CN101240284B - Recombination staphylokinase and highly effective secretion expression method thereof - Google Patents

Abstract

A staphylococci kinase and a method for high efficiency secreting and expressing recombination staphylococci kinase in biotechnology are provided. It obtains glucose kinase (Sak) containing signal peptide by using total DNA of staphylococcus aureus as form through PCR amplification. The 33th, 34th and 36th bite amino acid of the gene order are different from the reported Sak gene. Connecting the gene to carrier Pgex-4T-1, then transforming host bacterium to obtain recombination engineering bacterium. The recombination engineering bacterium can secreted express recombination glucose kinase having the function of fiber dissolvedzymogen activation by IPTG induction. The recombination glucose kinase expressed in the method of the invention has high gene expression and expressed outcome mainly is secretion type albumen which exists in thalli outer cytoplasm or outer cell. Thus, purifying process is simple, yield is high and production cost is low.

Description

The method of a kind of STAPHYLOKINASE and efficient secretory expression thereof

Technical field

The present invention relates to a kind of biological bacterial strain and preparation method thereof, specifically a kind of staphylokinase and utilize the method for biotechnology efficient secretory expression reorganization staphylokinase.

Background technology

(Staphylokinase is a kind of exocrine protein that is produced by some streptococcus aureus (Staphylococcus aureus) SAK) to staphylokinase, and relative molecular weight is 15.5-18.0kDa.As far back as 1948; Lack finds have a kind of protein (SAK) can dissolve human thrombomodulin in the streptococcus aureus culture supernatants; Just caused many scholars' interest, but because natural SAK originates less, not high further research and the application that has always limited it of preparation purity.Recent study person has successfully cloned the sak gene; And in intestinal bacteria, Bacillus subtilus, suis and lactococcus spp, expressed SAK albumen preferably, from then on be that the thrombolytic effect mechanism of the relevant STAPHYLOKINASE of purpose, the research of 26S Proteasome Structure and Function and the protein engineering research of setting up on this basis become one of focus of the research of novel thrombolytic agent in recent years with the clinical application.Research about SAK makes people recognize that it is a kind of very promising thrombolysis medicine; (the tissue-type plasminogen activator of its thrombolysis effect and tissue plasminogen activator; T-PA) close; But the selectivity to thrombus is higher than t-PA, and in intestinal bacteria, can realize low-cost scale operation, and market value is far below t-PA.As thrombolytic drug of new generation, SAK is high with its specificity, and the characteristics that hemorrhage side effect is little get more and more people's extensive concerning.In animal model, SAK shows than urokinase (UK) and streptokinase (SK) better application effect, so its application prospect is expected very much.Exist with inclusion body but reorganization SAK is many in the early stage report, must be through just obtaining activated expression product after sex change, the renaturation; Domestic early stage achievement in research has been taken the New Drug Certificate that the first in the world is opened reorganization SAK in 2005, but this and fail to promote the application of SAK, the paying capacity that this and its price still is higher than common patient is undivided; From streptococcus aureus, separate glucokinase gene so this research is intended, make up the engineering bacteria that its prokaryotic expression plasmid and expectation obtain its secreting, expressing in intestinal bacteria, for production cost and the clinical application that reduces staphylokinase lays the first stone.Sako equals nineteen eighty-three and has at first cloned the sak gene, and in intestinal bacteria, realizes expressing.Increasing about the research of sak clone and expression aspect subsequently.(High level secretion of recombinant staphylokinase into periplasm of Escherichia coli such as Sang Jun Lee; Biotechnology Letters; 1998) disclose a kind of in intestinal bacteria the method for secreting, expressing staphylokinase; Its formation is: from the golden bacterial strain NCTC10033 of Portugal that contains the sak gene, obtain a large amount of saks through pcr amplification with round pcr; And insert the expression vector pKK-ompA that contains a tac promotor and an ompA signal sequence, and being transformed into e. coli jm109, JM109 carries the recombinant plasmid secretion and produces reorganization SAK:15mg/L to periplasm; 5mg/L is outside born of the same parents, and weak point is that output is lower.(new type glucokinase Δ NMSak the efficiently expressing and separation and purification in intestinal bacteria of reduced immunogenicity such as Song Gang; Chinese biological chemistry and molecular biosciences journal, 2000) a kind of method that in intestinal bacteria, efficiently expresses the new type glucokinase of low epidemic focus property is disclosed, its formation is: obtain Δ NMSak after staphylokinase N end deletion mutant (Δ NSak) cDNA that has made up is transformed; Transform JF1125 after inserting prokaryotic expression carrier pLY-4; Through temperature-induced, recombinant protein accounts for 60% of bacterial protein, exists with the inclusion body form; Weak point is to exist with the inclusion body form, must be through just obtaining activated expression product after sex change, the renaturation.

