CN102604906A - Bombyx mori glutathione-S-transferase BmGSTD4 and genes thereof - Google Patents

Abstract

The invention discloses bombyx mori glutathione-S-transferase BmGSTD4 of the following (a) or (b), wherein (a) is protein composed of 23rd to 245th amino acids of SEQ ID No.2; and (b) is derived from (a) by replacement, deletion or insertion of one or more amino acids in an amino acid sequence limited by the (a) and has same or similar activity with the protein limited by the (a). The invention also discloses a gene sequence coding GmGSTD4 and a mRNA full-length sequence and a pronucleus preparation method for recombinant BmGSTD4 protein; research shows that BmGSDd4 genes belong to a delta kind of GSTs genes; the BmGSDd4 genes are only expressed in male moth antennas with specific and high quantity; the recombinant BmGSTD4 protein prepared by the method disclosed by the invention has GST activity; by using the BmGSDd4 genes as target points, hopefully biological prevention medicines for killing lepidoptera pests specifically without harm to other beneficial insects can be designed.

Description

Cultivated silkworm glutathione-S-transferase BmGSTD4 and gene thereof

Technical field

The invention belongs to gene engineering technology field, relate to a kind of glutathione-S-transferase and gene thereof.

Background technology

Because the organic synthesis sterilant has characteristics such as wide spectrum, efficient, quick, easy to use and remarkable in economical benefits, in field widespread uses such as agriculture prodn and preventive and curative healths.But along with sterilant reaches frequent use in a large number, for a long time, the insect resistance development has drug-fast pest species and is doubled and redoubled rapidly, and is wherein in the majority with Diptera and lepidopterous insects.

(glutathione S-transferases GSTs) is the one type of important toxenzyme of separating that extensively exists in the organism to glutathione-S-transferase.The intravital GSTs of insect has the effect of metabolism toxicide to sterilant, in the anti-medicine of the sterilant of insect forms, plays a significant role.Discover that the GSTs relevant with insect resistance mainly is special delta of insect and epsilon class.Silkworm is cloned and functional study silkworm GSTs as the lepidopterous insects model animals, illustrates the relation between GSTs and the sterilant resistance, and helping provides theory and practice to instruct to the biological control of insect in the agriculture prodn.

Summary of the invention

In view of this; One of the object of the invention is to provide a kind of cultivated silkworm glutathione-S-transferase; Two of purpose is to provide the gene of said glutathione-S-transferase; Three of purpose is to provide the recombinant expression vector that contains said glutathione-s-transferase gene, and four of purpose is to provide the engineering bacteria that contains said recombinant expression vector, and five of purpose is to provide the preparation method of said glutathione-S-transferase.

For achieving the above object, the present invention provides following technical scheme:

1, as follows (a) or cultivated silkworm glutathione-S-transferase BmGSTD4 (b):

(a) by the 23rd protein of forming to the 245th amino acids among the SEQ ID No.2;

(b) in the aminoacid sequence that (a) limits through replacing, lack or adding one or more amino acid and have same or similar active by (a) deutero-protein with protein that (a) limits.

Further, the proteinic N-terminal that (a) limits also has signal peptide sequence, said signal peptide sequence by among the SEQ ID No.2 the 1st form to the 22nd amino acids.

Further, the protein that (b) limits by among the SEQ ID No.2 the 25th form to the 245th amino acids.

2, the gene of coding (a) or cultivated silkworm glutathione-S-transferase BmGSTD4 (b).

Further, the gene of the cultivated silkworm glutathione-S-transferase BmGSTD4 of coding (a) qualification is made up of the 157th to the 828th Nucleotide among the SEQ ID No.1.

Further, 5 ' terminal signal coding sequence in addition of the gene of the cultivated silkworm glutathione-S-transferase BmGSTD4 that coding (a) limits is made up of the 91st to the 156th Nucleotide among the SEQ ID No.1.

Further, the gene mRNA full length sequence of the cultivated silkworm glutathione-S-transferase BmGSTD4 of coding (a) qualification is made up of the 1st to the 1049th Nucleotide among the SEQ ID No.1.

