CN101974558A - Genetic engineering bacterium for expressing elastase 2B and construction method and application thereof - Google Patents

Abstract

The invention provides a genetic engineering bacterium for efficiently expressing recombinant elastase 2B, which is characterized by cloning the gene of elastase 2B (ELA2B) in human renal tissues through reverse transcription, constructing the expression vector, transforming the expression vector into the pichia pastoris and sieving the strain for efficient secretory expression from the pichia pastoris. The expression quantity of the recombinant elastase fermented by the genetic engineering strain is about 375mu g/mL, and the activity of the fermented crude enzyme can reach 980U/mL, which is 24 times higher than that of the product fermented by pseudomonas aeruginosa.

Description

A kind of genetic engineering bacterium, its construction process and application of expressing elastoser 2B

Technical field

The invention belongs to biological technical field, be specifically related to genetic engineering bacterium, its construction process and the application of a kind of people of efficiently expressing source elastoser.

Background technology

Elastoser (elastase) is to be a class wide spectrum proteolytic ferment of feature with the insoluble hydrolysate elastin, in food, medicine, daily use chemicals and environmental protection field important DEVELOPMENT PROSPECT is arranged.The production method of present domestic elastoser mainly is to extract from the pancreas of animal, is subjected to the restriction of internal organs material and production technique, and cost is higher, and output is lower, lags behind the market requirement greatly, has limited the application of elastoser.Extensively carry out both at home and abroad and utilize microbial fermentation production elastoser, alleviated industrial enzymes to a certain extent, demand as aspects such as tenderization agent, cosmetics additives, but great limitation is still arranged as medical biochemical drug, this mainly is because the elastoser of microbial fermentation production is also having certain deficiency aspect biological safety and the toxicity, the elastin enzymatic property in different microorganisms source has certain difference in addition, and the medical biochemical drug commodity production of distance has certain distance.

The present invention utilizes genetic engineering technique, cloned elastoser 2B (elastase 2B in people's renal tissue by reverse transcription, ELA2B) gene, utilize pichia yeast expression system that it is expressed, make the people source elastoser of scale operation safety become possibility, structure and the property research for people source elastoser provides experiment material simultaneously.

Summary of the invention

The purpose of this invention is to provide a kind of genetic engineering bacterium that can efficiently express people source reorganization elastoser 2B.

Another object of the present invention provides the construction process of said gene engineering bacteria.

Further purpose of the present invention provides the application of said gene engineering bacteria in producing people source reorganization elastoser.

In order to realize the object of the invention, a kind of genetic engineering bacterium of expressing elastoser 2B of the present invention, its starting strain is a pichia spp.Preferably, its starting strain is pichia spp GS115.

The construction process of said gene engineering bacteria, it comprises step: 1) extract total RNA from the human kidney tissue, obtain the ELA2B gene through the RT-PCR amplification; 2) the ELA2B gene that obtains in the step 1) is inserted in the expression vector; 3) with step 2) the middle expression vector that makes up changes pichia spp over to, and screening positive clone, obtains the recombinant yeast pichia pastoris engineering bacteria.

Aforesaid method, the carrier that sets out of wherein said expression vector is pPIC9K, is about to described elastoser 2B gene clone to the multiple clone site of pPIC9K and obtain.

The method that above-mentioned expression vector is changed over to pichia spp can be an electrotransformation, and that can adopt also that those skilled in the art are familiar with anyly changes expression vector over to the method for yeast cell, for example lithium chloride conversion method, PEG method etc.

Aforesaid method, wherein screening positive clone comprises described in the step 3): with MD substratum preliminary screening positive colony, carry out postsearch screening with the G418 resistance again, obtain having the clone of elastin gene high copy number, adopt the dull and stereotyped revulsion of casein-MM at last, screening obtains the recombinant bacterial strain of high secreting, expressing.

Aforesaid method, wherein said described elastoser 2B gene has the nucleotide sequence shown in the SEQ ID NO.1.This sequence can (elastase 2B, ELA2B) gene be that the template clone obtains by the elastoser 2B in the people's renal tissue that produces elastoser.

The present invention makes up people source elastoser 2B pichia yeast expression system by engineered method, is about to the elastoser 2B gene clone of people source in carrier, utilizes the Yeast expression carrier system, expresses the external source elastoser in yeast cell.Pichia yeast expression system has very high biological safety, obtains to comprise the extensive approval of U.S. FDA.In addition, it has very high expression amount and high secretion property to foreign protein, can translate post-treatment, makes it more to approach the conformation and the activity of native protein, is a kind of eukaryotic expression system of widespread use.

More particularly, can make up the genetic engineering bacterium that efficiently expresses people source elastoser 2B by the following method:

Adopt reverse transcription-polymerase chain reaction (RT-PCR) technology, amplification obtains the ELA2B gene among the total RNA that extracts from the human kidney tissue.Then the PCR product is carried out the T-A clone, obtain plasmid pGEM T-easy/ELA2B.Compare with the ELA2B gene order among the GenBank in the order-checking back.Sequence on sequencing result and the GenBank is in full accord.Respectively pGEM T-easy/ELA2B and yeast expression vector pPIC9K are carried out double digestion with EcoRI and Not I, the elastoser gene is downcut, be transferred among the expression vector pPIC9K, make up plasmid pPIC9K/ELA2B.Through double digestion and sequence verification, prove that direction of insertion correctly and do not cause the variation of base sequence.