Summary of the invention

The object of the invention is exactly the defective to prior art, a kind of staphylokinase is provided and utilizes the method for biotechnology efficient secretory expression reorganization staphylokinase, and it has overcome the shortcoming that the prior art expression amount is low and yield poorly.

Biological sample preserving number of the present invention is CCTCC NO:M207112; The biological sample name is called: e. coli bl21-EpSS/pGEX-4T-1; Depositary institution is Chinese typical culture collection center, the address: in the Wuhan University of Wuhan City, Hubei Province, and postcode: 430072; Preservation date is on July 23rd, 2007, and its preparation method comprises following step:

(1) amplification glucokinase gene

Buying streptococcus aureus (Staphylococcus aureus) AS1.879 from Chinese DSMZ, is template with this strain chromosome DNA, is used for the upstream primer of PCR according to the sequences Design of known glucokinase gene (containing signal peptide):

5’-GC GAATTCATGCTCAAAAGAAGTTTAT-3’；

And downstream primer:

5’-CC GTCGACTTATTTCTTTTCTATAACAAC-3’。

Wherein GAATTC is the EcoRI restriction enzyme site, and GTCGAC is the SalI restriction enzyme site, and ATG is an initiator codon, and TTA is a terminator codon.Through the PCR glucokinase gene that directly increases, after the reorganization of pMD-18T plasmid vector, obtain following sequence through nucleotide sequence analysis:

tca agt tca ttc gac aaa gga aaa tat aaa aaa ggc gat gac gcg agt 48

Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala Ser

1 5 10 15

tat ttt gaa cca aca ggc ccg tat ttg atg gta aat gtg act gga gtt 96

Tyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val

20 25 30

20 25 30

gag ggt aaa gaa aat gaa ttg cta tcc cct cat tat gtc gag ttt cct 144

Glu Gly Lys Glu Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro

35 40 45

att aaa cct ggg act aca ctt aca aaa gaa aaa att gaa tac tat gtc 192

Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val

50 55 60

gaa tgg gca tta gat gcg aca gca tat aaa gag ttt aga gta gtt gaa 240

Glu Trp Ala Leu Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu

65 70 75 80

tta gat cca agc gca aag atc gaa gtc act tat tat gat aag aat aag 288

Leu Asp Pro Ser Ala Lys Ile Glu Val Thr Tyr Tyr Asp Lys Asn Lys

85 90 95

aaa aaa gaa gaa acg aag tct ttc cct ata aca gaa aaa ggt ttt gtt 336

Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val

100 105 110

gtc cca gat tta tca gag cat att aaa aac cct gga ttc aac tta att 384

Val Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile

115 120 125

aca aag gtt gtt ata gaa aag aaa taa 411

Thr Lys Val Val Ile Glu Lys Lys

130 135

Do not exist together with the sequence of other patent reports: the 99th Nucleotide is G; The 100th Nucleotide is G; The 107th Nucleotide is A.The 33rd amino acids is GLU; The 34th amino acids is GLY; The 36th amino acids is GLU.

(2) glucokinase gene with amplification is connected with carrier pGEX-4T-1, is built into expression plasmid

After above-mentioned clone's glucokinase gene and prokaryotic expression plasmid pGEX-4T-1 used the two enzymes of EcoRI/SalI respectively, connect then, be built into expression plasmid and called after pSS.

(3) with constructed expression plasmid transformed into escherichia coli E.coli (Escherichia coli) BL21

Expression plasmid transformed into escherichia coli E.coli BL21 with above-mentioned structure induces the r-Sak expression of gene with IPTG, engineering bacteria called after EpSS.

(4) intestinal bacteria of culture transformation

EpSS at the initial pH6.6 of substratum, sucrose 2%, induce before cell density be 0.8, the IPTG final concentration is 0.8mmol/L, 25 ℃ induce under the 9h condition that the STAPHYLOKINASE activity all reaches mxm. outside the kytoplasm and born of the same parents.

The present invention makes up gene with PCR, and gene expression dose is high, and r-SAK expresses with the secretion mode, is present in outside outer kytoplasm of thalline and the born of the same parents with active condition, and purification process is simple, and output is high, and cost is low.At expression in escherichia coli STAPHYLOKINASE albumen, expression product is expressed with the secretion mode with method of the present invention, about 20% (300mg/L) in the wherein outer kytoplasm, and outer about 50% (40mg/L) of born of the same parents, and ultimate production is about 340mg/L.Present method not only at expression in escherichia coli solubility, activated staphylokinase, and the expression amount of secreting, expressing and output all are higher than currently known methods.

Figure of description

Fig. 1. be the pSS construction of recombinant plasmid.