Further, the gene of the cultivated silkworm glutathione-S-transferase BmGSTD4 of coding (b) qualification is made up of the 163rd to the 828th Nucleotide among the SEQ ID No.1.

3, the recombinant expression vector that contains above-mentioned arbitrary gene.

Further, said recombinant expression vector is a carrier is carrier with prokaryotic expression carrier p28, and said prokaryotic expression carrier p28 gets the MCS transformation of pET28a plasmid, and improved MCS sequence is shown in SEQ ID No.11.

4, the engineering bacteria that contains above-mentioned recombinant expression vector.

Further, said engineering bacteria is the host bacterium with intestinal bacteria Rosetta (DE3) bacterial strain.

5, the preparation method of the cultivated silkworm glutathione-S-transferase BmGSTD4 of (b) qualification; May further comprise the steps: will go into the MCS of prokaryotic expression carrier p28 by the 163rd to the 828th gene clone that Nucleotide is formed among the SEQ ID No.1, and obtain recombinant expression vector BmGSTd4-p28 changes it over to intestinal bacteria Rosetta (DE3) bacterial strain again, obtains engineering bacteria BmGSTd4-p28-Rosetta (DE3), use final concentration as sec.-propyl-β-D-sulfo-galactopyranoside of 0.2mM in 16 ℃ of abduction deliverings 20 hours, collect the thalline behind the abduction delivering, ultrasonication, centrifugal, collect supernatant, use Ni ²⁺-NTA affinitive layer purification, desalination, ultrafiltration and concentration promptly makes by the 25th cultivated silkworm glutathione-S-transferase BmGSTD4 that forms to the 245th amino acids among the SEQ ID No.2; Said prokaryotic expression carrier p28 gets the MCS transformation of pET28a plasmid, and improved MCS sequence is shown in SEQ ID No.11.

Beneficial effect of the present invention is: the present invention is according to bibliographical information BmGSTd4The gene order fragment is carried out 5 ' RACE and 3 ' RACE amplification, and the clone obtains BmGSTd4Gene mRNA full length sequence and complete ORFs, and utilize the escherichia coli prokaryotic expression system right BmGSTd4Gene coded protein carries out heterogenous expression, has successfully obtained soluble recombining BmGSTD4 albumen.The research demonstration, BmGSTd4Gene belongs to delta class GSTs gene, the only special a large amount expression in male moth feeler of this gene; The reorganization BmGSTD4 albumen that adopts the inventive method to make has the GST activity.Because GSTs plays a significant role in the anti-medicine of the sterilant of insect forms, and silkworm is the lepidopterous insects model animals, therefore, with BmGSTd4Gene is a target spot, is expected to design the specific killing lepidoptera pest and the biological control medicine harmless to other useful insect.

Description of drawings

Fig. 1 does BmGSTd4The period expression characteristic of gene in silkworm.

Fig. 2 does BmGSTd4The tissue expression characteristic of gene in the 3rd day 5 ages silkworm female and male larvae.

Fig. 3 does BmGSTd4The tissue expression characteristic of gene in silkworm male and female adult.

Fig. 4 does BmGSTd4The aminoacid sequence of gene mRNA full length sequence and prediction, wherein the ATG of black matrix mark and TGA are respectively initiator codon and terminator codon, and underscore partly is a signal peptide sequence.

Fig. 5 is the proteic prokaryotic expression of reorganization BmGSTD4.

Fig. 6 is the proteic Ni of reorganization BmGSTD4 ²⁺-NTA affinitive layer purification.

Fig. 7 is active for the proteic GST of reorganization BmGSTD4.

Embodiment

In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that the present invention is made further detailed description below.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example; J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press; 2002) described in condition, or the condition of advising according to manufacturer.

The cultivated silkworm breed variety that uses among the embodiment is provided by Southwest China university domestic silkworm gene resources bank for making greatly.