With Sac I expression vector pPIC9K/ELA2B is carried out linearizing, the electricity consumption method for transformation changes plasmid among the pichia spp GS115 over to, utilizes the MD substratum to screen nearly 460 transformants, through the postsearch screening of G418 resistance, obtain the recombinant yeast pichia pastoris transformant that 50 strains contain high copy again.Adopt the dull and stereotyped inductive method of casein-MM, successfully filter out the recombinant bacterial strain of efficient secretory expression.

Bacterial strain is rule on fresh YPD flat board,, be inverted and cultivated 2 days in 30 ℃.Single bacterium colony of picking bacterial strain is inoculated in the 20mL BMGY substratum respectively, cultivates with the 500mL triangular flask, reaches OD until cell concn ₆₀₀=2～6.Culture condition is: 28 ℃, shaking speed are 200rpm.Centrifugal 5min under the room temperature 6000rpm condition collects mycetocyte, and abandoning supernatant is with 20mL BMMY substratum re-suspended cell, beginning abduction delivering.The culture condition of abduction delivering is: 30 ℃, shaking speed is 200rpm, cultivates 4 days.Get fermented liquid in 4 ℃, the centrifugal 5min of 10000rpm removes thalline, with supernatant liquor dialysed overnight in Tris-HCL damping fluid (pH8.0), promptly obtains crude enzyme liquid.By DEAE-Sepharose FF column purification, can obtain pure enzyme.

The fermentation supernatant is directly carried out the SDS-PAGE electrophoresis, can access and the close band (28kDa) of natural elastic proteolytic enzyme size, with the elastin is the dull and stereotyped hydrolysising experiment of substrate, find that the fermentation supernatant can single-minded hydrolysis substrate elastin, confirmed that expression product has certain biological activity.With Congo red-elastin is substrate, specifically measures the enzyme of reorganization elastoser and lives, and finds that the enzyme work of thick enzyme in the fermented liquid is higher 24 times than Pseudomonas aeruginosa tunning.

It is as follows to measure enzyme method alive:

Accurately take by weighing 10mg Congo red-elastin, add pH7.4,0.2mol/L borate buffer 2mL, add the last clear enzyme solution of 1mL again, behind 28 ℃ of oscillatory reaction 3h, use pH6.0 immediately through suitably diluting, concentration is the phosphoric acid buffer 2mL termination reaction of 0.7mol/L, behind the centrifugal 5min of 10000r/min, get the mensuration optical density value of supernatant liquor in the 495nm place, with the last clear enzyme solution that do not add substrate as blank.Under this reaction conditions, every 5mL reaction mixture 495nm place increases by 0.01 required enzyme amount of absorbancy and is defined as 1 elastase activity unit (u):

Elastase activity (U/mL)=(OD of sample ₄₉₅The OD of-blank ₄₉₅) * 100 * extension rate

By technique scheme, the present invention has following advantage and beneficial effect at least:

(1) in the eucaryon yeast expression system, efficiently expressed recombination human source elastoser first with very high enzyme work.

(2) output of recombinant protein is up to 375 μ g/mL, and the enzyme of thick enzyme is lived and is 980U/mL in the fermented liquid, is 24 times that Pseudomonas aeruginosa tunning enzyme is lived.

(3) but the recombinant protein direct secretion in substratum, the content that need not foreign protein in smudge cells and the substratum is few, purification step is simple, only needs can obtain pure enzyme by single step purification.

(4) be suitable for large-scale high density fermentation production, simple to operate, cost is low, and product has very high biological safety, is applicable to fields such as food and medicine.

Description of drawings

Fig. 1 carries out agarose gel electrophoresis figure behind the double digestion for plasmid pGEM-T easy/ELA2B of the present invention with EcoR I and Not I, and wherein M is DL2000DNA Marker.

Fig. 2 cuts the evaluation electrophorogram for the enzyme of recombinant plasmid pPIC9K/ELA2B of the present invention, and wherein M1 and M2 are DNA Marker, and 1～3 represents plasmid pPIC9K/ELA2B respectively; The pPIC9K/ELA2B that EcoRI restriction enzyme list is cut; The two pPIC9K/ELA2B that cut of EcoR I and Not I restriction enzyme.

Fig. 3 is the sequencing result of inventor source elastoser gene ELA2B and the result schematic diagram that the ELA2B sequence on the GenBank is compared.

Fig. 4 is His of the present invention ⁺Transformant is at the The selection result synoptic diagram that contains on the YPD flat board of G418.

Fig. 5 utilizes the result schematic diagram of type recombination yeast for the present invention utilizes MM/MD plate screening methyl alcohol.

Fig. 6 for recombinant pichia yeast strain of the present invention through the different number of days abduction delivering after, SDS-PAGE electrophorogram in the supernatant, wherein, M is low molecular weight protein (LMWP) Marker, 1 for transforming the yeast of pPIC9K empty carrier, 2～5 respectively representative induced 1 day, 2 days, 3 days, 4 days.

Recombinate for the present invention the contains gradient elution figure of crude enzyme liquid by DEAE-Sepharose FF column purification of elastoser of Fig. 7.

