CN102604906B - 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 the characteristics such as wide spectrum, efficient, quick, easy to use and remarkable in economical benefits, in field widespread uses such as agriculture production and preventive and curative healths.But a large amount of, the long-term and use frequently along with sterilant, 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-transferase (glutathione S-transferases, GSTs) is an important detoxication enzyme of class that extensively exists in organism.GSTs in insect body has the effect of metabolic detoxification to sterilant, play a significant role in the anti-medicine of the sterilant of insect forms.The research discovery, the GSTs relevant to insect resistance is mainly the 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 insecticide resistance, and helping provides theory and practice to instruct to the biological control of insect in agriculture production.

Summary of the invention

In view of this, one of purpose of the present invention is to provide a kind of cultivated silkworm glutathione-S-transferase, two of purpose is to provide the gene of described glutathione-S-transferase, three of purpose is to provide the recombinant expression vector that contains described glutathione-s-transferase gene, four of purpose is to provide the engineering bacteria that contains described recombinant expression vector, and five of purpose is to provide the preparation method of described glutathione-S-transferase.

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

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

(a) by the 23rd protein that forms to the 245th amino acids in SEQ ID No.2;

(b) in the aminoacid sequence that (a) limits through replacement, lack or add one or more amino acid and with protein that (a) limits have same or similar activity by (a) derivative protein.

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

Further, the protein that (b) limits by in 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) restriction is comprised of the 157th to the 828th Nucleotide in SEQ ID No.1.

Further, 5 ' end of the gene of the cultivated silkworm glutathione-S-transferase BmGSTD4 that coding (a) limits also has signal coding sequence, is comprised of the 91st to the 156th Nucleotide in SEQ ID No.1.

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

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

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

Further, described recombinant expression vector is take prokaryotic expression carrier p28 as carrier is carrier, and described prokaryotic expression carrier p28 gets the multiple clone site transformation of pET28a plasmid, and improved multiple clone site sequence is as shown in SEQ ID No.11.

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

Further, described engineering bacteria is take intestinal bacteria Rosetta (DE3) bacterial strain as Host Strains.

5, the preparation method of the cultivated silkworm glutathione-S-transferase BmGSTD4 of (b) restriction, comprise the following steps: will be entered the multiple clone site of prokaryotic expression carrier p28 by the 163rd to the 828th gene clone that Nucleotide forms in SEQ ID No.1, and obtain recombinant expression vector BmGSTd4-p28, then change it over to intestinal bacteria Rosetta (DE3) bacterial strain, obtain engineering bacteria BmGSTd4-p28-Rosetta (DE3), with final concentration be sec.-propyl-β-D-sulfo-galactopyranoside of 0.2mM in 16 ℃ of abduction deliverings 20 hours, collect the thalline after abduction delivering, ultrasonication, centrifugal, collect supernatant, use Ni ²⁺-NTA affinitive layer purification, desalination, ultrafiltration and concentration namely makes by the 25th cultivated silkworm glutathione-S-transferase BmGSTD4 that forms to the 245th amino acids in SEQ ID No.2; Described prokaryotic expression carrier p28 gets the multiple clone site transformation of pET28a plasmid, and improved multiple clone site sequence is as 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 open reading frame, and utilize escherichia coli prokaryotic expression system pair BmGSTd4Gene coded protein carries out heterogenous expression, has successfully obtained the restructuring BmGSTD4 albumen of solubility.Studies show that, BmGSTd4Gene belongs to delta class GSTs gene, the only special a large amount expression in male moth feeler of this gene; The restructuring 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 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 is BmGSTd4The period expression characteristic of gene in silkworm.

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

Fig. 3 is BmGSTd4The tissue expression feature of gene in silkworm male and female adult.

Fig. 4 is 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 is partly signal peptide sequence.

Fig. 5 is the prokaryotic expression of restructuring BmGSTD4 albumen.

Fig. 6 is the Ni of restructuring BmGSTD4 albumen ²⁺-NTA affinitive layer purification.

Fig. 7 is active for the GST of restructuring BmGSTD4 albumen.

Embodiment

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing.The experimental technique of unreceipted actual conditions in embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. the work such as Pehanorm 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 in embodiment is for making greatly, provided by Southwest China university domestic silkworm gene resources bank.

