CN100360674C - Prokaryotic expression carrier pASK-75-EX - Google Patents

Abstract

The present invention relates to a prokaryotic expression vector. The present invention uses designed and synthesized genes of a vector cloning expression area to form the new prokaryotic expression vector pASK-75-EX which is provided with a base sequence in a sequence list SEQ ID NO: 1 and is provided with multiple cloning sites and purifying check labels composed of common restriction enzyme. Results show that the pASK-75-EX can effectively express the foreign protein scFv-ML and B-L-SEC2, and an expression product can be conveniently purified and checked with low price by the purifying check labels additionally arranged in the vector.

Description

A kind of prokaryotic expression carrier pASK-75-EX

Technical field

The present invention relates to prokaryotic expression carrier, specifically adopt the carrier cloning that designs and synthesizes to express a kind of novel prokaryotic expression carrier pASK-75-EX of the gene constructed one-tenth in district.

Background technology

Carrier pASK-75 is a kind of efficient expression vector at the expression in escherichia coli foreign protein, the promotor tetA of its stringent type control has guaranteed under the situation of inductor disappearance expression of exogenous gene to be suppressed, be fit to express to the cytotoxic foreign protein of host bacterium, the tsiklomitsin of trace can fully be induced the startup of tetA promotor, makes it have higher expression efficiency.In addition, the tetA promoter expression system is not subjected to the control of any other cell regulation mechanism, therefore to the selection of expressive host bacterium without limits.But the multiple clone site of pASK-75 lacks the restriction endonuclease sites of common high vigor, and the purifying cost that the Strep-tag purification tag of its downstream fusion need be high, has limited the application of pASK-75.

Summary of the invention

The object of the present invention is to provide convenient, the cheap prokaryotic expression carrier pASK-75-EX of a kind of application.

For achieving the above object, the present invention transforms the pASK-75 carrier, has kept tetA promoter sequence and OmpA signal peptide sequence, has newly added the multiple clone site that contains restriction enzyme commonly used, and, be built into new expression vector pASK-75-EX with His-tag label replacement Strep-tag.Compare with original vector, pASK-75-EX makes the clone of foreign gene and the more convenient cheapness of purification assays of foreign protein;

The present invention designs and synthesizes carrier cloning and expresses district's gene, comprise StuI, NcoI, ShpI, EcoRI, NotI, BglII, XhoI and PstI restriction endonuclease sites, His-tag purifying appraisement label, it has base sequence among the sequence table SEQ ID NO:1;

It is as follows that the carrier cloning that designs and synthesizes is expressed district's gene base sequence:

CCTGGCCCAG CCGGCCCATG GCATGCGAAT?TCGCGGCCGC 40

AGATCTGCTC GAGCTGCAGC ATCATCATCA?TCATCATA 78

Utilize the enzyme of restriction endonuclease StuI and Hind III to cut interconnection technique, the synthetic carrier cloning is expressed district's gene integration go into expression vector pASK-75 clonal expression district (substituting the base sequence of 201 to 292 of former pASK-75 expression vectors), be built into new expression vector pASK-75-EX.

The present invention has following advantage:

1. the new expression vector pASK-75-EX of the present invention's structure has the multiple clone site that contains multiple restriction enzyme composition commonly used, greatly facilitates the clone of foreign gene.

2. the new expression vector pASK-75-EX of the present invention's structure replaces the Strep-tag purifying appraisement label of original vector with His-tag purifying appraisement label, simplified the foreign protein purifying of expression and the operation of evaluation, and significantly reduced the cost of purifying and evaluation.

3. the present invention has kept the tetA promoter sequence and the OmpA signal peptide sequence of original vector, has kept the expression vector pASK-75-EX of new structure that the stringent type of foreign gene is expressed control.

4. the new expression vector of Application Example preparation, convenient cheap realization to expression, purifying and the biological assay of single chain antibody protein (scFv-ML) and fusion immunotoxin albumen (B-L-SEC2).