Fig. 8 is elasticity protease activities detected result synoptic diagram in the recombination yeast fermented supernatant fluid of the present invention, wherein 1～4 fermented supernatant fluid of representing behind the recombination yeast abduction delivering of different batches; CK represents the not recombination microzyme fermented supernatant fluid of abduction delivering.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

The experimental technique of unreceipted actual conditions in following examples, usually operate according to normal condition, Sambrook etc. for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to the condition of manufacturer suggestion.

The amplification of embodiment 1 elastoser 2B gene

1. the extraction of total RNA in the renal tissue

(1) accurately takes by weighing the renal tissue sample that the 100mg liquid nitrogen is preserved, put into glass homogenizer, add the RNAiso Reagent of 1.5mL, place homogenate on ice;

(2) homogenate is transferred in the 1.5mL centrifuge tube, room temperature left standstill 5 minutes;

(3) 4 ℃ of centrifugal 15min of 12000rpm.Draw supernatant liquor, move into new centrifuge tube;

(4) add 400 μ L chloroforms, firmly concussion evenly, after treating that solution is fully emulsified and being milky white shape, room temperature leaves standstill 5min;

(5) 4 ℃ of centrifugal 15min of 12000rpm carefully draw supernatant liquor and are transferred to new centrifuge tube;

(6) add isopyknic Virahol in supernatant liquor, behind the abundant mixing of the centrifuge tube that turns upside down, room temperature leaves standstill 10min;

(7) 4 ℃ of centrifugal 10min of 12000rpm, careful supernatant discarded, slowly along 75% ethanol of centrifuge tube adding 1ml, the washing centrifuge tube tube wall that turns upside down gently, 4 ℃ of centrifugal ℃ of 5min of 12000rpm, supernatant discarded;

(8) drying at room temperature precipitation 4min adds the DEPC treating water, treat the RNA precipitation fully the dissolving back in-80 ℃ of preservations.

Detect the extraction effect of RNA by agarose gel electrophoresis.

2.cDNA first chain is synthetic

Use the High Fidelity PrimeScript of TaKaRa company ^TMThe reverse transcription test kit carries out the reverse transcription experiment.

(1) total RNA sex change is unwind, and adds following reaction solution in the PCR reaction tubes, and 65 ℃, after the 5min insulation, at pre-cooled 2min on ice;

Total RNA 1 μ L

Primer R 1 μ L

DNTP mixed solution 1 μ L

Random?6mers 1μL

DdH ₂O (not containing Rnase) 1 μ L

(2) reverse transcription reaction adds following reaction solution in the reaction system of unwinding behind ice bath, mixing gently, 42 ℃ of temperature bath 1h.

5 * PrimerScript RT damping fluid, 4 μ L

Rnase inhibitor 0.5 μ L

PrimeScript?Rtase 0.5μL

DdH ₂O (not containing Rnase) 5 μ L

After (3) 30 ℃ of temperature are bathed 10min, transfer 42 ℃ of temperature to and bathe 30min, at 95 ℃, cooled on ice behind the 5min, the product that obtains-20 a ℃ preservation is used for pcr amplification.

With the elastoser cDNA that extracts in the kidney is masterplate, carries out pcr amplification with the synthetic primer, thereby obtains target gene sequences.

(1) PCR 50 μ L reaction systems and amplification condition are as follows for the first time:

Primer P1, P2 are respectively:

5’-TACAGAACTCCCACGGACACAC-3’

5’-TTCCAAGTCTGAAACAGTCCCA-3’

Above-mentioned inverse transcription reaction liquid 2 μ L

5×PrimerSTAR 10μL

DNTP mixed solution 4 μ L

Primer P1 1 μ L

Primer P2 1 μ L

PrimerSTAR HS archaeal dna polymerase 0.5 μ L

ddH ₂O 31.5μL

Response procedures is: 94 ℃ of sex change 3min; 98 ℃ of sex change 10sec, 60 ℃ of annealing 5sec, 72 ℃ of annealing 90sec, 30 circulations; 72 ℃ are extended 90sec.

(2) PCR50 μ L reaction system and amplification condition are as follows for the second time:

Primer A1, A2 are respectively:

5’-TCCCACGGACACACCATGAT-3’

5’-GTTAGTTATTTGCAATCACCG-3’

Above-mentioned PCR reaction solution 1 μ L

5 * PrimerSTAR PCR damping fluid, 10 μ L

DNTP mixed solution 4 μ L

Primer A1 1 μ L

Primer A2 1 μ L

PrimerSTAR HS archaeal dna polymerase 0.5 μ L

ddH ₂O 32.5μL

Response procedures is: 94 ℃ of sex change 4min; 98 ℃ of sex change 10sec, 60 ℃ of annealing 15sec, 72 ℃ of annealing 60sec, 30 circulations; 72 ℃ are extended 10min.

Structure and the evaluation of embodiment 2 cloned plasmids pGEM-T easy/ELA2B

1. primer is synthetic

F:5 '-GCGC GAATTCTGTGGGGTCTCCACTTACG-3 ' (underscore is an EcoR I restriction enzyme site)

R:5 '-ATC GCGGCCGCTTAGTTATTTGCAATCACCGAATTG-3 ' (underscore is a Not I restriction enzyme site)

Primer is synthetic to be finished by the living worker in Shanghai company.