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 respectively the different times silkworm individual (after the 3rd day 5 ages, minute male and female are individual) of 1 the 1st day hibernating worm period to moth phase of age, and the 3rd day 5 age female and male larvae and each organization material of female male imago, adopt TRIzol reagent (Invitrogen company) to extract total RNA, utilize M-MLV reverse transcription test kit (Invitrogen company) to synthesize cDNA the 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 denaturations 10 minutes, and then 30 seconds, 72 ℃ of 40 seconds, 55 ℃ annealing of 95 ℃ of sex change were extended 90 seconds, totally 25 circulations, last 72 ℃ were extended 10 minutes eventually.The PCR product detects with agarose gel electrophoresis.

The results are shown in Figure 1 ~ 3, in the different times silkworm individuality of 1 the 1st day hibernating worm period to moth phase of age, only detect in the moth male of the 1st day BmGSTd4Genetic expression; All do not detect in each tissue of the 3rd day 5 ages female and male larvae and female insect 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 the synthetic 5 ' RACE of design and 3 ' RACE.Primer sequence is as follows:

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).

Take male moth feeler cDNA as template, adopt 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 is cloned into carrier pEasy-T1 simple(Transgen company), entrust Shanghai to give birth to work biotechnology company limited and check order.

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

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

Three, prokaryotic expression and the purifying of restructuring BmGSTD4 albumen

The signal peptide of BmGSTD4 albumen n end only is used for instructing the cross-film of protein to shift, it is cut when protein arrives the cross-film transfer position, ripe protein is not contain signal peptide sequence, and namely actual performance GST activity is the BmGSTD4 albumen (the 23rd ~ 245 amino acids in SEQ ID No.2) of removing after signal peptide sequence.Therefore, when recombinating the prokaryotic expression of BmGSTD4 albumen, do not need to 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 in SEQ ID No.1, the restructuring BmGSTD4 albumen reality of expressing is comprised of the 25th ~ 245 amino acids in SEQ ID No.2, namely remove signal peptide sequence and lacked front 2 amino acid (Gly-Pro, GP) that N holds.

According to removing signal peptide sequence BmGSTd4Gene coding region (the 163rd ~ 828 Nucleotide in SEQ ID No.1) design special primer: forward primer: 5'-gcta CatatgAaaaagctagtggcgcctataaaac-3'(SEQ ID No.9, underscore is partly Nde IRestriction enzyme site); Reverse primer: 5'-ccg CtcgagTcattcatcatccttatttataaac-3'(SEQ ID No.10, underscore is partly Xho IRestriction enzyme site).Take male moth feeler cDNA as template, adopt above-mentioned special primer to carry out pcr amplification.The PCR amplification condition is: 95 ℃ of denaturations 10 minutes, and then 40 seconds, 72 ℃ of 40 seconds, 55 ℃ annealing of 95 ℃ of sex change were extended 90 seconds, 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 p28 skeleton fragment for 1:3 with Solution I(Takara company) connect, connect product and transform the TOP10 competent cell, screening positive clone entrusts Shanghai living work biotechnology company limited to carry out sequence verification, obtains recombinant vectors BmGSTd4-p28.Prokaryotic expression carrier p28 by being so kind as to give by professor Zhou Congzhao of China Science ﹠ Technology University, is that the multiple clone site of pET28a plasmid (Novagen company) is transformed and got, and improved multiple clone site sequence is: 5'-ccatgggacaccatcaccatcac CatatgGccaaaaaggccgcggccgca Ctcgag-3'(SEQ ID No.11, underscore partly is respectively Nde IWith Xho IRestriction enzyme site).

With recombinant vectors BmGSTd4-p28 transforms intestinal bacteria Rosetta (DE3) bacterial strains (Novagen company) competent cell, and screening positive clone obtains engineering bacteria BmGSTd4-p28-Rosetta (DE3).Picking positive monoclonal bacterium colony, with the 2 * YT that contains kantlex and paraxin cultivate 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 after abduction delivering, (100mM NaCl+20mM Tris, pH8.0) is resuspended with resuspended damping fluid, puts ultrasonication on mixture of ice and water, centrifugal 30 minutes of 4 ℃, 16000rpm, and collecting precipitation and supernatant, carry out the SDS-PAGE analysis respectively.The results are shown in Figure 5, with IPTG in 16 ℃ of abduction deliverings 20 hours, engineering bacteria BmGSTd4-p28-Rosetta (DE3) can a large amount express restructuring 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 Ni through binding buffer liquid (binding buffer) balance ²⁺In-NTA medium, rinse with binding buffer liquid, then the imidazoles solution (20mM, 50mM, 100mM, 200mM, 500mM, 1M) of using gradient concentration wash-out successively, collect each elution peak solution, carry out SDS-PAGE and analyze, the results are shown in Figure 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), use again super filter tube (Millipore Amicon company) concentrated, protein concentrate solution is (containing 30% glycerine and 1mM DTT) the restructuring BmGSTD4 protein solution of purifying, and-80 ℃ save backup.

