CN1948461A - Expression method of human heparinase and its special engineering bacteria - Google Patents
Expression method of human heparinase and its special engineering bacteria Download PDFInfo
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- CN1948461A CN1948461A CN 200610065754 CN200610065754A CN1948461A CN 1948461 A CN1948461 A CN 1948461A CN 200610065754 CN200610065754 CN 200610065754 CN 200610065754 A CN200610065754 A CN 200610065754A CN 1948461 A CN1948461 A CN 1948461A
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Abstract
This invention relates to an expression method of human heparinase and its specially used engineering bacteria. The expressing method of human heparinase is that fermenting specially used expression engineering bacteria GS115/HPA1 CGMCC No.1585 for human heparinase, getting human heparinase. Using the invented engineering bacteria to express human heparinase, retrieves deficient of the traditional preparation of human heparinase and expression style of prokaryotic expression system.
Description
Technical field
The present invention relates to the expression method and the dedicated engineering bacteria thereof of enzyme, particularly relate to a kind of expression method and dedicated engineering bacteria thereof of human heparinase.
Background technology
Heparin sulfate, it is linear negatively charged ion glycosaminoglycan, extensively be present in various cell surfaces, interaction between the mediating protein, as participate in cytokine (FGF-1,2, HGF, TGF β, PDGF) with the combining of acceptor, regulate the effect between antithrombin and the zymoplasm, and be the important component part of basilar membrane under intercellular substance and the blood vessel endothelium.In neoplastic process, extracellular matrix parcel tumour that form with separation healthy tissues, and the basilar membrane blood vessel endothelium has under constituted and has stoped the physiologic barrier of tumour to adjacent tissue infiltration and hematogenous metastasis.
Heparinase-β-1,4 glucoside restriction endonuclease acts on the specific site of heparin sulfate specifically, and it is degraded to small molecule segment.The research of relevant heparinase is more than two decades, studies show that heparinase all plays an important role to growth, inflammatory process, vasculogenesis, the metastases of body.At present by people's common concern be the generation of malignant tumour and development all with the high expression level of heparinase, this means: 1. the integrity of basilar membrane is destroyed, tumour cell will be broken through to limit to and is transferred to it and locates or penetrate the basilar membrane intravasation; 2. heparin enzymatic angiogenic action provides the desired nutritional of settling down, grow for the tumour cell of invasion and attack, transfer.Therefore, heparinase has vital role to generation, the development of tumour.Heparin sulfate in the heparinase degraded basement membrane of blood vessel increases the basilar membrane permeability, is the mechanism that heparinase participates in inflammatory reaction, will cause autoimmune disorder when the heparinase overexpression.This shows that the generation of heparinase and tumour, development and transfer and autoimmune disorder are closely related.Therefore develop a kind of heparinase inhibitor and be significant, but the screening model of setting up a kind of heparinase inhibitor is a matter of utmost importance for the treatment of malignant tumour and autoimmune disease.The present domestic report that does not still have the heparinase inhibitor screening model, the research progress lags significantly behind the needs of clinical study.
At present, domestic, the outer method for preparing human heparinase mainly is from thrombocyte, extract in the tissue of high expression level heparinases such as placenta, Vlodavsky etc. utilize gene recombination expressing cho cell heparinase, it is low to have expression amount, purification difficult, the raw material biology growing cycle is long, shortcoming (Vlodavsky I such as production and extraction cost height, Friedmann Y, Elkin M, et al.Mammalian heparanase:gene cloning, expression and functionin tumor progression and metastasis.Nat Med.1999,5 (7): 793-802).
Summary of the invention
The dedicated engineering bacteria that the objective of the invention is a strain capable of high-efficiency expressing human heparinase.
The dedicated engineering bacteria that efficiently expresses human heparinase provided by the present invention, name is called GS115/HPA1, and this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 01 16th, 2006, and deposit number is CGMCC No.1585.