2. the pcr amplification of goal gene

The PCR reaction adopts 30 μ L systems to carry out, and is template with the pMD-T easy/ELA2B plasmid of recombinating, and adds successively in 0.5mL Eppendorf pipe:

Sterile distilled water 20.2 μ L

10 * Pyrobest damping fluid II (contains Mg ²⁺) 3 μ L

DNTP mixture (each 2.5mM) 2.4 μ L

Upstream primer (20pmol/ μ L) 1 μ L

Downstream primer (20pmol/ μ L) 1 μ L

Genomic dna (1 μ g/ μ L) 1 μ L

Pyrobest high-fidelity polysaccharase (5U/ μ L) 0.4 μ L

Amount to 30 μ L

Behind the instantaneous centrifugal mixing, increase: 94 ℃ of pre-sex change 4min by following reaction conditions; 98 ℃ of sex change 10s, 64 ℃ of renaturation 30s, 72 ℃ are extended 1min, 25 circulations; 72 ℃ are extended 10min and finish reaction.Detect the PCR product with 1% agarose gel electrophoresis.The result shows, the purpose band occurs at the 760bp place, conforms to expection.

3. reclaim the PCR product of ELA2B gene

PCR product with amplification ELA2B gene carries out electrophoresis with 1% sepharose.Under ultraviolet lamp, downcut the purpose band rapidly, reclaim the explanation of test kit (day root biochemical technology company limited) according to Tiangen DNA and reclaim.

4. be connected with pGEM-T easy carrier, make up plasmid pGEM-T easy/ELA2B

10 μ L systems are adopted in ligation, and all operation is all carried out on ice.In the Eppendorf of 0.5mL pipe, add successively:

Sterile distilled water 3 μ L

2 * Lig damping fluid, 5 μ L

pGEM-T?easy 0.5μL

Target DNA 0.5 μ L

T ₄Dna ligase 1 μ L

Amount to 10 μ L

More than each solution behind instantaneous centrifugal mixing, in the connection of spending the night of 4 ℃ of refrigerators.

5. connect product to the competent conversion of intestinal bacteria

It is centrifugal to managing at the end to connect product before uncapping, and adds in the Eppendorf pipe of 1.5mL in drawing 10 μ L connection product on ice; Take out-70 ℃ of frozen bacillus coli DH 5 alpha competent cells, put and make its slow thawing on ice, flick tube wall and make cytomixis even; Get 100 μ L competent cells and place pipe connecting, mixing places 30min on ice; Thermal shock 90s in 42 ℃ of water-baths, ice bath 2min immediately; Adding 800 μ L does not have additional antibiotic LB liquid nutrient medium, mixing, 37 ℃ of pre-45min that cultivate; Draw 200 μ L bacterium liquid and coat on the LB/Amp/IPTG/X-Gal flat board, cultivate 16-24h for 37 ℃, observe the colony growth situation, utilize blue hickie primary dcreening operation recombinant conversion body.

6. the evaluation of positive colony

(1) alkaline process extracts plasmid in a small amount

The single colony inoculation of picking white is in the 25mLLB/Kan liquid nutrient medium from the LB/Amp/IPTG/X-Gal flat board, 37 ℃ of shaken overnight.

Draw 1mL bacterium liquid in the Eppendorf of 1.5mL pipe, in 4 ℃, the centrifugal 5min of 12000 * g collects thalline.

With the resuspended bacterial sediment of 0.5mL STE solution (0.1M NaCl, 0.01M Tris-Cl (pH8.0), 0.001MEDTA (pH8.0)), in 4 ℃, the centrifugal 5min of 12000 * g, the supernatant that inclines, and make precipitation dry as far as possible.

100 μ L solution I (50mM glucose, 25mM Tris-Cl (pH8.0), 10mM EDTA (pH8.0)) with precooling (4 ℃ of refrigerators are placed and can directly be used) are resuspended, behind thermal agitation on the vibrator, leave standstill 5min on ice.

Add the solution II (0.2M NaOH, 1% (m/V) SDS) of the new configuration of 200 μ L, cover tight pipe cap, turn upside down gently fast 5 times, make it abundant mixing, will not vibrate, and place 5min on ice.

The solution III (5M sodium acetate 60.0mL, 11.5mL Glacial acetic acid, 28.5mL water) that adds 150 μ L precoolings, behind the gentle vibration 10s, ice bath 5min.

In 4 ℃, the centrifugal 5min of 15000 * g moves into supernatant in another clean centrifuge tube then.

Add isopyknic phenol-chloroform-primary isoamyl alcohol (25: 24: 1), behind the mixing, in 4 ℃, the centrifugal 5min of 15000 * g goes into the upper water phase transition in another clean centrifuge tube then gently.

With the isopropanol precipitating plasmid DNA of equal-volume or 2/3 volume, wait to turn upside down 4～5 times, fully behind the mixing, place 10min in-20 ℃.

In 4 ℃, the centrifugal 5min of 15000 * g inhales and removes supernatant.

70% ethanol vibration washing DNA with the 1mL precooling precipitates 1 time, in 4 ℃, and the centrifugal 2min of 15000 * g.

The supernatant that inclines behind the centrifugal again 2min, exhausts supernatant, eliminates the drop on the tube wall, and dry up 10min on Bechtop.

With 20 μ L sterilized water (RNaes that contains 20 μ g/mL) dissolving DNAs precipitation, behind 37 ℃ of insulation degraded 1h, behind 1% agarose electrophoresis detection plasmid, frozen standby in-20 ℃.