One, BmGSTd4 The specifically expressing of gene in the male moth feeler of silkworm

According to bibliographical information BmGSTd4The gene order fragment (Yu Q., etc al. Identification, genomic organization and expression pattern of glutathione S-transferase in the silkworm, Bombyx mori. 2008, Insect Biochem Mol Biol. 38,1158-1164) design special primer: upstream primer: 5 '-atgctgacagcg-agtgtcttgggag-3 ' (SEQ ID No.3); Downstream primer: 5 '-tcattcatcatccttatttataaac-3 ' (SEQ ID No.4).

Take from the different times silkworm individual (the branch male and female are individual after the 3rd day 5 ages) of 1 the 1st day hibernating worm period to moth phase of age respectively; And 5 the 3rd day age female and male larvae and each organization material of male and female adult; Adopt TRIzol reagent (Invitrogen company) to extract total RNA; Utilize M-MLV reverse transcription test kit (Invitrogen company) to synthesize cDNA first chain, carry out RT-PCR with above-mentioned special primer again, with the silkworm actin gene Actin3Be internal reference.The PCR amplification condition is: 95 ℃ of preparatory sex change 10 minutes, and 40 seconds, 55 ℃ annealing of 95 ℃ of sex change were extended 90 seconds for 30 seconds, 72 ℃ then, totally 25 circulations, last 72 ℃ were extended 10 minutes eventually.The PCR product detects with agarose gel electrophoresis.

The result sees Fig. 1 ~ 3, in the different times silkworm individuality of 1 the 1st day hibernating worm period to moth phase of age, only in the 1st day male of moth, detects BmGSTd4Genetic expression; In each tissue of the 3rd day 5 ages female and male larvae and female insect, all do not detect BmGSTd4Genetic expression only detects in the feeler of male insect BmGSTd4The a large amount of gene, specifically expressing.

Two, BmGSTd4 The clone of gene mRNA full length sequence

According to bibliographical information BmGSTd4The gene order fragment (Yu Q., etc al. Identification, genomic organization and expression pattern of glutathione S-transferase in the silkworm, Bombyx mori. 2008, Insect Biochem Mol Biol. 38,1158-1164), according to GeneRacer ^TMTest kit (Invitrogen company) specification sheets, gene specific primer and the nest bordetella gene special primer of synthetic 5 ' RACE of design and 3 ' RACE.Primer sequence is following:

5 ' RACE gene specific primer: 5 '-ctctgcaccggtctggtcgatgt-3 ' (SEQ ID No.5);

5 ' RACE nest bordetella gene special primer: 5 '-gggttcttagggtacaacgcatcgtt-3 ' (SEQ ID No.6);

3 ' RACE gene specific primer: 5 '-gaatcctcaacataccataccgact-3 ' (SEQ ID No.7);

3 ' RACE nest bordetella gene special primer: 5 '-atcgaccagaccggtgcagaga-3 ' (SEQ ID No.8).

With male moth feeler cDNA is template, adopts above-mentioned special primer, according to GeneRacer ^TMThe test kit specification sheets carries out 5 ' RACE and 3 ' RACE successively.The PCR product carries out agarose gel electrophoresis; Adopt gel to reclaim test kit (Shanghai China Shun Bioisystech Co., Ltd) and cut glue recovery purifying purpose fragment; Again gained purpose fragment cloning is gone into carrier pEasy-T1 simple (Transgen company), entrust Shanghai to give birth to worker's biotechnology ltd and check order.

Gained BmGSTd4The aminoacid sequence of gene mRNA full length sequence and prediction is seen Fig. 4, BmGSTd4Gene mRNA total length 1049bp (the 1st ~ 1049 Nucleotide among the SEQ ID No.1); The ORFs (the 91st ~ 828 Nucleotide among the SEQ ID No.1) that comprises 738bp; The length of encoding altogether 245 amino acid whose BmGSTD4 albumen (the 1st ~ 245 amino acids among the SEQ ID No.2), wherein albumen n end comprises a signal peptide sequence of being made up of 22 amino acid (the 1st ~ 22 amino acids among the SEQ ID No.2).