Description of drawings

Fig. 1 is that the single endonuclease digestion of pASK-75-EX multiple clone site is verified with 1.0% agarose gel electrophoresis analytical results figure, wherein: 1. the original pASK75 plasmid of cutting without enzyme, 2. the pASK75-EX plasmid of cutting without enzyme, 3-7. respectively through SphI, EcoRI, BglII, XhoI, pASK75-EX plasmid behind the PstI single endonuclease digestion, 4.DL2000 dna molecular amount standard

Fig. 2 is for utilizing the His-tag that introduces with the result of single-chain antibody ML albumen behind Ni affinity chromatography column purification who expresses.

Fig. 3 is for utilizing the His-tag that introduces with the result of fusion immunotoxin B-L-SEC2 albumen behind Ni affinity chromatography column purification who expresses.

Fig. 4 identifies the single-chain antibody ML albumen of expressing for the His-tag that utilizes introducing through protein immunization marking method result.

Fig. 5 identifies the fusion immunotoxin B-L-SEC2 albumen of expressing for the His-tag that utilizes introducing through protein immunization marking method result.

Embodiment

Embodiment 1

Carrier cloning is expressed the preparation of district's gene, and it has the base sequence shown in the sequence table SEQ ID NO:1:

ACCATCGAATGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCC

CTATCAGTGATAGAGAAAAGTGAAATGAATAGTTCGACAAAAATCTAGATAACGAGGGCAAAAAATGAAAAAGAC

AGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGG CCTGGCCCAGCCGGCCCATGGCATG

CGAATTCGCGGCCGCAGATCTGCTCGAGCTGCAGCATCATCATCATCATCATAAGCTTGACCTGTGAAGTGAAAA

ATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTACCGCTACTGCGTCACGGATCTCCACGCGCCCTGTA

GCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCG

CTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCC

CTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTG

GGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCC

AAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATT

GGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCAGGTG

GCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCA

TGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCG

CCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATG

CTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC

GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACG

CCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAA

AGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCA

ACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTC

GCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAA

TGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATAGACTGGA

TGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTG

GAGCCGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTA

TCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTA

AGCATTGGTAGGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATG

AGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGC

ATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCC

CTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATC

GCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCT

TTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCAGTGGGGCATTTTACTTTAGGTT

GCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCAT

TATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCA

TATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAAAAGCAGCATAACCTTTTTCCGTGATGGTAAC

TTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTT

CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCT

GCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC

CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT

TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA

AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTT

CGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG

CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGG

AGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT

TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTT

GCTGGCCTTTTGCTCACATGACCCGAC

The sequence of above carrier is to utilize the enzyme of restriction endonuclease StuI and Hind III to cut interconnection technique, the synthetic carrier cloning is expressed district's gene (the base sequence fragment of band underscore part in the above sequence) be integrated into the expression vector pASK-75 clonal expression district (base sequence that alternative former pASK-75 expression vector is 201 to 292, replacing the presequence fragment is CCTGAGACCAGAATTCGAGCTCGGTACCCGGGGATCCCTCGAGGTCGACCTGCAGG CAGCGCTTGGCGTCACCCGCAGTTCGGTGGTTAATA), be built into new expression vector pASK-75-EX.

(1) information of SEQ ID NO.1 (referring to sequence table)

(a) sequence signature:

* length: 3252 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: linearity

(b) molecule type: DNA

(c) suppose: not

(d) antisense: not

(e) initial source: carrier pASK-75

(2) carrier cloning is expressed the preparation of district's gene

1) it is synthetic that carrier cloning is expressed the design of distinguishing gene:

Intended purposes according to making up new expression vector designs and synthesizes two oligonucleotide chains:

Forward chain p-F:5 '-CC TGG CCC AGC CGG CCC ATG GCA TGC GAATTC GCG GCC GCA GAT CTG CTC GAG CTG CAG CAT CAT CAT CATCAT CATA-3 ';

Reverse strand p-R:5 '-A GCT ATG ATG ATG ATG ATG ATG GTC CAG CTCGAG CAG ATC TGC GGC CGC GAA TTC GCA TGC CAT GGG CCG GCTGGG CCA GG-3 '

2) annealing of oligonucleotide chain:

10 * annealing hybridization buffer (SSC) composition: 1.5mol/LNaCl

0.3M trisodium citrate 2H ₂O,

Transfer pH to 7.0 with HCl

The annealing reaction system:

10×SSC?buffer 1μl

Positive and negative each 5pmol of oligonucleotide chain

Aseptic ultrapure water polishing volume to 10 μ l

Annealing reaction condition: 94 ℃ of 10min; 0 ℃ of 10min; 72 ℃ of 20min;

Embodiment 2

The structure of new expression vector pASK-75-EX

(1) double digestion of pASK-75 carrier: the German Biometra pASK-75 of company vector plasmid DNA is fully digested through restriction endonuclease StuI and HindIII, and digestion product is through 0.8% agarose gel electrophoresis analysis, and glue reclaims the big fragment of purifying 3200bp;

(2) enzyme is cut the connection and the conversion of product: annealed oligonucleotide chain and the big fragment of the pASK-75 carrier molar ratio with 5: 1 is mixed, and spend the night with 16 ℃ of connections of T4 dna ligase, be built into expression vector pASK-75-EX.(conversion operation is pressed F. Ao Sibai, R. Brunt, R.E. James Kingston to connect product Transformed E .coli DH5 α competent cell, D.D. Moore, J.G Sai Deman, J.A. Smith, K. Si Telaer " fine works molecular biology experiment guide " USA New York John Wiley﹠amp; The nineteen ninety-five third edition P39-40 of Sons press), with acillin resistance screening transformant.

(3) checking of expression vector pASK-75-EX: the positive transformant of selecting is spread cultivation, (the plasmid DNA extracting method is pressed J. Sa nurse Brooker to extract plasmid DNA, E.F. Flitch, T. Manny A Disi " molecular cloning experiment guide " cold spring port, the USA New York calendar year 2001 third edition P27-30 of press), carrier cloning through existing only in design is expressed the restriction enzyme SphI in district respectively, EcoRI, BglII, XhoI, the PstI single endonuclease digestion is identified (accompanying drawing 1), and confirms with the terminal cessation method order-checking of the two deoxidations of Sanger.

Embodiment 3

The practical application of new expression vector pASK-75-EX and effect assessment

(1) exogenous genetic fragment is connected into new expression vector pASK-75-EX: pASK-75-EX plasmid DNA and the pET-28a-ml plasmid DNA that contains single-chain antibody ML gene are used Nco I, Not I double digestion respectively, through 1.0% agarose gel electrophoresis, glue reclaims ml fragment and the pASK-75-EX plasmid 3200bp fragment that test kit reclaims 770bp, spend the night with 16 ℃ of connections of T4 dna ligase, make up single chain antibody protein expression vector pASK-75-EX-ml.Connect product Transformed E .coli BL21 (DE3) competent cell.With acillin resistance screening transformant, select recombinant clone and spread cultivation, extract plasmid DNA, identify correct recombinant clone through Nco I, Not I double digestion.

With NcoI, XhoI double digestion pASK-75-EX plasmid with contain the pET-28a-b-l-sec2 plasmid DNA of fusion immunotoxin gene, make up fusion immunotoxin protein expression vector pASK-75-EX-b-l-sec2 with quadrat method.

(2) expression of foreign protein: respectively inoculation transformed recombinant plasmid pASK-75-EX-ml and pASK75-EX-b-l-sec2 the single bacterium colony of BL21 (DE3) in the liquid LB that contains 100 μ g/ml acillins 37 ℃ spend the night, next day, 37 ℃ were cultured to OD by switching in 1: 50 ₆₀₀0.8 the adding final concentration is that the tsiklomitsin of 0.2ug/ml is induced 6h for 37 ℃.

(3) utilize the new His-tag label purifying foreign protein of introducing: the thalline behind the centrifugal collection abduction delivering, the thalline of every 100ml stock culture is resuspended in the damping fluid of 10ml 20mmol/L Tris-HCl pH7.5, add 0.025% N,O-Diacetylmuramidase in 37 ℃ of lysing cell, in 0 ℃ of ultrasonication until bacterium liquid become limpid, the centrifugal 20min of 12000g collects supernatant, be splined on the good Ni affinity column of level pad balance (available from U.S. Novagen company), with balance in conjunction with damping fluid (5mmol/L imidazoles, 0.5MNaCl, 20mmol/L Tris-HCl pH7.5) balance is to baseline, with the balance that contains the 100mmol/L imidazoles in conjunction with the buffer solution elution target protein, and with SDS-PAGE purity assay (accompanying drawing 2,3).(the SDS-PAGE concrete operation method is pressed Guo Yaojun " protein electrophorese experimental technique " first version P132 in 1999 of Science Press).The molecular weight of single-chain antibody ML albumen and fusion immunotoxin protein B-L-SEC2 is respectively 35kD and 60kD.

