μ g/ml except Cys in M9 substratum) resuspended, and in substratum, supplement Rifampin and 600 μMs of SeCys of final concentration 400 μ g.Continue to cultivate 2-12h, GPX2 is expressed in the pericentral siphon chamber of thalline with soluble form.Collect thalline (6000rpm, 10min) with isopyknic bufferT(50mM Tris buffer, pH7.5, containing 1mM EDTA) wash twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collects supernatant.By specification process GSH affinity post, application damping fluid (50mmol/L Tris, pH7.5) balance, adds on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein by supernatant liquor.By elutriant dialysis also freeze-drying, obtain people GPX2 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and is combined with anti-GPX2 antibodies specific, proves to have successfully been obtained target protein, sees the 3rd swimming lane of Fig. 1.Its GPX vigor is 512U/ μm of ol, reaches the order of magnitude level of natural GPX.

Auxotrophic E. coli BL21(DE3) Cys derives from exercise question for the article of " Structure of the Cathelicidin Motif ofProtegrin-3Precursor:Structural Insights into the Activation Mechanism of an Antimicrobial Protein ", within 2002, be published in Structure the 10th volume, 1363 – 1370 pages.The acquisition of this bacterial classification can be granted by author or professor AugustBock.

Embodiment 4: prepare genetically engineered people GPX2 mutant protein with gene amplification and sudden change method and auxotroph prokaryotic expression system

With mRNA Mini Kit (Sigma, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, mRNA is extracted, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist under, be transcribed into cDNA by RT-PCR (RT-polymerase chain reaction).Disclosed in gene library, the gene of people GPX2 is (see NCBI, NM_002083.3) primers increases its encoding gene, guarantee that 5 ' end of gene is containing initiator codon (ATG) and NdeI restriction enzyme site, 3 ' end is containing terminator codon and Hind III digestion site; 5 ' concrete end primer is 5 '-GGAATTC CATATGGCTTTCATTGCCAAGTCCT-3 ', and 3 ' end primer is 5 '-CGG AAGCTTCTATATGGCAACTTTAAGGAGG-3 '.After restriction endonuclease Nde I and Hind III double digestion, be connected to DNA ligase pCold III(TAKARA, the Cat.#3369 that same enzyme cuts) on carrier.Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers of the complete isometric complementation of amino acid whose gene order design near No. 40 SeCys, the long 25-50bp of primer, centered by the codon of No. 40 SeCys (TGA).With primer and Fast Fixed-point mutagenesis kit (the Invitrogen company of these two complete complementaries, by the operation of test kit specification sheets), the encoding sequence TGA of the GPX2 gene be structured on prokaryotic expression carrier pCold III No. 40 SeCys is mutated into the codon (TGC) of Cys, determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, with same method, the sequence encoding mutant of other all Cys in people GPX2 gene is become the codon of Ser, it is the codon (TCC) of Ser by the sequence encoding mutant of 117 Ala, corresponding sense primer is respectively: C55S 5 '-GCTCAACGAGCTGCAAT ccCGCTTTCCCAGGCGCCTG-3 ', C68S5 '-GTGGTCCTTGGCTTCCCTT ccAACCAATTTGGCCATCAGGAG-3 ', C77S 5 '-CAGGAGAACT ctCAGAATGAGGAGATCCTGAA taGTCTCAAGTATG-3 ', C105S5 '-CTTCACCCTTGTCCAAAAAT ctGAGGTGAATGGGCAGAAC-3 ' and A117S5 '-GAACGAGCATCCTGTCTTC tcCTACCTGAAGGATAAG-3 '.Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene encodes sequence 2(SEQ ID of the present invention No:2 after suddenling change) described in GPX2 mutant protein.