Will BmGSTd4The aminoacid sequence of gene open reading frame sequence and prediction carries out the BLAST comparison on NCBI, result's demonstration, BmGSTd4Gene belongs to delta class GSTs gene.

Three, reorganization proteic prokaryotic expression of BmGSTD4 and purifying

The signal peptide of BmGSTD4 albumen n end only is used to instruct the proteinic film of striding to shift;, protein arrival excised when striding the film transferring position; Sophisticated protein is not contain signal peptide sequence, and the promptly actual GST of performance is active to be the BmGSTD4 albumen (the 23rd ~ 245 amino acids among the SEQ ID No.2) behind the removal signal peptide sequence.Therefore, when recombinating the proteic prokaryotic expression of BmGSTD4, need not express the signal peptide sequence of BmGSTD4 albumen n end.In order to improve the prokaryotic expression effect; The present invention is with sequence construct BmGSTD4 prokaryotic expression carrier shown in the 163rd ~ 828 Nucleotide among the SEQ ID No.1; The reorganization BmGSTD4 albumen reality of expressing is made up of the 25th ~ 245 amino acids among the SEQ ID No.2; Promptly removed signal peptide sequence and lacked the N end preceding 2 amino acid (Gly-Pro, GP).

According to removing signal peptide sequence BmGSTd4Gene coding region (the 163rd ~ 828 Nucleotide among the SEQ ID No.1) design special primer: forward primer: 5'-gcta Catatg(SEQ ID No.9, underscore partly does aaaaagctagtggcgcctataaaac-3' Nde IRestriction enzyme site); Reverse primer: 5'-ccg Ctcgag(SEQ ID No.10, underscore partly does tcattcatcatccttatttataaac-3' Xho IRestriction enzyme site).With male moth feeler cDNA is template, adopts above-mentioned special primer to carry out pcr amplification.The PCR amplification condition is: 95 ℃ of preparatory sex change 10 minutes, and 40 seconds, 55 ℃ annealing of 95 ℃ of sex change were extended 90 seconds for 40 seconds, 72 ℃ then, totally 35 circulations, last 72 ℃ were extended 10 minutes eventually.

The PCR product carries out agarose gel electrophoresis, cuts glue with AxyGen PCR cleaning agents box and reclaims purifying purpose fragment.Gained purpose fragment and prokaryotic expression carrier p28 use respectively Nde IWith Xho ICarry out double digestion, enzyme is cut product and is carried out agarose gel electrophoresis, cuts glue with AxyGen PCR cleaning agents box and reclaims purifying BmGSTd4Gene fragment and p28 skeleton fragment.With gained BmGSTd4Gene fragment is connected with Solution I (Takara company) for 1:3 with p28 skeleton fragment in molar ratio; Connect product and transform the TOP10 competent cell; Screening positive clone is entrusted Shanghai to give birth to worker's biotechnology ltd and is carried out sequence verification, obtains recombinant vectors BmGSTd4-p28.Prokaryotic expression carrier p28 is that the MCS of pET28a plasmid (Novagen company) is transformed and got by being so kind as to give by professor Zhou Congzhao of China Science & Technology University, and improved MCS sequence is: 5'-ccatgggacaccatcaccatcac CatatgGccaaaaaggccgcggccgca Ctcgag(SEQ ID No.11, underscore partly is respectively-3' Nde IWith Xho IRestriction enzyme site).

With recombinant vectors BmGSTd4-p28 transformed into escherichia coli Rosetta (DE3) bacterial strain (Novagen company) competent cell, screening positive clone obtains engineering bacteria BmGSTd4-p28-Rosetta (DE3).Picking positive monoclonal bacterium colony; With the 2 * YT cultivation that contains kantlex and paraxin based on 37 ℃ of shaking culture to cell density OD600 be 0.6 ~ 1.0; Adding sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) to final concentration is 0.2mM, carries out abduction delivering.Collect the thalline behind the abduction delivering, with resuspended damping fluid (100mM NaCl+20mM Tris, pH8.0) resuspended, put ultrasonication on the mixture of ice and water, centrifugal 30 minutes of 4 ℃, 16000rpm, collecting precipitation and supernatant carry out the SDS-PAGE analysis respectively.The result sees Fig. 5, with IPTG in 16 ℃ of abduction deliverings 20 hours, engineering bacteria BmGSTd4-p28-Rosetta (DE3) can a large amount express recombinant BmGSTD4 albumen, and this albumen is soluble proteins.