Fig. 2. be that EpSS born of the same parents express product evaluation, wherein M outward: albumen Marker, 1: through inductive Ep6EX; The extracellular protein that 2:EpSS expresses, soluble proteins in the born of the same parents that 3:EpSS expresses.

Fig. 3. be expression product evaluation, wherein M in the outer kytoplasm of EpSS: albumen Marker, 1: through inductive EpGEX; Soluble proteins in the born of the same parents that 2:EpSS expresses.

Fig. 4. be that the external thrombolysis activity of expression product is identified.1.2:SAK standard substance wherein, 3.4: negative control, 5.6: soluble proteins in the born of the same parents, 7.8: fermented liquid supernatant (extracellular protein)

Embodiment

Below through embodiment the present invention is described in detail:

Embodiment one: the structure of Sak genetic engineering bacterium

1, design of primers and pcr amplification

Contain the primer of EcoRI restriction enzyme site, promotor and staphylokinase signal peptide according to the sequences Design upper reaches of known glucokinase gene (containing signal peptide), its sequence is:

5’-GCGAATTCATGCTCAAAAGAAGTTTAT-3’；

Contain the primer of SalI restriction enzyme site and terminator with downstream:

5’-CC GTCGACTTATTTCTTTTCTATAACAAC-3’。

Wherein GAATTC is the EcoRI restriction enzyme site, and GTCGAC is the SalI restriction enzyme site, and ATG is an initiator codon, and TTA is a terminator codon.

After primer is synthetic, be template with golden yellow staphylococcus AS1.879 strain chromosome DNA, loop parameter is: 94 ℃ 45 seconds-45 ℃ 60 seconds-70 ℃ 70 seconds, through 30 circulations, obtain the sak gene fragment.

2, cDNA clone and order-checking

The sak gene fragment that pcr amplification is obtained is after 1% sepharose reclaims purifying, and with vector plasmid pMD-18T reorganization, transformed into escherichia coli filters out the positive strain that contains the sak gene, called after pTSS (Fig. 1).It is carried out determined dna sequence, consistent with expection.

3, the expression of cDNA in intestinal bacteria

The pTSS plasmid reclaims the sak gene fragment through the EcoRI/SalI double digestion.Prokaryotic expression carrier pGEX-4T-1 reclaims big fragment through the EcoRI/SalI double digestion, with the sak recombination of reclaiming, transformed into escherichia coli (Fig. 2).With penbritin-LB plate screening, the single bacterium colony of picking, the preparation plasmid carries out PCR and enzyme and cuts evaluation, contains the plasmid called after pSS of sak gene expression characteristics, bacterial strain called after EpSS.EpSS is cultured to OD at 37 ℃ ₆₀₀After about 0.6, after adding IPTG 0.1mM (final concentration) and inducing 4-6 hour, centrifugal collection bacterium also keeps fermented liquid supernatant, respectively bacterial sediment (Fig. 3) and fermented liquid supernatant (Fig. 4) is carried out SDS-PAGE and analyzes.

4, expression product is measured:

(1) existence is measured: the supernatant of culture medium after engineering bacteria is induced is carried out the biological activity determination positive, and the differential protein band of the 15.5kDa that has an appointment in the SDS-PAGE demonstration supernatant of culture medium, explains that expression product exists with secretor state; Engineering bacteria ultrasonication to after inducing is centrifugal, and centrifugal supernatant carries out determination of activity and SDS-PAGE respectively to be analyzed, and the result shows that expression product exists with soluble, active condition.

(2) biological activity determination:

A. the preparation of typical curve: take by weighing the 0.1g agarose and add in the 18.6mL sterile saline, balance in the rearmounted 56 ℃ of water-baths of heating for dissolving adds 100IU/mL zymoplasm 11.2 μ L; 0.5mg/mL human plasminogen 224 μ L behind the human fibrinogen 1.76mL of adding 6mg/mL, do not stop to shake up and treat to pour into immediately in the ready protein electrophoresis plate after the muddiness behind the mixing; After fully solidifying; After placing 30min in 4 ℃ of refrigerators, beat the 2mm hole, every hole adds the SAK standard substance of the different weaker concns of 2.5 μ L; Do multiple hole, put in the wet box 37 ℃ and hatch 12h.To measuring the solusphere diameter, average in length and breadth, the logarithm of corresponding solusphere diameter is carried out straight-line regression, try to achieve regression equation with the logarithm of each dilution BA of standard solution.