Auxotrophic E. coli BL21(DE3 is transformed with the carrier (pCold III-GPX2 dashes forward) that GPX2 mutant gene is housed) Cys, be coated with the nutrient agar plate containing Cys, screening positive transformant.Positive transformant is inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.1, be cultured to OD600 and be about 0.3-1, add the IPTG of final concentration 0.1-1mM, after 10min, add the paraxin of final concentration 10 μ g/ml.Add paraxin medium centrifugal after five minutes, centrifugal 5 minutes of low temperature 6000rpm, twice is washed with the physiological salt solution of ice bath, each 1.2L, then use productive culture base (adding the 19 seed amino acid each 50-400 μ g/ml except Cys in M9 substratum) resuspended, and in substratum, supplement Rifampin and 600 μMs of SeCys of final concentration 400 μ g.Continue to cultivate 2-12h, GPX2 is expressed in the pericentral siphon chamber of thalline with soluble form.Collect thalline (6000rpm, 10min) with isopyknic bufferT(50mM Tris buffer, pH7.5, containing 1mM EDTA) wash twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collects supernatant.By specification process GSH affinity post, application damping fluid (50mmol/L Tris, pH7.5) balance, adds on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein by supernatant liquor.By elutriant dialysis also freeze-drying, obtain people GPX2 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and is combined with anti-GPX2 antibodies specific, proves to have successfully been obtained target protein, sees the 4th swimming lane of Fig. 1.Its GPX vigor is 507U/ μm of ol, reaches the order of magnitude level of natural GPX.The GPX2 mutant of the aminoacid sequence of (present method also can be used for preparation 87 Ala do not sport Serine namely there is sequence 1(SEQ ID No:1), concrete grammar except save the sequence encoding mutant of 87 Ala be Ser this step of codon except, other is identical with this law.）

Embodiment 5: prepare people GPX2 mutant protein with the gene synthesized and the transfection of mammalian cell expression system one step

1. the structure of people GPX2 mutant protein expression vector: according to sequence 1(SEQ ID No:1 of the present invention) described in the aminoacid sequence of GPX2 mutant at biological reagent company DNA synthesizer synthetic GPX2 mutant gene, guarantee that 5 ' end of gene is containing initiator codon (ATG) and Hind III digestion site, 3 ' hold people GPX2 mutant gene terminator codon downstream GPX2 gene 3 ' end non-translational region whole base sequences (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A, concrete sequence is see NCBI, NM_002083.3), and BamHI restriction enzyme site is inserted after the previous base of poly oligonucleotide A of GPX2 gene, concrete target-gene sequence is (see NCBI by GPX2 gene, NM_002083.3) in the 55th, 68, the codon of the halfcystine of 77 and 105 replaces with the encoding sequence of Serine (successively: AGC, AGC, AGT, what AGT) obtain can code book invention sequence 1(SEQ ID No:1) described in the gene of GPX2 mutant protein, and whole base sequences of the 3 ' end non-translational region of 3 ' of target gene end containing GPX2 gene are (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A, concrete sequence is see NCBI, NM_002083.3), and BamHI restriction enzyme site is inserted after the previous base of poly oligonucleotide A of GPX2 gene.Cut latter linked method with first enzyme, by Hind III/BamHI restriction enzyme site, GPX2 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 associated proteins 2(SBP2 in gene library; NCBI; NM_024077.3) 512 amino acid whose gene orders of carboxyl terminal 343-854 position are at biotech firm's its encoding gene of DNA synthesizer synthetic; guarantee that 5 ' end of gene is containing initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end is containing terminator codon and EcoRI restriction enzyme site.Cut latter linked method with first enzyme, carboxyl terminal (343-854aa) genome of SBP2 installed on born of the same parents' inner mold cells of mamma animals expression vector pcDNA3.1/myc-HisA (Invitrogen company) that same enzyme cuts by XbaI/EcoRI restriction enzyme site.

2. the screening of people GPX2 mutant gene positive cell strain: when Growth of Cells is to 1 × 10 ⁶during the density of/ml, with the expression vector containing SBP2 and GPX2 mutant gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower cotransfection 293F cell (Invitrogen of mediation, R79007), cell proceeded in culture dish in after transfection 72 hours and start adherent culture, the usual 2-50 of adherent culture 48() hour after change nutrient solution containing G418 and Zeocin, screening has the positive cell clone of two kinds of resistances and Simultaneous Stabilization expression SBP2 and GPX2 mutant two kinds of foreign proteins, period G418 and Zeocin concentration are progressively successively decreased from 800-200 μ g/ml and 400-100 μ g/ml respectively, subtract 200 μ g/ml and 100 μ g/ml respectively at every turn, each concentration uses 5 days, within 2-3 days, change a nutrient solution, screening and culturing after 20 days by positive cell clone with 96, 48, 24, 12 and 6 well culture plates amplification culture step by step.Detect the expression amount of GPX2 mutant protein with anti-GPX2 polyclonal antibody and elisa technique, select the high cell strain of target protein expression amount and support with frozen for spreading cultivation.