The supernatant of collecting is carried out Ni ²⁺-NTA (GE healthcare company) affinity chromatography: supernatant is added through binding buffer liquid (binding buffer) equilibrated Ni ²⁺In-NTA the medium, with binding buffer liquid flushing, the imidazoles solution (20mM, 50mM, 100mM, 200mM, 500mM, 1M) of using gradient concentration again is wash-out successively, collects each elution peak solution, carries out SDS-PAGE and analyzes, and the result sees Fig. 6.The protein solution of 500mM imidazoles eluant solution gained is carried out desalination and damping fluid conversion with HiLoad 16/60 desalting column (GE healthcare company); Concentrate with ultrafiltration pipe (Millipore Amicon company) again; Protein concentrate solution (containing 30% glycerine and 1mM DTT) is purified recombinant BmGSTD4 protein solution, and-80 ℃ of preservations are subsequent use.

Four, the proteic GST determination of activity of reorganization BmGSTD4

Get purified recombinant BmGSTD4 albumen, according to literature method (Habig, W. H.; Etc al. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. 1974; J Biol Chem. 249 7130-7139) measures the GST activity, with 1-chloro-2; 4-dinitrobenzene (CDNB) is the pattern substrate, and the concentration of fixing another substrate gsh is 1mM.The result sees Fig. 7, and the enzyme that records parameter value alive is respectively: KM=0.020 ± 0.001 mM, VMax=176.302 ± 4.943 μ mol/mg/min, KCat=(4.760 ± 0.133) * 10 ³Min ^-1, explain that reorganization BmGSTD4 albumen has the GST activity.

Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art should be appreciated that and can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

< 110>Southwestern University

< 120>cultivated silkworm glutathione-S-transferase BmGSTD4 and gene thereof

<160> 11

<210> 1

<211> 1049

<212> DNA

< 213>silkworm (Bombyx mori Linnaeus)

<220>

<221> CDS

<223> (91)…(828)

<400> 1

gaaaaatgat tcccaagagt cttcgcgcgt gttgttgaag cacgttcatt gcttaatatc 60

tttttaaaat atatctgatt ccatttaaat atgaacgatt tcatggtcgt aacatctata 120

ttgttattta ttaacggctt aggtgaatcg gcagcaggcc ccaaaaagct agtggcgcct 180

ataaaactgt attttcttcc accgtcgcca ccatgcaggg ccgtcatgct gacagcgagt 240

gtcttgggag ttgaacttga gttgatagct gtgaacattt tagacaatga acataaaaca 300

ccggaatacc ttaagatgaa tcctcaacat accataccga ctatggacga caatggattt 360

attctatggg aaagtcgagc cattcaagct tatttggtaa atgcgtatgg aaagaacgat 420

gcgttgtacc ctaagaaccc gcgcttaaga gcgataatcg atcaaaggct taactttgat 480

cttggtacct tatcaagaag atggatagat ctatatgtgc caatgttgat aaaaggggaa 540

ccatttgacg acgaaaaagg agaaaaatta aatgaggccc tggaattgct caatattttc 600

cttgaaggcc acgctttcgt ggcaggagag aatatgtcca ttgctgatct atcaattgtc 660

gttaccatct ctaatttaga tgcagttgaa tatgacctta gctcttatga taatgtgaga 720

aaatggttcg agaggatgaa gattgctcta aaaccttacg attatgagga catcgaccag 780

accggtgcag agatacttgc ctcgtttata aataaggatg atgaatgata cttgcctcgt 840

ttataaataa ggatgatgaa tgatcacaaa aacatatcac caccaaaaca tttgtcatat 900

gtgtatcatg gaataacgag aatcatctac tagggactat ttaattatat ctcaatcatg 960

ttatatgcac attgaaacat tatgtaatga aagtgactta tttgaaaata aaaataccat 1020

tacatcataa aaaaaaaaaa aaaaaaaaa 1049

<210> 2

<211> 245

<212> PRT

< 213>silkworm (Bombyx mori Linnaeus)