The dedicated engineering bacteria GS115/HPA1 CGMCC No.1585 of expressing human heparinase is a gram negative bacillus, no gemma, and the tool end is given birth to flagellum or peritrichous, can move.The thalline size is (0.5-0.9) * (1.0-3.0) micron, and the thalli morphology of growing in varying environment is different.Under culture condition, this bacterium is little rod-short, and thalline dyeing is inhomogeneous, forms painted and the ring bodies of coloured part not, non-staining part lipid content height.The bacterium colony of growing on yeast water mannite agar substratum plane is rounded, neat in edge, and projection slightly, colourless translucent or shallow oyster white, lawn is denseer.
Second purpose of the present invention provides the construction process of a kind of above-mentioned engineering bacteria GS115/HPA1 CGMCC No.1585.
Construction process provided by the present invention may further comprise the steps:
1) pcr amplification is removed the encoding sequence of the human heparinase of signal peptide, and the human heparinase of described removal signal peptide has the amino acid residue sequence of sequence 2 in the sequence table;
2) encoding sequence of the human heparinase of the removal signal peptide that step 1) is obtained imports among the carrier pPIC9K, obtains the restructured Pichia pastoris in expression carrier;
3) with step 2) the restructured Pichia pastoris in expression carrier that makes up transforms Pichi strain GS115, and screening obtains the dedicated expression engineered bacteria GS115/HPA1 CGMCC No.1585 of human heparinase.
In above-mentioned construction process, step 2) the restructured Pichia pastoris in expression carrier in can make up according to the ordinary method in the bioengineering field; The method that in the step 3) restructured Pichia pastoris in expression carrier is transformed pichia spp is preferably the electric shock conversion method, but also can adopt method commonly used in other bioengineering field, for example, and lithium chloride conversion method, protoplast transformation method etc.
Another object of the present invention provides a kind of expression method of human heparinase.
The expression method of human heparinase provided by the present invention is the dedicated expression engineered bacteria GS115/HPA1 CGMCC No.1585 of the above-mentioned human heparinase of fermentation, obtains human heparinase.
Need add the methanol induction agent when fermenting above-mentioned recombinant yeast pichia pastoris, add methyl alcohol concentration be 0.8-1.2%, be preferably 1%.
For the volatilization loss of compensation methyl alcohol, also to add methyl alcohol in per 24 hours, make the methanol concentration in the bacterium liquid remain on 0.8-1.2%, inducing temperature is 28 ℃.
Described percentage concentration is concentration expressed in percentage by volume.
The invention provides a kind of expression method and dedicated engineering bacteria thereof of human heparinase.Carry out the expression of human heparinase with engineering bacteria of the present invention, remedied the deficiency of traditional human heparinase preparation method and prokaryotic expression system phraseology; In addition, if apply the present invention to the production of industrialization human heparinase, then have raw material biology growing cycle weak point, industrial scale is big, and extraction cost is low, and the advantage that enzymic activity is high has important prospects for commercial application and practical significance.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is that people's ditetrazolium chloride of different positive colonies is surveyed the slip-knot fruit
Concrete enforcement side
Method therefor is ordinary method if no special instructions among the following embodiment, and the solvent in the described substratum is water, and described primer synthesizes and examining order is finished by three rich Bioisystech Co., Ltd.
The acquisition of the dedicated engineering bacteria GS115/HPA1 CGMCC No.1585 of embodiment 1, expressing human heparinase and the expression of human heparinase
One, removes the clone of the human heparinase encoding sequence of signal peptide
1, primer design and synthetic
Inquire about the cDNA sequence (amino acid residue sequence of sequence 1 in the code sequence tabulation that obtains the human heparinase total length through Blast, reference: Vlodavsky I, Friedmann, Y, Elkin M, et al Mammalianheparanase:gene cloning, expression and function in tumor progression andmetastasis Nat Med, 1999,5:793-802), cDNA sequence (sequence 2 in the sequence table) according to the human heparinase of removing coded signal peptide base designs primer again, and introduces the recognition site of restriction enzyme EcoR I and AVR II in primer sequence, and primer sequence is as follows:
P1 (upstream primer): 5 '-cc
GaattcCaggacgtcgtggacc-3 ' (band underscore base is the recognition site of restriction enzyme EcoR I)
P2 (downstream primer): 5 '-cg
CctaggTcagatgcaagcagca-3 ' (band underscore base is the recognition site of restriction enzyme AVR II).