Four, the GST determination of activity of restructuring BmGSTD4 albumen

Get the restructuring BmGSTD4 albumen of purifying, 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 results are shown in Figure 7, 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, illustrate that restructuring 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 by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and not depart from the spirit and scope of the present invention that appended claims limits.

＜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

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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

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<212> PRT

＜213〉silkworm (Bombyx mori Linnaeus)

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

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

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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

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

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

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

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

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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

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＜213〉artificial sequence

<220>

＜223〉amplification Bmgstd4The upstream primer of gene order fragment

<400> 3

atgctgacag cgagtgtctt gggag 25

<210> 4

<211> 25

<212> DNA

＜213〉artificial sequence

<220>

＜223〉amplification Bmgstd4The downstream primer of gene order fragment

<400> 4

tcattcatca tccttattta taaac 25

<210> 5

<211> 23

<212> DNA

＜213〉artificial sequence

<220>

＜223〉5 ' RACE gene specific primer

<400> 5

ctctgcaccg gtctggtcga tgt 23

<210> 6

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉5 ' RACE nest bordetella gene special primer

<400> 6

gggttcttag ggtacaacgc atcgtt 26

<210> 7

<211> 25

<212> DNA

＜213〉artificial sequence

<220>

＜223〉3 ' RACE gene specific primer

<400> 7

gaatcctcaa cataccatac cgact 25

<210> 8

<211> 22

<212> DNA

＜213〉artificial sequence

<220>

＜223〉3 ' RACE nest bordetella gene special primer

<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 multiple clone site sequence of p28a plasmid

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ccatgggaca ccatcaccat caccatatgg ccaaaaaggc cgcggccgca ctcgag 56

Claims (8)

1. cultivated silkworm glutathione-S-transferase BmGSTD4, is characterized in that, by in SEQ ID No.2 the 25th form to the 245th amino acids.

2. the encode gene of cultivated silkworm glutathione-S-transferase BmGSTD4 claimed in claim 1.

3. gene according to claim 2, is characterized in that, is comprised of the 163rd to the 828th Nucleotide in SEQ ID No.1.

4. the recombinant expression vector that contains claim 2 or 3 described genes.

5. recombinant expression vector according to claim 4, it is characterized in that, take prokaryotic expression carrier p28 as carrier is carrier, described prokaryotic expression carrier p28 transforms the multiple clone site of pET28a plasmid and get, and improved multiple clone site sequence is as shown in SEQ ID No.11.

6. the engineering bacteria that contains claim 4 or 5 described recombinant expression vectors.

7. engineering bacteria according to claim 6, is characterized in that, take intestinal bacteria Rosetta (DE3) bacterial strain as Host Strains.

8. the preparation method of the described cultivated silkworm glutathione-S-transferase BmGSTD4 of claim 1, it is characterized in that, comprise the following steps: will be entered the multiple clone site of prokaryotic expression carrier p28 by the 163rd to the 828th gene clone that Nucleotide forms in SEQ ID No.1, and obtain recombinant expression vector BmGSTd4-p28, then change it over to intestinal bacteria Rosetta (DE3) bacterial strain, obtain engineering bacteria BmGSTd4-p28-Rosetta (DE3), with final concentration be sec.-propyl-β-D-sulfo-galactopyranoside of 0.2mM in 16 ℃ of abduction deliverings 20 hours, collect the thalline after abduction delivering, ultrasonication, centrifugal, collect supernatant, use Ni ²⁺-NTA affinitive layer purification, desalination, ultrafiltration and concentration namely makes by the 25th cultivated silkworm glutathione-S-transferase BmGSTD4 that forms to the 245th amino acids in SEQ ID No.2; Described prokaryotic expression carrier p28 gets the multiple clone site transformation of pET28a plasmid, and improved multiple clone site sequence is as shown in SEQ ID No.11.

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