B. sample determination: in kind prepare scleroproein plate, punching.Engineering bacteria after inducing is centrifugal, leave and take the engineering bacteria supernatant after supernatant of culture medium and the ultrasonication, and 10 times of the dilutions of the engineering bacteria supernatant after the ultrasonication is subsequent use.Every hole adds ready protein sample 2.5 μ L, puts in the wet box 37 ℃ and measures the solusphere diameter after hatching 12h.Each sample is done three times and is averaged, and asks the BA of trial-product according to the logarithm of the solusphere diameter of trial-product.The gross activity that records is the 2435AU/mL engineering bacteria.

Embodiment two: the preliminary study of EpSS engineering bacteria inductive condition

According to the factor that relates in the engineering bacteria EpSS culturing process, select IPTG concentration (mmol/L), induction time (h), OD ₆₀₀Sucrose content and inducing temperature in the pH value of absorbancy, substratum, the substratum (℃) six factors, every factor selects several levels to do experiment of single factor according to practical situation.The protein sample that the experiment of single factor of being done is obtained carries out determination of activity; Obtain EpSS at the initial pH6.6 of substratum, sucrose 2%, induce before cell density be 0.8, the IPTG final concentration is 0.8mmol/L, 25 ℃ induce under the 9h condition that the STAPHYLOKINASE activity all reaches mxm. outside the kytoplasm and born of the same parents, every liter of fermented liquid can be gathered in the crops 2.435*10 ⁶The STAPHYLOKINASE albumen of AU.

SSAK-DNAAA-SEQUENCE.ST25.seq

SEQUENCE LISTING

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<170>PatentIn version 3.3

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tca agt tca ttc gac aaa gga aaa tat aaa aaa ggc gat gac gcg agt 48

Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala Ser

1 5 10 15

tat ttt gaa cca aca ggc ccg tat ttg atg gta aat gtg act gga gtt 96

Tyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val

20 25 30

gag ggt aaa gaa aat gaa ttg cta tcc cct cat tat gtc gag ttt cct 144

Glu Gly Lys Glu Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro

35 40 45

att aaa cct ggg act aca ctt aca aaa gaa aaa att gaa tac tat gtc 192

Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val

50 55 60

gaa tgg gca tta gat gcg aca gca tat aaa gag ttt aga gta gtt gaa 240

Glu Trp Ala Leu Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu

65 70 75 80

tta gat cca agc gca aag ate gaa gtc act tat tat gat aag aat aag 288

Leu Asp Pro Ser Ala Lys Ile Glu Val Thr Tyr Tyr Asp Lys Asn Lys

85 90 95

aaa aaa gaa gaa acg aag tct ttc cct ata aca gaa aaa ggt ttt gtt 336

Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val

100 105 110

gtc cca gat tta tca gag cat att aaa aac cct gga ttc aac tta att 384

Val Pro Asp Leu Set Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile

115 120 125

aca aag gtt gtt ata gaa aag aaa taa 411

Thr Lys Val Val Ile Glu Lys Lys

130 135

Claims (3)

1. the method for efficient secretory expression reorganization staphylokinase, it comprises following step:

(1) the strain chromosome DNA with streptococcus aureus (Staphylococcus aureus) AS1.879 is a template, with 5 '-GC GAATTCATGCTCAAAAGAAGTTTAT-3 ' is as upstream primer, with 5 '-CC GTCGACTTATTTCTTTTCTATAACAAC-3 ' carries out pcr amplification as downstream primer; Wherein GAATTC is the EcoRI restriction enzyme site, and GTCGAC is the SalI restriction enzyme site, and ATG is an initiator codon; TTA is a terminator codon; After primer is synthetic, be template, obtain the glucokinase gene gene fragment with golden yellow staphylococcus AS1.879 strain chromosome DNA;

(2) above-mentioned clone's glucokinase gene and prokaryotic expression plasmid pGEX-4T-1 are used the two enzymes of EcoRI/SalI respectively after, connect then, be built into expression plasmid and called after pSS;

(3) with expression plasmid transformed into escherichia coli (Escherichia coli) BL21 of above-mentioned structure, induce the r-Sak expression of gene with IPTG, engineering bacteria called after EpSS;

(4) filter out the positive strain that contains the sak gene, obtain staphylokinase.

2. the method for a kind of efficient secretory expression reorganization staphylokinase according to claim 1; After wherein primer synthesizes; With golden yellow staphylococcus AS1.879 strain chromosome DNA is template; Loop parameter is: 94 ℃ 45 seconds-45 ℃ 60 seconds-70 ℃ 70 seconds, through 30 circulations, obtain the sak gene fragment.

3. the method for a kind of efficient secretory expression reorganization staphylokinase according to claim 1, wherein EpSS at the initial pH6.6 of substratum, sucrose 2%, induce before cell density be OD ₆₀₀=0.8, the IPTG final concentration is that 0.8mmol/L, 25 ℃ induce under the 9h condition and express.

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