2, the acquisition of template
Extract total RNA of people's liver cancer HePG2 cell earlier with TRIZOL reagent, concrete grammar is: with the full bottle of people's liver cancer HePG2 cell cultures to, collect, clean 2 times with PBS, blot, add TRIZOL 1.5mL, change 2mL EP pipe over to, add 300 μ l chloroforms, jolting 30 minutes was left standstill 10 minutes, centrifugal 15 minutes of 12000rpm, change supernatant over to 1.5mL EP pipe, add the different propyl alcohol mixing of equal-volume, room temperature left standstill 5 minutes, centrifugal 10 minutes of 12000rpm, clean with 75% ethanol, 500 μ l, centrifugal 5 minutes of 12000rpm, dry back adds 20 μ l DEPC water.Extract is carried out 1% agarose gel electrophoresis detect, detected result shows and has obtained the higher RNA of purity.Be template with the RNA that extracts again, use reverse transcription test kit and synthetic its cDNA of reference reagent box specification sheets reverse transcription of INVITROGEN company.
3, pcr amplification is removed the encoding sequence of the human heparinase of signal peptide
CDNA is a template with step 2 reverse transcription synthetic, and under the guiding of primer P1 and P2, pcr amplification is removed the human heparinase encoding sequence of signal peptide, and 25 μ l PCR reaction systems are: 10 * PCR damping fluid, 2.5 μ l, 25mM MgCl
21.5 μ l, 10mM dNTPs 0.5 μ l, each 1 μ l of primer P1 and P2, Taq enzyme 0.2 μ l, cDNA template 1 μ l, DEPC water 17.3 μ l.The PCR reaction conditions is: 95 ℃ of 2min of elder generation; 94 ℃ of 30sec then, 58 ℃ of 35sec, 72 ℃ of 90sec, totally 30 circulations; Last 72 ℃ of 7min, 4 ℃ of preservations.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis detect, obtained the dna fragmentation that length is about 1527bp, conform to expected results through pcr amplification.
4, structure contains the segmental cloning vector of purpose
Reference reagent box specification sheets is operated, and the direct subclone of purpose fragment of step 3 pcr amplification is gone among the carrier pGEM T-Easy (Promega company).
5, the screening and the order-checking of transformed into escherichia coli and positive colony transformant
Connection product transformed into escherichia coli DH5 α competent cell with step 4 acquisition, concrete grammar is: with the connection product and the 20 μ l bacillus coli DH 5 alpha competent cell mixings of 10 μ l steps 4, ice bath 30min, 42 ℃ of heat shock 90sec, ice bath 10min, add 800 μ l then and contain LB liquid nutrient medium (the tryptone 3g of 0.05g/mL penbritin, yeast extract 1.5g, NaCl 3g, water 285mL) in, 180rpm, 37 ℃ of jolting 90min are applied to LB resistance culture plate (the tryptone 3g that contains the 0.05g/mL penbritin, yeast extract 1.5g, NaCl 3g, agar powder 4.5g, water 285mL, 16 μ l X-gal and 4 μ l IPTG/ flat boards) carry out the white screening of indigo plant, cultivated 12-24 hour for 37 ℃.Picking hickie and as template carries out bacterium colony PCR with primer P1 and P2 and identifies that PCR reaction system and reaction conditions are identical with step 3.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis detect, can contain the positive colony of transformant.The positive colony that screens acquisition is gone to 4mL to be contained in the LB liquid nutrient medium of 0.05mg/mL penbritin, 37 ℃, 300rpm jolting 12-24 hour, bacterium liquid is transferred to three rich Bioisystech Co., Ltd to check order, sequencing result shows that the sequence of the encoding sequence of human heparinase of the removal signal peptide of being cloned and Blast report is in full accord, contains the segmental cloned plasmids called after of purpose pGEM-hpa with what step 4 made up.
Two, the structure of restructured Pichia pastoris in expression carrier
Use the DNA purification kit and the reference reagent box specification sheets of Promega company to extract the T plasmid pGEM-hpa that contains goal gene.Use restriction enzyme EcoR I and AVR II (TaKaRa company) that plasmid pGEM-hpa and pPIC9K are carried out double digestion.Enzyme is cut product carry out the detection of 1% agarose gel electrophoresis, downcut the blob of viscose that contains goal gene and linear plasmid pPIC9K respectively, the agarose gel purification kit that uses the Shen, Shanghai can widely collect company reclaims goal gene and linear plasmid pPIC9K respectively.Then with goal gene and linear plasmid pPIC9K T
4Dna ligase (Promega company) connects, and 4 ℃ connect 12-24 hour, obtain the restructured Pichia pastoris in expression carrier, called after pPIC9K-hpa.