(4) utilize the new His-tag label of introducing to identify foreign protein: the single-chain antibody ML albumen behind the purifying is separated with SDS-PAGE with fusion immunotoxin protein B-L-SEC2 with protein immunization marking method, half-dried commentaries on classics film instrument goes to nitrocellulose membrane, the sealing of 2% skimmed milk, goat anti-rabbit antibody with the anti-His-tag antibody of rabbit, alkali phosphatase enzyme mark is handled successively, NBT/BCIP colouring reagents box colour developing (accompanying drawing 4,5).(used damping fluid and concrete operation method are pressed Wang Jiazheng, model bright " protein technical manual " the 2002 first version P166 of Science Press)

SEQUENCE?LISTING

＜110〉Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences

＜120〉a kind of prokaryotic expression carrier pASK-75-EX

<130>

<160>1

<170>PatentIn?version?3.1

<210>1

<211>3252

<212>DNA

＜213〉carrier pASK-75

<220>

<221>misc_feature

<222>(1)..(3252)

<223>

<400>1

accatcgaat?ggccagatga?ttaattccta?atttttgttg?acactctatc?attgatagag 60

ttattttacc?actccctatc?agtgatagag?aaaagtgaaa?tgaatagttc?gacaaaaatc 120

tagataacga?gggcaaaaaa?tgaaaaagac?agctatcgcg?attgcagtgg?cactggctgg 180

tttcgctacc?gtagcgcagg?cctggcccag?ccggcccatg?gcatgcgaat?tcgcggccgc 240

agatctgctc?gagctgcagc?atcatcatca?tcatcataag?cttgacctgt?gaagtgaaaa 300

atggcgcaca?ttgtgcgaca?ttttttttgt?ctgccgttta?ccgctactgc?gtcacggatc 360

tccacgcgcc?ctgtagcggc?gcattaagcg?cggcgggtgt?ggtggttacg?cgcagcgtga 420

ccgctacact?tgccagcgcc?ctagcgcccg?ctcctttcgc?tttcttccct?tcctttctcg 480

ccacgttcgc?cggctttccc?cgtcaagctc?taaatcgggg?gctcccttta?gggttccgat 540

ttagtgcttt?acggcacctc?gaccccaaaa?aacttgatta?gggtgatggt?tcacgtagtg 600

ggccatcgcc?ctgatagacg?gtttttcgcc?ctttgacgtt?ggagtccacg?ttctttaata 660

gtggactctt?gttccaaact?ggaacaacac?tcaaccctat?ctcggtctat?tcttttgatt 720

tataagggat?tttgccgatt?tcggcctatt?ggttaaaaaa?tgagctgatt?taacaaaaat 780

ttaacgcgaa?ttttaacaaa?atattaacgc?ttacaatttc?aggtggcact?tttcggggaa 840

atgtgcgcgg?aacccctatt?tgtttatttt?tctaaataca?ttcaaatatg?tatccgctca 900

tgagacaata?accctgataa?atgcttcaat?aatattgaaa?aaggaagagt?atgagtattc 960

aacatttccg?tgtcgccctt?attccctttt?ttgcggcatt?ttgccttcct?gtttttgctc 1020

acccagaaac?gctggtgaaa?gtaaaagatg?ctgaagatca?gttgggtgca?cgagtgggtt 1080

acatcgaact?ggatctcaac?agcggtaaga?tccttgagag?ttttcgcccc?gaagaacgtt 1140

ttccaatgat?gagcactttt?aaagttctgc?tatgtggcgc?ggtattatcc?cgtattgacg 1200

ccgggcaaga?gcaactcggt?cgccgcatac?actattctca?gaatgacttg?gttgagtact 1260

caccagtcac?agaaaagcat?cttacggatg?gcatgacagt?aagagaatta?tgcagtgctg 1320