3. the expression and purification of people GPX2 mutant protein: the 293F containing 1-10 μM of Sodium Selenite at Large Copacity expresses in substratum the cell strain that the Simultaneous Stabilization that screens acquisition in foster 2 of spreading cultivation expresses SBP2 and GPX2 mutant two kinds of foreign proteins.In SBP2, SECIS and 293 cells other protein factor common participation under, GPX2 mutant protein is expressed with soluble form and is secreted in substratum, every 48 hr collections nutrient solution.Nutrient solution, after concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, with GSH affinity chromatography column purification GPX2 mutant protein, collects the elutriant containing GPX4, and dialysis removing small-molecule substance, namely freeze-drying obtains people GPX2 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and is combined with anti-GPX2 antibodies specific, proves to have successfully been obtained target protein, sees the 5th swimming lane of Fig. 1.Its GPX vigor is 2653U/ μm of ol, reaches the same order of magnitude of natural GPX.

Embodiment 6: prepare people GPX2 mutant protein with the gene synthesized and the transfection of mammalian cell expression system substep

2. the screening of people GPX2 mutant gene positive cell strain: when Growth of Cells is to 1 × 10 ⁶during the density of/ml, with the expression vector containing SBP2 gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower transfection 293F cell (Invitrogen of mediation, R79007), cell proceeded in culture dish in after transfection 72 hours and start adherent culture, the usual 2-50 of adherent culture 48() hour after change nutrient solution containing G418, screening has the positive cell clone of G418 resistance and stably express SBP2, period G418 concentration is progressively decremented to 200 μ g/ml from 800 μ g/ml, subtract 200 μ g/ml at every turn, each concentration uses 5 days, within 2-3 days, change a nutrient solution, screening and culturing after 20 days by positive cell clone with 96, 48, 24, 12 and 6 well culture plates amplification culture step by step.Again with containing the expression vector of GPX2 mutant gene at lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the 293F cell (Invitrogen of the lower transfection stably express SBP2 of mediation, R79007), cell proceeded in culture dish in after transfection 72 hours and start adherent culture, the usual 2-50 of adherent culture 48() hour after the liquid medium-selection changed containing G418 and Zeocin there is the positive cell clone that two kinds of resistances and Simultaneous Stabilization express SBP2 and GPX2 mutant two kinds of foreign proteins, period G418 concentration maintains 200 μ g/ml, Zeocin concentration is progressively decremented to 100 μ g/ml from 400 μ g/ml, subtract 100 μ g/ml at every turn, each concentration uses 5 days, within 2-3 days, change a nutrient solution, 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 amplification culture step by step.With the polyclonal antibody of anti-GPX2 and the expression amount of elisa technique detection GPX2 mutant protein, select the high cell strain of target protein expression amount and support with frozen for spreading cultivation.

Aforesaid method also can first transfection GPX2 mutant gene, rear transfection SBP2 gene.

3. the expression and purification of people GPX2 mutant: the 293F containing 1-10 μM of Sodium Selenite at Large Copacity expresses in substratum the cell strain that the Simultaneous Stabilization that screens acquisition in foster 2 of spreading cultivation expresses SBP2 and GPX2 mutant two kinds of foreign proteins.In SBP2, SECIS and 293F cell other protein factor common participation under, GPX2 mutant is expressed with soluble form and is secreted in substratum, every 48 hr collections nutrient solution.Nutrient solution, after concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, with GSH affinity chromatography column purification GPX2 mutant, collects the elutriant containing GPX2 mutant, and dialysis removing small-molecule substance, namely freeze-drying obtains people GPX2 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and is combined with anti-GPX2 antibodies specific, proves to have successfully been obtained target protein, sees the 6th swimming lane of Fig. 1.Its GPX vigor is 2538U/ μm of ol, reaches the same order of magnitude of natural GPX.