Three, transformed yeast bacterium and high copy transform the screening of bacterial strain
GS115 is cultured to OD with Pichia yeast
600Be about 1.5,4 ℃, the centrifugal 6min of 1500rpm, abandon supernatant, add 50mL distilled water centrifugal (condition is the same), abandon supernatant, add 50mL water again, centrifugal (condition is the same), abandon supernatant, add the ice-cold Sorbitol Solution USP (1M) of 4mL, centrifugal (condition is the same) abandons supernatant, adds 200 μ l sorbyl alcohols (1M), get 80 μ l and add 17 μ l contain goal gene through restriction enzyme Sal I restructured Pichia pastoris in expression carrier pPIC9K-hpa, shock by electricity conversion (1500 volts, 400 Europe, 9 milliseconds, Biorad company instrument), add the ice-cold sorbyl alcohol of 1mL then, mixing is got 200 μ l and is applied to MD culture plate (3g agar powder, after the cooling of 160mL distilled water high pressure, add filtered 13.4%YPD substratum (take by weighing 134g Yeast Nitrogen Basew/o Amino Acid and be dissolved in the 1L water, filter) 20mL, 20%D-glucose 20mL with 0.22 μ m filter membrane (MILLIPORE), 20%B-vitamin H 0.4mL, the 0.05g/mL penbritin) cultivate.Single bacterium colony that picking grows on the MD culture plate, be inoculated in G418 culture plate (yeast extract 1g, peptone 2g, distilled water 70mL, agar powder 1.5g, high pressure cooling back adds the phosphate buffered saline buffer of 10mL 1M pH6.0, the nitrogenous substratum 10mL of 13.4% yeast, 20%B-vitamin H 0.2mL, 10% glycerine 10mL, 4mg/mL G418) the high copy conversion of screening bacterial strain.
Four, the expression of heparinase
Picking is eugonic 34 and No. 56 single bacterium colonies on the G418 culture plate, the negative contrast of recombinant pichia yeast strain of blank carrier pPIC9K is arranged with conversion, be inoculated in 5mL BMGY substratum (yeast extract 1g, peptone2g, distilled water 70mL, agar powder 1.5g high pressure cooling back adds the phosphate buffered saline buffer of 10mL 1M pH6.0,10 * YNB10mL, 20%B-vitamin H 0.2mL, 10% glycerine 10mL) in, 30 ℃, 300rpm jolting 12-24 hour (preserving a part of bacterial classification), the centrifugal supernatant of abandoning of 3000rpm, add 5mL BMMY substratum (yeast extract 1g, peptone2g, distilled water 70mL, agar powder 1.5g, high pressure cooling back adds the phosphate buffered saline buffer of 10mL 1M pH6.0, the nitrogenous substratum 10mL of 13.4% yeast, 20%B-vitamin H 0.2mL, methyl alcohol 10mL), 28 ℃, 300rpm jolting 12-24 hour adds 50 μ l methyl alcohol (making the concentration expressed in percentage by volume of methyl alcohol remain on 1%) afterwards every day and induces, induced 72 hours, the centrifugal 10min of 9000rpm leaves and takes supernatant then.
Five, the evaluation of expression product
1, ditetrazolium chloride is surveyed and is lived
After the fermented liquid supernatant ultrafiltration of 34 and No. 56 bacterial strains that step 4 is obtained, the negative contrast of fermented liquid supernatant of the recombinant pichia yeast strain of blank carrier pPIC9K is arranged with conversion, ultrafiltration is replaced into the enzymolysis damping fluid, mix back 37 ℃ again hatched 3 hours with heparin sodium, add 60 ℃ of insulations of ditetrazolium chloride 3 hours then, reaction system (enzymolysis damping fluid: 20mM phosphoric acid salt-citrate buffer solution, 1mM CaCl as shown in table 1
2Solution, 1mM NaCl solution (pH5.4)), survey OD at last
570Value is depicted as typical curve, as shown in Figure 1, shows that the fermentation expression product of above-mentioned bacterial strains has higher heparanase activity, particularly No. 56 bacterial strains.