ccataaccat?gagtgataac?actgcggcca?acttacttct?gacaacgatc?ggaggaccga 1380

aggagctaac?cgcttttttg?cacaacatgg?gggatcatgt?aactcgcctt?gatcgttggg 1440

aaccggagct?gaatgaagcc?ataccaaacg?acgagcgtga?caccacgatg?cctgtagcaa 1500

tggcaacaac?gttgcgcaaa?ctattaactg?gcgaactact?tactctagct?tcccggcaac 1560

aattgataga?ctggatggag?gcggataaag?ttgcaggacc?acttctgcgc?tcggcccttc 1620

cggctggctg?gtttattgct?gataaatctg?gagccggtga?gcgtggctct?cgcggtatca 1680

ttgcagcact?ggggccagat?ggtaagccct?cccgtatcgt?agttatctac?acgacgggga 1740

gtcaggcaac?tatggatgaa?cgaaatagac?agatcgctga?gataggtgcc?tcactgatta 1800

agcattggta?ggaattaatg?atgtctcgtt?tagataaaag?taaagtgatt?aacagcgcat 1860

tagagctgct?taatgaggtc?ggaatcgaag?gtttaacaac?ccgtaaactc?gcccagaagc 1920

taggtgtaga?gcagcctaca?ttgtattggc?atgtaaaaaa?taagcgggct?ttgctcgacg 1980

ccttagccat?tgagatgtta?gataggcacc?atactcactt?ttgcccttta?gaaggggaaa 2040

gctggcaaga?ttttttacgt?aataacgcta?aaagttttag?atgtgcttta?ctaagtcatc 2100

gcgatggagc?aaaagtacat?ttaggtacac?ggcctacaga?aaaacagtat?gaaactctcg 2160

aaaatcaatt?agccttttta?tgccaacaag?gtttttcact?agagaatgca?ttatatgcac 2220

tcagcgcagt?ggggcatttt?actttaggtt?gcgtattgga?agatcaagag?catcaagtcg 2280

ctaaagaaga?aagggaaaca?cctactactg?atagtatgcc?gccattatta?cgacaagcta 2340

tcgaattatt?tgatcaccaa?ggtgcagagc?cagccttctt?attcggcctt?gaattgatca 2400

tatgcggatt?agaaaaacaa?cttaaatgtg?aaagtgggtc?ttaaaagcag?cataaccttt 2460

ttccgtgatg?gtaacttcac?tagtttaaaa?ggatctaggt?gaagatcctt?tttgataatc 2520

tcatgaccaa?aatcccttaa?cgtgagtttt?cgttccactg?agcgtcagac?cccgtagaaa 2580

agatcaaagg?atcttcttga?gatccttttt?ttctgcgcgt?aatctgctgc?ttgcaaacaa 2640

aaaaaccacc?gctaccagcg?gtggtttgtt?tgccggatca?agagctacca?actctttttc 2700

cgaaggtaac?tggcttcagc?agagcgcaga?taccaaatac?tgtccttcta?gtgtagccgt 2760

agttaggcca?ccacttcaag?aactctgtag?caccgcctac?atacctcgct?ctgctaatcc 2820

tgttaccagt?ggctgctgcc?agtggcgata?agtcgtgtct?taccgggttg?gactcaagac 2880

gatagttacc?ggataaggcg?cagcggtcgg?gctgaacggg?gggttcgtgc?acacagccca 2940

gcttggagcg?aacgacctac?accgaactga?gatacctaca?gcgtgagcta?tgagaaagcg 3000

ccacgcttcc?cgaagggaga?aaggcggaca?ggtatccggt?aagcggcagg?gtcggaacag 3060

gagagcgcac?gagggagctt?ccagggggaa?acgcctggta?tctttatagt?cctgtcgggt 3120

ttcgccacct?ctgacttgag?cgtcgatttt?tgtgatgctc?gtcagggggg?cggagcctat 3180

ggaaaaacgc?cagcaacgcg?gcctttttac?ggttcctggc?cttttgctgg?ccttttgctc 3240

acatgacccg?ac 3252

Claims (1)

1. prokaryotic expression carrier pASK-75-EX, it is characterized in that: sequence is shown in base sequence among the sequence table SEQ ID NO:1.

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