<400> 2

Met Asn Asp Phe Met Val Val Thr Ser Ile Leu Leu Phe Ile Asn

1 5 10 15

Gly Leu Gly Glu Ser Ala Ala Gly Pro Lys Lys Leu Val Ala Pro

20 25 30

Ile Lys Leu Tyr Phe Leu Pro Pro Ser Pro Pro Cys Arg Ala Val

35 40 45

Met Leu Thr Ala Ser Val Leu Gly Val Glu Leu Glu Leu Ile Ala

50 55 60

Val Asn Ile Leu Asp Asn Glu His Lys Thr Pro Glu Tyr Leu Lys

65 70 75

Met Asn Pro Gln His Thr Ile Pro Thr Met Asp Asp Asn Gly Phe

80 85 90

Ile Leu Trp Glu Ser Arg Ala Ile Gln Ala Tyr Leu Val Asn Ala

95 100 105

Tyr Gly Lys Asn Asp Ala Leu Tyr Pro Lys Asn Pro Arg Leu Arg

110 115 120

Ala Ile Ile Asp Gln Arg Leu Asn Phe Asp Leu Gly Thr Leu Ser

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Arg Arg Trp Ile Asp Leu Tyr Val Pro Met Leu Ile Lys Gly Glu

140 145 150

Pro Phe Asp Asp Glu Lys Gly Glu Lys Leu Asn Glu Ala Leu Glu

155 160 165

Leu Leu Asn Ile Phe Leu Glu Gly His Ala Phe Val Ala Gly Glu

170 175 180

Asn Met Ser Ile Ala Asp Leu Ser Ile Val Val Thr Ile Ser Asn

185 190 195

Leu Asp Ala Val Glu Tyr Asp Leu Ser Ser Tyr Asp Asn Val Arg

200 205 210

Lys Trp Phe Glu Arg Met Lys Ile Ala Leu Lys Pro Tyr Asp Tyr

215 220 225

Glu Asp Ile Asp Gln Thr Gly Ala Glu Ile Leu Ala Ser Phe Ile

230 235 240

Asn Lys Asp Asp Glu

245

<210> 3

<211> 25

<212> DNA

< 213>artificial sequence

<220>

<223>Amplification Bmgstd4The segmental upstream primer of gene order

<400> 3

atgctgacag cgagtgtctt gggag 25

<210> 4

<211> 25

<212> DNA

< 213>artificial sequence

<220>

<223>Amplification Bmgstd4The segmental downstream primer of gene order

<400> 4

tcattcatca tccttattta taaac 25

<210> 5

<211> 23

<212> DNA

< 213>artificial sequence

<220>

< 223>5 ' RACE gene specific primers

<400> 5

ctctgcaccg gtctggtcga tgt 23

<210> 6

<211> 26

<212> DNA

< 213>artificial sequence

<220>

< 223>5 ' RACE nest bordetella gene special primers

<400> 6

gggttcttag ggtacaacgc atcgtt 26

<210> 7

<211> 25

<212> DNA

< 213>artificial sequence

<220>

< 223>3 ' RACE gene specific primers

<400> 7

gaatcctcaa cataccatac cgact 25

<210> 8

<211> 22

<212> DNA

< 213>artificial sequence

<220>

< 223>3 ' RACE nest bordetella gene special primers

<400> 8

atcgaccaga ccggtgcaga ga 22

<210> 9

<211> 35

<212> DNA

< 213>artificial sequence

<220>

<223>Signal peptide sequence is removed in amplification Bmgstd4The forward primer of gene coding region

<400> 9

gctacatatg aaaaagctag tggcgcctat aaaac 35

<210> 10

<211> 34

<212> DNA

< 213>artificial sequence

<220>

<223>Signal peptide sequence is removed in amplification Bmgstd4The reverse primer of gene coding region