Table 1 reaction system
| Numbering |
| 1 | 2 | 3 | 4 | 5 | 6 | |
| Enzymolysis damping fluid (μ l) heparin sodium (μ l) fermented liquid supernatant (μ l) | 90 5 5 | 85 5 10 | 80 5 15 | 75 5 20 | 70 5 25 | 65 5 30 |
2, heparinase antibody test
Method with ELISA is done further detection to the heparinase of fermentation expression: at first the fermented liquid supernatant ultrafiltration with 34 and No. 56 bacterial strains is replaced into coating buffer (NaCO
31.59g, NaHCO
32.93g adding distil water is settled to 1L), carry out following step then:
1) wrap by elisa plate: will be replaced into the fermented liquid supernatant that bag is cushioned liquid with 50mM carbonate buffer solution (pH 9.5), every hole 100 μ l placed 12-24 hour for 4 ℃;
2) wrapped the elisa plate of quilt with the sealing of 10% calf serum, 100 μ l/ holes, 37 ℃ of temperature were bathed 30 minutes;
3) add (Wu Xiongwen, Liang Zhihui, " practical immunological experiment technology ", chapter 1,1-2 page or leaf) rabbit anti human heparinase polyclonal antiserum of preparation according to a conventional method, 100 μ l/ holes, 37 ℃ of temperature were bathed 1 hour, gave a baby a bath on the third day after its birth time with 10mM PBS then;
4) add the goat-anti rabbit second antibody (available from Military Medical Science Institute) of horseradish peroxidase-labeled, 50 μ l/ holes, 37 ℃ of temperature were bathed 30 minutes, gave a baby a bath on the third day after its birth time with 10mM PBS then;
5) add the horseradish peroxidase substrate solution, 50 μ l/ holes, 37 ℃ were developed the color 15 minutes;
6) add 2N H
2SO
4Termination reaction, 50 μ l/ holes;
7) measure OD
450Value.
Detected result shows the OD of the fermented liquid supernatant of No. 56 bacterial strains
450Value is 1.288, prove that prepared fermented liquid supernatant contains heparanase protein really, to change the active higher bacterial strain called after GS115/HPA1 of strain, this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 01 16th, 2006, and deposit number is CGMCC No.1585.
Sequence table
<160>2
<210>1
<211>543
<212>PRT
＜213〉Genus Homo people (Homo sapiens)
<400>1
Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu Leu
1 5 10 15
Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro
20 25 30
Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro
35 40 45
Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn
50 55 60
Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu
65 70 75 80
Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly
85 90 95
Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe
100 105 110
Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys
115 120 125
Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp
130 135 140
Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe
145 150 155 160
Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe
165 170 175
Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu
180 185 190
Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu
195 200 205
Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn
210 215 220
Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser
225 230 235 240
Gln Leu Gly Glu Asp Phe Ile Gln Leu His Lys Leu Leu Arg Lys Ser
245 250 255
Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg
260 265 270
Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu
275 280 285
Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr
290 295 300
Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile
305 310 315 320
Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly
325 330 335
Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala
340 345 350
Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys
355 360 365
Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val
370 375 380
Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro
385 390 395 400
Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr
405 410 415
Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg
420 425 430
Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly
435 440 445
Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr Leu
450 455 460
Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu
465 470 475 480
Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn
485 490 495
Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met
500 505 510
Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser
515 520 525
Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile
530 535 540
<210>2
<211>508
<212>PRT
＜213〉Genus Homo people (Homo sapiens)
<400>2
Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro Leu His Leu
1 5 10 15
Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn Leu Ala Thr
20 25 30
Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu Arg Thr Leu
35 40 45
Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly Thr Lys Thr
50 55 60
Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe Glu Glu Arg
65 70 75 80
Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys Tyr Gly Ser
85 90 95
Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp Pro Tyr Gln
100 105 110
Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe Lys Asn Ser
115 120 125
Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe Ala Asn Cys
130 135 140
Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr Ala
145 150 155 160
Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu Asp Tyr Cys
165 170 175
Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn
180 185 190
Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser Gln Leu Gly