Embodiment 7: prepare people GPX2 mutant protein with the target gene increased with the transfection of mammalian cell expression system one step

1. the structure of people GPX2 mutant protein expression vector: with mRNA Mini Kit (Sigma, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, mRNA is extracted, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist under, be transcribed into cDNA by RT-PCR (RT-polymerase chain reaction).Disclosed in gene library, the gene order of people GPX2 is (see NCBI, NM_002083.3) encoding gene of primer amplification GPX2 and the base sequence of 3 ' end non-translational region thereof is designed, when designing primer, guarantee that 5 ' end of target gene is containing initiator codon (ATG) and HindIII restriction enzyme site, 3 ' holds whole base sequences of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream (from first base after the terminator codon of GPX2 gene to the previous base of poly oligonucleotide A, concrete sequence is see NCBI, and EcoRI restriction enzyme site NM_002083.3), the target gene of specific restriction enzyme site and cells of mamma animals expression vector is had (as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), then by specific restriction enzyme site, GPX2 gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region with DNA ligase, 5 ' concrete end primer is 5 '-CCC AAG CTT CAT CAT CAT CAT CAT CAT ATG GCT TTC ATT GCC-3 ', and 3 ' end primer is 5 '-CGGAA TTC GGT CTC TTC TAG CAG AGT G-3 '.Cut latter linked method with first enzyme, by Hind III/EcoRI restriction enzyme site, GPX2 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 prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit, the sequence encoding mutant of the whole halfcystines in the people GPX2 gene be structured on prokaryotic expression carrier is become the codon of Serine; Concrete sense primer is respectively: C55S 5 '-GCTCAACGAGCTGCAAT ccCGCTTTCCCAGGCGCCTG-3 ', C68S5 '-GTGGTCCTTGGCTTCCCTT ccAACCAATTTGGCCATCAGGAG-3 ', C77S 5 '-CAGGAGAACT ctCAGAATGAGGAGATCCTGAA taGTCTCAAGTATG-3 ', C105S5 '-CTTCACCCTTGTCCAAAAAT ctGAGGTGAATGGGCAGAAC-3 '; Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene encodes sequence 1(SEQ ID of the present invention No:1 after suddenling change) described in GPX2 mutant protein.According to human selenocysteine insertion sequence associated proteins 2(SBP2 in gene library, NCBI, NM_024077.3) 512 amino acid whose gene orders of carboxyl terminal 343-854 position, its encoding gene of design primer amplification, guarantee that 5 ' end of gene is containing initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end is containing terminator codon and EcoRI restriction enzyme site; 5 ' concrete end primer is 5 '-GCTCTAGAATGGAAGCTTTATCTTC-3 ', and 3 ' end primer is 5 '-CGGAATTCTAAATTCAAATTC-3 '.Cut latter linked method with first enzyme, carboxyl terminal (343-854aa) genome of SBP2 installed on born of the same parents' inner mold cells of mamma animals expression vector pcDNA3.1/myc-HisA (Invitrogen company) that same enzyme cuts by XbaI/EcoRI restriction enzyme site.

3. the expression and purification of people GPX2 mutant protein: the 293F containing 1-10 μM of Sodium Selenite at Large Copacity expresses in substratum the cell strain that the Simultaneous Stabilization that screens acquisition in foster 2 of spreading cultivation expresses SBP2 and GPX2 mutant two kinds of foreign proteins.In SBP2, SECIS and 293 cells other protein factor common participation under, GPX2 mutant protein is expressed with soluble form and is secreted in substratum, every 48 hr collections nutrient solution.Nutrient solution, after concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, with GSH affinity chromatography column purification GPX2 mutant protein, collects the elutriant containing GPX4, and dialysis removing small-molecule substance, namely freeze-drying obtains people GPX2 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and is combined with anti-GPX2 antibodies specific, proves to have successfully been obtained target protein, sees the 7th swimming lane of Fig. 1.Its GPX vigor is 2631U/ μm of ol, reaches the same order of magnitude of natural GPX.