(2) the recombinant plasmid enzyme is cut evaluation

Sterile distilled water 9 μ L

DNA 7μL

10 * K damping fluid (TaKaRa company), 2 μ L

EcoR I (TaKaRa company) 1 μ L

Not I (TaKaRa company) 1 μ L

Amount to 20 μ L

More than each solution through instantaneous centrifugal after, in 37 ℃ of water-baths digestion, 1～3h, get 10 μ L products with 1% agarose gel electrophoresis detection enzyme slitting band, the result as shown in Figure 1.

(3) determined dna sequence and analysis

One of picked at random people source ELA2B recon is transferred to the Chinese Academy of Agricultural Sciences and is carried out sequencing, and utilizes DNAMAN software analysis sequencing results such as (Version5.0).ELA2B sequence on this sequencing result and the GenBank is compared, and the result as shown in Figure 3.

Structure and the evaluation of embodiment 3 recombinant expression plasmid pPIC9K/ELA2B

The PCR product of people source ELA2B gene and expression vector pPIC9K all are cut into two sticky ends with EcoR I and Not I, spend the night transformed into escherichia coli DH5 α with 4 ℃ of connections of T4 dna ligase.By the screening of ammonia benzyl resistance substratum, picking list bacterium colony is cultivated respectively, extracts plasmid, identifies the segmental insertion situation of external source through EcoR I and Not I double digestion.The clone of gene, recombinant screen all carry out with reference to the method for " molecular cloning) " (second edition) of Sambrook

1. the PCR primer design of band restriction enzyme site is synthetic

According to single endonuclease digestion site EcoRI on the expression plasmid pPIC9K multiple clone site (MCS) and the ELA2B sequence on Not I and the GenBank (Gene ID.51032) design primer, primer is synthetic to be finished by the living worker in Shanghai company.

Upstream primer: 5 '-GCGC GAATTCTGTGGGGTCTCCACTTACG-3 (underscore is an EcoR I restriction enzyme site)

Downstream primer: 5 '-ATC GCGGCCGCTTAGTTATTTGCAATCACCGAATTG-3 ' (underscore is a Not I restriction enzyme site)

2. adopt the high-fidelity enzyme to carry out pcr amplification

Adopt Pyrobest high-fidelity polysaccharase and primer to increase, the electrophoresis detection amplified production reclaims the PCR product.Method is with the description among the embodiment 2.

3. two doubly-linkeds of cutting make up pPIC9K/ELA2B

Pcr amplification product and expression plasmid pPIC9K with the ELA2B that reclaims, carry out double digestion with restriction enzyme EcoR I and Not I respectively, endonuclease reaction adopts 20 μ L systems, adds an amount of template according to template concentrations, each reacts 5 pipes, adds successively in 0.5mL Eppendorf pipe:

Sterilized water 7 μ L

10 * H damping fluid (TaKaRa company), 2 μ L

0.1％BSA 2μL

EcoR I (TaKaRa company) 1 μ L

NotI (TaKaRa company) 1 μ L

Each 7 μ L of plasmid pGEM-T/ELA2B/ plasmid pPIC9k

Amount to 20 μ L

More than each solution behind instantaneous centrifugal mixing, cut 3h in 37 ℃ of enzymes, two kinds of enzymes are cut product and are carried out 1% preparative agarose gel electrophoresis, reclaim dna segment respectively, the concentration of product is respectively reclaimed in the agarose gel electrophoresis guestimate, according to pPIC9K carrier segments and ELA2B gene fragment mol ratio 1: the ratio of (3～10) is carried out ligation, is undertaken by following reaction system:

Sterilized water 6.5 μ L

10 * connection buffer (Promage company), 2.5 μ L

PPIC9K carrier segments 5 μ L

ELA2B gene fragment 10 μ L

T4DNA ligase enzyme (Promage company) 2.5 μ L

More than each solution behind instantaneous centrifugal mixing, in 16 ℃ spend the night connect after, get 10 μ L and connect product transformed into escherichia coli DH5 α.The conversion operation step is with the description among the embodiment 2.

4. the evaluation of recombinant expression plasmid pPIC9K/ELA2B

(1) recombinant plasmid bacterium liquid PCR identifies

With the bacterium liquid of recombinant conversion bacterium template, increase with primer as the PCR reaction.Method is with the description among the embodiment 2.The recombinant expression plasmid that extracts detects by PCR method, has located the purpose band and increase out about 760bp, conforms to expected results.

(2) enzyme is cut evaluation

Carry out enzyme with restriction enzyme Not I and EcoR I and cut evaluation, enzyme is cut system with the description among the embodiment 2, and 37 ℃ of enzymes are cut 3h rear electrophoresis detection enzyme and cut situation.The result obtains two fragments of size for about 9300bp and about 760bp respectively as shown in Figure 2.

(3) order-checking and analysis

To cut correct recon through PCR and enzyme serves the sea and gives birth to the order-checking of worker biotech firm.Use DNAMAN (Version 5.0) software analysis sequencing result.Sequencing result is with cloning unanimity as a result, and base sequence does not change.

Embodiment 4 recombinant plasmid pPIC9K/ELA2B transform Pichi strain GS115

1. prepare the GS115 competent cell

(1) gets 100 μ L and insert in the fresh YEPD substratum of 2mL, after 28～30 ℃ of shaking table 180rpm incubated overnight, on fresh YEPD flat board, rule, cultivate 2 days to single bacterium colony appearance for 28～30 ℃ in-70 ℃ of frozen GS115 bacterial strains (American I nvitrogen company).