<400> 10

ccgctcgagt cattcatcat ccttatttat aaac 34

<210> 11

<211> 56

<212> DNA

< 213>artificial sequence

<220>

< 223>the MCS sequence of p28a plasmid

<400> 11

ccatgggaca ccatcaccat caccatatgg ccaaaaaggc cgcggccgca ctcgag 56

Claims (13)

1. (a) or cultivated silkworm glutathione-S-transferase BmGSTD4 (b) as follows:

(a) by the 23rd protein of forming to the 245th amino acids among the SEQ ID No.2;

(b) in the aminoacid sequence that (a) limits through replacing, lack or adding one or more amino acid and have same or similar active by (a) deutero-protein with protein that (a) limits.

2. cultivated silkworm glutathione-S-transferase BmGSTD4 according to claim 1 is characterized in that, the proteinic N-terminal that (a) limits also has signal peptide sequence, said signal peptide sequence by among the SEQ ID No.2 the 1st form to the 22nd amino acids.

3. cultivated silkworm glutathione-S-transferase BmGSTD4 according to claim 1 is characterized in that, the protein that (b) limits by among the SEQ ID No.2 the 25th form to the 245th amino acids.

4. the gene of coding claim 1 described cultivated silkworm glutathione-S-transferase BmGSTD4.

5. gene according to claim 4 is characterized in that, the gene of the cultivated silkworm glutathione-S-transferase BmGSTD4 that coding (a) limits is made up of the 157th to the 828th Nucleotide among the SEQ ID No.1.

6. gene according to claim 5 is characterized in that, 5 ' terminal signal coding sequence in addition of the gene of the cultivated silkworm glutathione-S-transferase BmGSTD4 that coding (a) limits is made up of the 91st to the 156th Nucleotide among the SEQ ID No.1.

7. gene according to claim 6 is characterized in that, the gene mRNA full length sequence of the cultivated silkworm glutathione-S-transferase BmGSTD4 that coding (a) limits is made up of the 1st to the 1049th Nucleotide among the SEQ ID No.1.

8. gene according to claim 4 is characterized in that, the gene of the cultivated silkworm glutathione-S-transferase BmGSTD4 that coding (b) limits is made up of the 163rd to the 828th Nucleotide among the SEQ ID No.1.

9. the recombinant expression vector that contains each said gene of claim 4 to 8.

10. recombinant expression vector according to claim 9; It is characterized in that; With prokaryotic expression carrier p28 is carrier is carrier, and said prokaryotic expression carrier p28 transforms the MCS of pET28a plasmid and get, and improved MCS sequence is shown in SEQ ID No.11.

11. contain the engineering bacteria of claim 9 or 10 said recombinant expression vectors.

12. engineering bacteria according to claim 11 is characterized in that, is the host bacterium with intestinal bacteria Rosetta (DE3) bacterial strain.

13. the preparation method of the said cultivated silkworm glutathione-S-transferase BmGSTD4 of claim 1; It is characterized in that; May further comprise the steps: will go into the MCS of prokaryotic expression carrier p28 by the 163rd to the 828th gene clone that Nucleotide is formed among the SEQ ID No.1, and obtain recombinant expression vector BmGSTd4-p28 changes it over to intestinal bacteria Rosetta (DE3) bacterial strain again, obtains engineering bacteria BmGSTd4-p28-Rosetta (DE3), use final concentration as sec.-propyl-β-D-sulfo-galactopyranoside of 0.2mM in 16 ℃ of abduction deliverings 20 hours, collect the thalline behind the abduction delivering, ultrasonication, centrifugal, collect supernatant, use Ni ²⁺-NTA affinitive layer purification, desalination, ultrafiltration and concentration promptly makes by the 25th cultivated silkworm glutathione-S-transferase BmGSTD4 that forms to the 245th amino acids among the SEQ ID No.2; Said prokaryotic expression carrier p28 gets the MCS transformation of pET28a plasmid, and improved MCS sequence is shown in SEQ ID No.11.

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