Embodiment 8: prepare people GPX2 mutant protein with the target gene increased with the transfection of mammalian cell expression system substep

3. the expression and purification of people GPX2 mutant: the 293F containing 1-10 μM of Sodium Selenite at Large Copacity expresses in substratum the cell strain that the Simultaneous Stabilization that screens acquisition in foster 2 of spreading cultivation expresses SBP2 and GPX2 mutant two kinds of foreign proteins.In SBP2, SECIS and 293F cell other protein factor common participation under, GPX2 mutant is expressed with soluble form and is secreted in substratum, every 48 hr collections nutrient solution.Nutrient solution, after concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, with GSH affinity chromatography column purification GPX2 mutant, collects the elutriant containing GPX2 mutant, and dialysis removing small-molecule substance, namely freeze-drying obtains people GPX2 mutant protein.Identify target protein with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and is combined with anti-GPX2 antibodies specific, proves to have successfully been obtained target protein, sees the 8th swimming lane of Fig. 1.Its GPX vigor is 2533U/ μm of ol, reaches the same order of magnitude of natural GPX.

Embodiment 9:

Preparation method is identical with embodiment 1, when being the encoding gene of synthesis GPX2 mutant, the encoding sequence of the 117th L-Ala is replaced with the codon (TCC) of Serine, the 117th of the GPX2 mutant protein finally obtained is Serine, instead of L-Ala, other aminoacid sequence is identical with first method, i.e. sequence 2(SEQ ID No:2 of the present invention) described in GPX2 mutant protein.This mutant molecule amount only has small change, as broad as long in SDS-PAGE and Western Blot result, sees the 9th swimming lane of Fig. 1.But its GPX vigor is 2417U/ μm of ol, a little more than embodiment 1, reach the same order of magnitude of natural GPX.

Embodiment 10:

Preparation method is identical with embodiment 1, when being the encoding gene of synthesis GPX2 mutant, the encoding sequence of the 77th Serine is replaced with the codon of L-Ala, the 77th of the GPX2 mutant protein finally obtained is L-Ala, instead of Serine, other aminoacid sequence is identical with first method, i.e. sequence 3(SEQ ID No:3 of the present invention) described in GPX2 mutant protein.This mutant molecule amount only has small change, as broad as long in SDS-PAGE and Western Blot result, sees the 10th swimming lane of Fig. 1.But its GPX vigor is 2695U/ μm of ol, higher than embodiment 1, reach the same order of magnitude of natural GPX.

Embodiment 11:

Remove expression vector pCold III(TAKARA used for embodiment 1, Cat.#3369) pColdI(TAKARA of band Histidine purification tag is changed into, Cat.#3367), all the other are identical with embodiment 1, namely the most at last obtain sequence 4(SEQ ID No:4 of the present invention) described in GPX2 mutant protein.This mutant introduces 16 exogenous amino acids carrier comprising 6 Histidines at aminoterminal, therefore molecular weight increases to some extent, SDS-PAGE and Western Blot result can be seen, see the 11st swimming lane of Fig. 1, its advantage also can use the affine series of strata target protein of nickel.Its GPX vigor is 2336U/ μm of ol, and a little less than embodiment 1, prove that the vigor of purification tag to albumen does not have a significant effect, vigor reaches the same order of magnitude of natural GPX.

Claims

1. a high vigor Selenoperoxidase GPX2 mutant, its aminoacid sequence is as shown in SEQ ID No:1.

2. the preparation method of high vigor Selenoperoxidase GPX2 mutant according to claim 1, its step is as follows:

1), the structure of expression vector: the aminoacid sequence of GPX2 mutant according to claim 1, the gene of GPX2 mutant protein can be expressed in auxotrophic strain at biotech firm's DNA synthesizer synthetic, guarantee that 5 ' end of target gene is containing initiator codon ATG, 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends, to guarantee in target gene the 55th, 68, the encoding sequence of the halfcystine of 77 and 105 replaces with the codon of Serine, the codon of the 40th seleno-cysteine replaces with the codon of halfcystine, and according to the degeneracy of codon, under the prerequisite not changing aminoacid sequence, ACA sequences all in gene is all replaced with the encoding gene that non-ACA sequence obtains, or other any one can express GPX2 mutant protein in auxotrophic strain, and the encoding gene of total length not containing ACA sequence, contain GPX2 mutant gene and the secretor type prokaryotic expression carrier of specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type prokaryotic expression carrier with DNA ligase, described specific restriction enzyme site be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in GPX2 mutant gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier,