(2) single colony inoculation of picking is in the 5mLYEPD substratum, and 28～30 ℃ of shaking table 180rpm incubated overnight are taken out 1mL and are inoculated in that enlarged culturing is to OD600=1.3-1.5 in the 100mL YEPD substratum, and all the other glycerine of available 20% carry out culture presevation.

(3) in 4 ℃, the centrifugal 5min of 1500 * g collects somatic cells, and ices the resuspended somatic cells of precooling sterilized water with 100mL.

(4) in 4 ℃, the centrifugal 5min of 1500 * g collects somatic cells, again with the resuspended somatic cells of 50mL ice precooling sterilized water.

(5) in 4 ℃, the centrifugal 5min of 1500 * g collects somatic cells, ices the aseptic resuspended somatic cells of 1mol/L sorbyl alcohol of precooling again with 4mL.

(6) in 4 ℃, the centrifugal 5min of 1500 * g collects somatic cells, ices the aseptic resuspended somatic cells of 1mol/L sorbyl alcohol of precooling again with 0.2mL.Place standby on ice.

2. the preparation of electric transfering DNA sample

PPIC9K/ELA2B is carried out the linearizing endonuclease reaction with Sac I, promptly add successively in the Eppendorf of 0.5mL pipe: 2 μ L10 * enzyme cutting buffering liquids, 0.5 μ LBSA solution, 3 μ L recombinant expression plasmids, 1 μ L restriction enzyme Sac I add sterilized water at last to total system 20 μ L.Can do several pipes simultaneously more.Above solution behind instantaneous centrifugal mixing, is cut in 37 ℃ of enzymes and to be spent the night.Detect enzyme with 1% agarose gel electrophoresis and cut effect, if endonuclease reaction is complete, then each pipe is merged, adds isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, the centrifugal 5min of 10000g, get supernatant liquor, add the 3M sodium acetate soln (pH5.2) of 1/10 volume and the dehydrated alcohol of 2 times of volumes and carry out concentrating of linear plasmid, in-20 ℃ of precipitation 1h, the centrifugal 5min of 10000g, ethanolic soln washing precipitation with 70% ,-20 ℃ store for future use.

3. the electricity of pichia spp competent cell transforms

On Bio-Rad Gene Pulse electroporation, carry out electric conversion reaction.The electricity conversion condition is: voltage 1500V, and electric capacity 25 μ F, resistance 200 Ω, transformation time changes with the difference of DNA sample, and is provided automatically by instrument, usually in the 3.5-4.0s scope.

(1) linearizing DNA sample and the 0.2cm electricity conversion cup with pichia pastoris phaff competent cell, recombinant expression plasmid pPIC9K/ elastoser gene places on ice precooling 10min.

(2) in 0.2cm electricity conversion cup, 80 μ L competent cells and 10 μ g linear DNAs are mixed immediately, behind the ice bath 5min, dry electricity and transform cup, and place on the electroporation electric shock rapidly 1 time, the aseptic Sorbitol Solution USP that adds the precooling of 1mL 1mol/L ice then immediately, behind the mixing, change in the 1.5mL micro-centrifuge tube, place on ice again.

(3) getting 0.4mL electricity conversion product evenly coats on the MD flat board.

(4) according to the method described above, transform bacterial strain GS115, as the negative control of expressing with empty plasmid pPIC9K.

The screening of the proteic pichia yeast genetic engineering bacteria of embodiment 5 high expression level elastoser 2B

1. high copy pichia spp His ⁺The rapid screening of transformant

Plasmid pPIC9K has the kalamycin resistance gene of bacterium, makes it produce resistance to microbiotic G418 for yeast cell thereby can transmit resistance.The Pichi strain that transforms is cultivated in the substratum that contains different concns G418 respectively, and The selection result as shown in Figure 4.Along with the continuous increase of microbiotic G418 concentration, the Pichi strain that can survive reduces gradually, obtains the bacterial strain of the anti-high density G418 of 50 strains at last, and these bacterial strains contain high copy transformant probably.The concrete operations step comprises:

(1) with His ⁺The transformant point is connected on the fresh MD flat board, cultivates until transformant in 30 ℃ to occur.With transformant place 4 ℃ standby.

(2) utilize aseptic technique, in each hole of Tissue Culture Plate, add 200 μ LYPD substratum respectively.

(3) with aseptic toothpick with the His on the MD flat board ⁺Transformant inserts respectively in each hole, stirs.It should be noted that in operation to prevent living contaminants, also should avoid the crossed contamination between the transformant simultaneously.

(4) build the loam cake of Tissue Culture Plate, with first His ⁺Transform in 30 ℃ and leave standstill cultivation 2d.

(5) get the fresh Tissue Culture Plate of another piece, and in every hole, add 190 μ LYPD substratum respectively.

(6) in each hole, add 10 first His of μ L respectively according to order ⁺Transformant, with pipettor with its abundant mixing.

(7) build the loam cake of Tissue Culture Plate, with second crowd of His ⁺Transform in 30 ℃ and leave standstill cultivation 1d.

(8) loam cake of Tissue Culture Plate is built, with the 3rd crowd of His in repeating step (4) and (5) ⁺Transformant leaves standstill in 30 ℃ cultivates 1d.

(9), thereby make the 3rd crowd of His with 100 μ L pipettors pressure-vaccum pichia spp cell culture repeatedly up and down ⁺Transformant obtains fully to suspend.