195 200 205
Glu Asp Phe Ile Gln Leu His Lys Leu Leu Arg Lys Ser Thr Phe Lys
210 215 220
Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg Arg Lys Thr
225 230 235 240
Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu Val Ile Asp
245 250 255
Ser Val Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro Leu
1 5 10
His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn Leu
15 20 25 30
Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu Arg
35 40 45
Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly Thr
50 55 60
Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe Glu
65 70 75
Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys Tyr
80 85 90
Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp Pro
95 100 105 110
Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe Lys
115 120 125
Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe Ala
130 135 140
Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu Arg
145 150 155
Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu Asp
160 165 170
Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn Glu
175 180 185 190
Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser Gln
195 200 205
Leu Gly Glu Asp Phe Ile Gln Leu His Lys Leu Leu Arg Lys Ser Thr
210 215 220
Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg Arg
225 230 235
Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu Val
240 245 250
Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr Ala
255 260 265 270
Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile Ser
275 280 285
Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly Lys
290 295 300
Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala Pro
305 310 315
Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys Leu
320 325 330
Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val Phe
335 340 345 350
Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro Leu
355 360 365
Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr Lys
370 375 380
Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg Val
385 390 395
Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly Asp
400 405 410
Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr Leu Arg
415 420 425 430
Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu Arg
435 440 445
Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn Gly
450 455 460
Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met Glu
465 470 475
Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser Tyr
480 485 490
Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile
495 500 505
Claims (6)
1, the dedicated engineering bacteria GS115/HPA1 CGMCC No.1585 of expressing human heparinase.
2, the construction process of a kind of engineering bacteria GS115/HPA1 CGMCC No.1585 may further comprise the steps:
1) pcr amplification is removed the encoding sequence of the human heparinase of signal peptide, and the human heparinase of described removal signal peptide has the amino acid residue sequence of sequence 2 in the sequence table;
2) encoding sequence of the human heparinase of the removal signal peptide that step 1) is obtained imports among the carrier pPIC9K, obtains the restructured Pichia pastoris in expression carrier;
3) with step 2) the restructured Pichia pastoris in expression carrier that makes up transforms Pichi strain GS115, and screening obtains the dedicated expression engineered bacteria GS115/HPA1 CGMCC No.1585 of human heparinase.
3, a kind of expression method of human heparinase is the dedicated expression engineered bacteria GS115/HPA1 CGMCC No.1585 of the described human heparinase of fermentation claim 1, obtains human heparinase.
4, expression method according to claim 3 is characterized in that: add the methanol induction agent during described fermentation, the concentration expressed in percentage by volume of methyl alcohol is 0.8-1.2%.
5, expression method according to claim 4 is characterized in that: the concentration expressed in percentage by volume of described methanol induction agent is 1%.
6, according to claim 4 or 5 described expression methods, it is characterized in that: described inducing temperature is 28 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533628A (en) * | 2012-02-24 | 2012-07-04 | 山东大学 | Strain of bacillus subtilis engineering bacteria and application thereof in producing heparinase I |
| CN108841738A (en) * | 2018-07-02 | 2018-11-20 | 江南大学 | A method of building efficient secretory expression source of people heparin hydrolase recombinant bacterium |
| CN108841739A (en) * | 2018-07-02 | 2018-11-20 | 江南大学 | A method of building efficient secretory expression bacterium heparin hydrolase recombinant bacterium |
-
2006
- 2006-03-15 CN CN 200610065754 patent/CN1948461A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533628A (en) * | 2012-02-24 | 2012-07-04 | 山东大学 | Strain of bacillus subtilis engineering bacteria and application thereof in producing heparinase I |
| CN102533628B (en) * | 2012-02-24 | 2013-12-11 | 山东大学 | Strain of bacillus subtilis engineering bacteria and application thereof in producing heparinase I |
| CN108841738A (en) * | 2018-07-02 | 2018-11-20 | 江南大学 | A method of building efficient secretory expression source of people heparin hydrolase recombinant bacterium |
| CN108841739A (en) * | 2018-07-02 | 2018-11-20 | 江南大学 | A method of building efficient secretory expression bacterium heparin hydrolase recombinant bacterium |
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