By step 1) the middle competent cell containing expression vector conversion auxotrophic strain-BL21 (DE3) Cys of GPX2 mutant gene built, be coated with the nutrient agar plate containing Cys, screening positive strain; Again by the pMazF Plastid transformation positive strain containing express nucleic acid restriction endonuclease MazF, screen positive transformant with containing Double M9 solid medium; Positive transformant is spread cultivation after supporting, in the substratum containing SeCys, essential growth factor and nutrient substance, through IPTG low temperature 4-25 DEG C of abduction delivering, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX2 mutant containing SeCys at the combining site of substrate GSH, zymoprotein is expressed with soluble form and is secreted in the pericentral siphon chamber of thalline, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein; First cultivation screening high expression level strain in a small amount, then enlarged culturing and abduction delivering; Under collection, washing, ice bath, ultrasonication thalline, release zymoprotein, centrifugal by liquid cryogen, removes bacterial sediment, obtains supernatant liquor; With gsh GSH affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain GPX2 mutant protein sterling;

Described uses gsh GSH affinitive layer purification GPX2 mutant protein, is by pH7.5,50mmol/LTris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH;

Described is centrifugal by liquid cryogen, is at 4 DEG C, the centrifugal 15-30min of 8000-12000g;

Or,

1), the structure of expression vector:

The gene order design primer of GPX2 disclosed in gene library, increase its encoding gene, and when designing primer, guarantee that 5 ' end of gene is containing initiator codon ATG, 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends; After identical restriction endonuclease cut vector and target gene, then by specific restriction enzyme site, GPX2 genome is installed on secretor type prokaryotic expression carrier with DNA ligase; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport 40 seleno-cysteines of halfcystine and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit, the sequence encoding mutant of the seleno-cysteine in the GPX2 gene be structured on prokaryotic expression carrier is become the codon of halfcystine; In like manner, guaranteeing under the prerequisite not introducing ACA sequence by the method for above-mentioned rite-directed mutagenesis, the sequence encoding mutant of other halfcystine in GPX2 gene is become the codon of Serine, again according to the degeneracy of codon, under the prerequisite not changing aminoacid sequence, ACA series jumps all in GPX2 gene are fallen; Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee that the gene after suddenling change can express GPX2 mutant protein in auxotrophic strain; Described specific restriction enzyme site be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

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

By step 1) the middle competent cell containing expression vector conversion auxotrophic strain-BL21 (DE3) Cys of GPX2 mutant gene built, be coated with the nutrient agar plate containing halfcystine, screening positive strain, again the plasmid pMazF of express nucleic acid restriction endonuclease MazF is transformed positive strain, screen positive transformant with containing Double M9 solid medium, positive transformant is spread cultivation after supporting, containing seleno-cysteine, in the substratum of essential growth factor and nutrient substance, through isopropylthio-β-D-galactoside low temperature 4-25 DEG C of abduction delivering, auxotrophic strain-BL21 (DE3) Cys can synthesize seleno-cysteine with the codon of halfcystine, directly give expression to the restructuring GPX2 mutant containing seleno-cysteine at the combining site of substrate gsh, zymoprotein is expressed with soluble form and is secreted in the pericentral siphon chamber of thalline, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein, first cultivation screening high expression level strain in a small amount, then enlarged culturing and abduction delivering, under collection, washing, ice bath, ultrasonication thalline, release zymoprotein, centrifugal by liquid cryogen, removes bacterial sediment, obtains the supernatant liquor containing GPX2 mutant, with gsh GSH affinitive layer purification GPX, namely obtain zymoprotein after dialysis freeze-drying pure,

Described uses gsh GSH affinitive layer purification GPX2 mutant, is by pH7.5,50mmol/L Tris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH;

Or

1), the structure of expression vector:

The aminoacid sequence of GPX2 mutant according to claim 1, the gene of GPX2 mutant protein can be expressed in auxotrophic strain at biotech firm's DNA synthesizer synthetic, guarantee that 5 ' end of target gene is containing initiator codon ATG, 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 40 seleno-cysteine SeCys of target gene replaces with the codon of halfcystine Cys; Concrete gene order is the codon encoding sequence of halfcystine of the 55th, 68,77 and 105 in GPX2 gene being replaced with Serine, the encoding gene that the codon that the codon of the 40th seleno-cysteine replaces with halfcystine obtains; Or other any one can express the encoding gene of GPX2 mutant protein in auxotrophic strain; Contain GPX2 mutant gene and the secretor type prokaryotic expression carrier of specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type prokaryotic expression carrier with DNA ligase; Described specific restriction enzyme site be in the multiple clone site of expression vector containing and any one restriction enzyme site non-existent in GPX2 mutant gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