(10) on different YPD/G418 flat boards (final concentration of G418 be respectively 0,0.50,1.00,2.00,4.00mg/mL) meets the identical His of 2 μ L respectively ⁺The transformant culture.

(11) leave standstill 30min, treat that bacteria suspension is blotted after, the YPD/G418 flat board be inverted is cultivated in 30 ℃, observed G418 resistance transformant at the 2nd, 3,4,5 day.

2. the rapid screening that has the high expression level pichia spp transformant of elastase activity

The elastoser of utilizing transformed yeast to express has protease activity widely, can contain the characteristic that forms transparent circle on the caseic flat board, adopts casein-MM flat board (to contain 1.34%YNB, 0.5% methyl alcohol, 4 * 10 ^-5% vitamin H, 1.5% skim-milk, 1.5% agar) method screen and produce the enzyme high transformant of living.The result has the yeast strain of empty carrier pPIC9K to grow on casein-MM flat board though change as shown in Figure 5, can not form transparent circle.According to the transparent circle size, can tentatively judge the height of yield of enzyme.

(1) with aseptic toothpick with the His on the MD flat board ⁺Transformant is put respectively and is connected on the fresh casein MM flat board, is inverted in 30 ℃ and cultivates.It should be noted that in operation and should prevent that living contaminants from also should avoid simultaneously the crossed contamination between the transformant.

(2) add methyl alcohol every 24h, the amount of adding methyl alcohol is the 2v/v% of culture volume, and the method for adding is pure methyl alcohol to be joined on the lid of substratum the inversion cultivation.

Observed caseic degraded situation in (3) the 2nd, 3,4,5 days.

The abduction delivering of embodiment 6 recombinant yeast pichia pastoris bacterium

1. the abduction delivering of recombinant pichia yeast strain

Pichi strain is rule on fresh YPD flat board,, be inverted and cultivated 2 days in 30 ℃.

Single bacterium colony of picking bacterial strain respectively is inoculated in 25mL BMGY and (in the substratum, cultivates until cell concn with the 500mL triangular flask and to reach OD ₆₀₀=2～6.Culture condition is: 28 ℃, shaking speed are 200rpm.Centrifugal 5min under room temperature 4000rpm condition collects mycetocyte, abandoning supernatant.

With 20mL BMMY substratum re-suspended cell, the beginning abduction delivering.The culture condition of abduction delivering is: 30 ℃, shaking speed is 250rpm, cultivates 6 days.

The 0.5mL that takes a sample every day, and to add 100% methyl alcohol to final concentration be 1v/v%.It should be noted that in adding methanol process the fermentating liquid volume that sample volume and evaporation bring changes, so that add amount of methanol.

0.5mL fermented liquid liquid is placed the 1.5mL micro-centrifuge tube, 4 ℃ of centrifugal 5min of 10000rpm.Supernatant liquor is transferred in another new centrifuge tube ,-20 ℃ of preservations are equipped with inspection again.

2. the SDS-PAGE of fermentation supernatant detects

The albumen sensibility reciprocal of SDS-PAGE is about tens microgram scopes, as long as this albumen has a certain amount of basal expression, no matter and its be activated or the albumen of non-activity, all can be verified by this method.

Prepare sample and low molecular weight protein (LMWP) Marker:

(1) supernatant liquor of each strain cultured solution is placed on ice thaws.

(2) in the 1.5mL micro-centrifuge tube,, in boiling water bath, boil 5min with 100 μ L supernatant liquors and 25 μ L, 5 * SDS sample buffer thorough mixing.

Make sample become skim in the bottom in hole when (3) going up sample, the sample applied sample amount is 20 μ L as far as possible, and low molecular weight protein (LMWP) Marker applied sample amount is 5 μ L.Vacant well need add isopyknic blank 1 * SDS sample buffer, to prevent the diffusion of neighbouring lane sample.

(4) carry out electrophoresis with the 80V constant voltage, after sample enters concentrated glue, voltage is risen to 120V.

(5) treat that bromjophenol blue runs the bottom to glue, take out gel, place sizeable container, cover gel, slowly shake 1～2h with staining fluid.

(6) staining fluid that inclines covers gel with destainer, slowly shakes about 2h, changes destainer therebetween 3～4 times, until blue clearly band of acquisition and clean background.

The SDS-PAGE electrophoresis result as shown in Figure 6.As can be seen from the figure, be that a protein band is clearly arranged about 28kDa at molecular weight, consistent with known elastin enzyme molecular weight, and foreign protein is seldom, and this will be very beneficial for proteic purifying.With band leader 3.0 softwares protein band is carried out scanning analysis, draw expressing protein and account for more than 90% of supernatant total protein.

3. the mensuration of fermented supernatant fluid protein content

Adopt the Brandford method to measure total protein content in the substratum.

(1) drafting of typical curve

A) standard protein solution: 100 μ g/mL bovine serum albumins (BSA)

B) Coomassie brilliant blue G-250 solution: claim 100mg Coomassie brilliant blue G-250 to be dissolved in 50mL90% ethanol, add the phosphoric acid of 100mL 85w/v%, be settled to 1L with distilled water, place brown bottle, normal temperature can be preserved 1 month.

C) drafting of typical curve

Get 6 test tube with ground stoppers, press table 1 and add following reagent:

Table 1

After above-mentioned each pipe mixed, Xiang Guanzhong added 5mL Coomassie brilliant blue G-250 solution, shakes up, and places 3min, measures light absorption value with 1cm optical path cuvette under 595nm, is X-coordinate with the protein concn, is ordinate zou drawing standard curve with the absorbancy.

(2) sample determination

Draw an amount of sample, adding distil water is mended to 1mL, adds 5mL Coomassie brilliant blue G-250 solution again, surveys absorbancy behind the reaction 3min under 595nm.Absorbancy and typical curve draw protein content in the sample solution per sample, and the content that draws the total protein in the substratum is 421 μ g/mL.The content that can estimate elastoser is about 375 μ g/mL.

The activity of embodiment 7 reorganization elastoser 2B detects

(1) elastin flat band method

Detect the activity of elastoser with the specific substrate elastin of elastoser 2B.Whether containing on the agar plate of elastin the Oxford cup of placing the bacterium of going out, adding the fermented supernatant fluid of 200 μ L with rifle, placing 48h at 37 ℃ of incubators, observing has transparent degraded circle to form on the elastin agar plate.Reorganization elastoser 2B to the degraded of elastin plate as shown in Figure 8,1～4 fermented supernatant fluid of representing behind the recombination yeast abduction delivering of different batches wherein; CK represents the not recombination microzyme fermented supernatant fluid of abduction delivering.

(2) absorbance method

Accurately take by weighing 10mg Congo red-elastin, add pH7.4,0.2mol/L borate buffer 2mL, add the last clear enzyme solution of 1mL again, behind 28 ℃ of oscillatory reaction 3h, use pH6.0 immediately through suitably diluting, concentration is the phosphoric acid buffer 2mL termination reaction of 0.7mol/L, behind the centrifugal 5min of 10000r/min, get the mensuration optical density value of supernatant liquor in the 495nm place, with the last clear enzyme solution that do not add substrate as blank.Under this reaction conditions, every 5mL reaction mixture 495nm place increases by 0.01 required enzyme amount of absorbancy and is defined as 1 elastin activity unit (u):

Elastase activity (U/mL)=(OD of sample ₄₉₅The OD of-blank ₄₉₅) * 100 * extension rate

Embodiment 5 obtains the enzyme of bacterial strain thick enzyme in fermented liquid of the anti-high density G418 of 50 strains and lives to being up to 980U/mL, and minimum is 260U/mL, lives high 24 times and 6 times than the enzyme of Pseudomonas aeruginosa tunning respectively.

The purifying of embodiment 8 reorganization elastoser 2B

1, the preparation of sample

(1) activation of dialysis tubing

Dialysis tubing is placed alkaline EDTA solution (NaHCO ₃20g/L, EDTA 1mmol/L, pH 8.0) in boil 30min, clean with distilled water wash then.

(2) dialysis of fermented liquid supernatant

Collect fermented liquid supernatant, be loaded in the dialysis tubing, use distill water dialysis 24h, during replacing distilled water 3～4 times.

(3) fermented liquid supernatant after the collection dialysis is adjusted Tris concentration to 0.02M according to dialysis back volume, and pH is 7.2 (identical with level pad), sample in the preparation.

2, go up sample

Ready protein sample is flow through liquid with the flow velocity of 1.3mL/min with peristaltic pump, and whether flexible protease activity judges whether elastoser is attached on the post in the effluent liquid thereby detect.

3, drip washing

With level pad drip washing chromatography column, detect the absorbancy of effluent liquid at the 280nm place, reach below 0.1 until A280.

4, gradient elution

Successively with containing 0.2M NaCl, 0.3M NaCl, 0.4M NaCl, 0.5M NaCl, the elution buffer of 0.8M NaCl carries out the stage gradient wash-out, 2～3 column volumes of each concentration wash-out, the control flow velocity is 1.3mL/min, collects (every pipe 2～3mL) step by step with clean test tube.Detect every pipe A280 and elastase activity.

Wash-out purifying figure verifies that by the SDS-PAGE electrophoresis proteic purity reaches more than 97% as shown in Figure 7.

Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (7)

1. contain the ELA2B expression carrier, described ELA2B gene has the nucleotide sequence shown in the Seq ID No:1.

2. expression vector as claimed in claim 1 is characterized in that, the carrier that sets out is pPIC9K.

3. contain the engineering bacteria of the described expression vector of claim 2, it is characterized in that, starting strain is a pichia spp.

4. engineering bacteria as claimed in claim 3 is characterized in that, starting strain is pichia spp GS115.

5. make up the method for claim 3 or 4 described engineering bacterias, it is characterized in that it comprises step:

1) from the human kidney tissue, extracts total RNA, obtain the ELA2B gene through the RT-PCR amplification;

2) the ELA2B gene that obtains in the step 1) is inserted in the expression vector;

3) with step 2) the middle expression vector that makes up changes pichia spp over to, and screening positive clone, obtains the recombinant yeast pichia pastoris engineering bacteria.

6. method according to claim 5, it is characterized in that, screening positive clone comprises described in the step 3): with MD substratum preliminary screening positive colony, carry out postsearch screening with the G418 resistance again, obtain having the clone of elastin gene high copy number, adopt the dull and stereotyped revulsion of casein-MM at last, screening obtains the recombinant bacterial strain of high secreting, expressing.

7. claim 3 or the application of 4 described engineering bacterias in production elastoser 2B.

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