By step 1) the middle competent cell containing expression vector conversion auxotrophic strain-BL21 (DE3) Cys of GPX2 mutant gene built, be coated with the nutrient agar plate containing Cys, screening positive transformant; Positive transformant is spread cultivation after supporting, in the substratum containing SeCys, essential growth factor and nutrient substance, induce through isopropylthio-β-D-galactoside IPTG, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, finally directly give expression to the GPX2 mutant protein containing SeCys at the combining site of substrate GSH, zymoprotein is expressed with soluble form and is secreted in the pericentral siphon chamber of thalline; First cultivation screening high expression level strain in a small amount, then enlarged culturing and abduction delivering; Under collection, washing, ice bath, ultrasonication thalline, release zymoprotein, centrifugal by liquid cryogen, removes bacterial sediment, obtains supernatant liquor; With gsh GSH affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain GPX2 mutant protein sterling;

Or

1), the structure of expression vector:

The gene order design primer of GPX2 disclosed in gene library, increase its encoding gene, and when designing primer, guarantee that 5 ' end of gene is containing initiator codon ATG, 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends; After identical restriction endonuclease cut vector and target gene, then by specific restriction enzyme site, GPX2 genome is installed on secretor type prokaryotic expression carrier with DNA ligase; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport 40 SeCys of halfcystine Cys and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer; With rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit, the sequence encoding mutant of the SeCys in the GPX2 gene be structured on prokaryotic expression carrier is become the codon of Cys; In like manner, by the method for above-mentioned rite-directed mutagenesis, the sequence encoding mutant of other halfcystine in GPX2 gene is become the codon of Serine; Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee that the gene after suddenling change can express GPX2 mutant protein in auxotrophic strain; Described specific restriction enzyme site be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

Or

1), the structure of expression vector:

The aminoacid sequence of GPX2 mutant according to claim 1, encoding gene in biotech firm with DNA synthesizer synthetic GPX2 mutant, guarantee that 5 ' end of gene is containing initiator codon ATG and specific restriction enzyme site, 3 ' holds whole base sequence of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream or selenocysteine insertion sequence SECIS and specific restriction enzyme site; Concrete gene order is the encoding gene obtained by the codon that the encoding sequence of halfcystine of the 55th, 68,77 and 105 in GPX2 gene replaces with Serine, or the gene of other any one coding GPX2 mutant protein, but 3 ' of target gene end must hold whole base sequence or the selenocysteine insertion sequence SECIS of non-translational region containing 3 ' of GPX2 gene; Contain GPX2 mutant gene and the cells of mamma animals expression vector of specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence with DNA ligase; According to 512 amino acid whose gene orders of selenocysteine insertion sequence associated proteins 2 carboxyl terminal 343-854 position in gene library at biotech firm's its encoding gene of DNA synthesizer synthetic, guarantee that 5 ' end of gene is containing initiator codon ATG and specific restriction enzyme site, SBP2 genome, containing terminator codon and specific restriction enzyme site, installs on born of the same parents' inner mold cells of mamma animals expression vector with specific restriction enzyme site by 3 ' end; Described specific restriction enzyme site be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

The cell strain amplification culture of two kinds of foreign proteins will be expressed in shaking flask simultaneously, GPX2 mutant is being expressed with cells of mamma animals containing in the substratum of Sodium Selenite, zymoprotein is secreted in substratum with soluble form, with gsh GSH affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain zymoprotein sterling;

The expression amount of described detection GPX2 mutant protein is to detect by the polyclonal antibody of anti-GPX2 and ELISA or GPX vitality test technology;

Or

1), the structure of expression vector:

The encoding gene of aminoacid sequence in biotech firm with DNA synthesizer synthetic GPX2 mutant of GPX2 mutant according to claim 1, guarantee that 5 ' end of gene is containing initiator codon ATG and specific restriction enzyme site, 3 ' holds whole base sequence of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream or selenocysteine insertion sequence SECIS and specific restriction enzyme site; Concrete gene order is the encoding gene obtained by the codon that the encoding sequence of halfcystine of the 55th, 68,77 and 105 in GPX2 gene replaces with Serine, or the gene of other any one coding GPX2 mutant protein or selenocysteine insertion sequence SECIS; Contain GPX2 mutant gene and the cells of mamma animals expression vector of specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence with DNA ligase; According to 512 amino acid whose gene orders of the 343-854 position of selenocysteine insertion sequence associated proteins 2 carboxyl terminal in gene library at biotech firm's its encoding gene of DNA synthesizer synthetic, guarantee that 5 ' end of gene is containing initiator codon ATG and specific restriction enzyme site, SBP2 genome, containing terminator codon and specific restriction enzyme site, installs on born of the same parents' inner mold cells of mamma animals expression vector with specific restriction enzyme site by 3 ' end; Described specific restriction enzyme site be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

To express the cell strain amplification culture of two kinds of foreign proteins in shaking flask, expressing GPX2 mutant containing in the substratum of Sodium Selenite with cells of mamma animals, zymoprotein is secreted in substratum with soluble form simultaneously; With gsh affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain zymoprotein sterling;

The expression amount of described detection GPX2 mutant protein detects by the polyclonal antibody of anti-GPX2 and ELISA or GPX vitality test technology;

Described uses gsh affinitive layer purification GPX2 mutant, is by pH7.5,50mmol/L Tris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH;

Or

1), the structure of expression vector:

According to the encoding gene of GPX2 gene order design primer amplification GPX2 in gene library and the base sequence of 3 ' end non-translational region thereof, when designing primer, guarantee that 5 ' end of target gene is containing initiator codon ATG and specific restriction enzyme site, 3 ' of target gene holds whole base sequence of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream 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; There are target gene and the cells of mamma animals expression vector of specific restriction enzyme site with identical sex-limited endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 gene are assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region with DNA ligase; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit, the sequence encoding mutant of the whole halfcystines in the GPX2 gene be structured on carrier for expression of eukaryon is become the codon of Serine; Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene encodes GPX2 mutant protein after suddenling change; According to 512 amino acid whose its encoding genes of gene order design primer amplification of the 343-854 position of selenocysteine insertion sequence associated proteins 2 carboxyl terminal in gene library, guarantee that 5 ' end of gene is containing initiator codon ATG and specific restriction enzyme site, 3 ' end is containing terminator codon and specific restriction enzyme site; SBP2 genome installs on born of the same parents' inner mold cells of mamma animals expression vector with specific restriction enzyme site by same cutting after connection through enzyme; Described specific restriction enzyme site be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein:

Or

1), the structure of expression vector:

According to the encoding gene of GPX2 gene order design primer amplification GPX2 in gene library and the base sequence of 3 ' end non-translational region thereof, when designing primer, guarantee that 5 ' end of target gene is containing initiator codon ATG and specific restriction enzyme site, 3 ' of target gene holds whole base sequence of the 3 ' end non-translational region introducing GPX2 gene in terminator codon downstream 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; There are target gene and the cells of mamma animals expression vector of specific restriction enzyme site with identical sex-limited endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 gene are assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region with DNA ligase; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX2, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point mutagenesis kit, the sequence encoding mutant of the whole halfcystines in the GPX2 gene be structured on carrier for expression of eukaryon is become the codon of Serine; Determine to suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee that the gene after suddenling change can be encoded GPX2 mutant protein; According to 512 amino acid whose its encoding genes of gene order design primer amplification of the 343-854 position of selenocysteine insertion sequence associated proteins 2 carboxyl terminal in gene library, guarantee that 5 ' end of gene is containing initiator codon ATG and specific restriction enzyme site, 3 ' end is containing terminator codon and specific restriction enzyme site; SBP2 genome installs on born of the same parents' inner mold cells of mamma animals expression vector with specific restriction enzyme site by same cutting after connection through enzyme; Described specific restriction enzyme site be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in target gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier;

2), the screening of positive cell strain:

3), the expression and purification of target protein: