CN1207414A

CN1207414A - Method for producing glucokinase by gene engrg. technique

Info

Publication number: CN1207414A
Application number: CN 98102132
Authority: CN
Inventors: 李倩
Original assignee: Institute of Microbiology and Epidemiology of AMMS
Current assignee: Institute of Microbiology and Epidemiology of AMMS
Priority date: 1998-05-11
Filing date: 1998-05-11
Publication date: 1999-02-10
Anticipated expiration: 2018-05-11
Also published as: CN1064406C

Abstract

A process for producing glucokinase with gene engineering technique features use of recombined bacterial strain to express glucokinase protein. Connecting cloned glucokinase gene with vector pBV220 can construct expression plasmids, which is then used to transform colibacillus to construct engineering bacterial strains of recombined glucokinase. The expressed amount of glucokinase is 60-70% of total protein of said bacterial strains.

Description

Utilize genetic engineering technique to produce the method for staphylokinase

The present invention relates to a kind of method of producing staphylokinase, specifically, the present invention relates to utilize genetic engineering technique to produce the method for staphylokinase, and the recombinant expression plasmid and the glucokinase gene that are used to express staphylokinase.

Staphylokinase (Staphylokinase, Sak) a kind of proteolytic ferment that produces for some streptococcus aureus.It is a kind of thrombolysis new drug, especially when hematoblastic coronary artery thrombosis is rich in dissolving, and better effects if.

The relevant genetic engineering technique that utilizes is produced the existing report of staphylokinase.For example, Sako etc. inserts the Sak gene and contains lambda particles phage P _RThe expression plasmid of promotor, transformed into escherichia coli, the expression amount of target protein accounts for 25% (Sako, T, et al, Eur.J.Biochem (1985) 149:557-563) of bacterial protein after inducing; Schlott, the staphylokinase engineering strain expression amount of structures such as B also only account for the 10-15% (Schlott, B et al, Bio-Technology (1992) 12:185-189) of bacterial protein; Swallow etc. is with staphylokinase cDNA and plasmid vector Ply-4 reorganization behind Song of Shanghai Medical Univ, and transformed into escherichia coli induces the back expression amount to account for 40% (CN 1096325A, CN 1096326A) of tropina.

It is higher to the purpose of this invention is to provide a kind of expression amount, active better, more stable engineering strain, thus a kind of method that efficiently expresses staphylokinase is provided.

Through discovering, glucokinase gene is connected with vector plasmid pBV220, and after transforming intestinal bacteria, can obtains to efficiently express the engineering strain of staphylokinase.

Method of the present invention may further comprise the steps:

(1) amplification glucokinase gene;

(2) kinase gene with amplification is connected with carrier pBV220, is built into expression plasmid;

(3) with constructed expression plasmid transformed into escherichia coli;

(4) cultivate the intestinal bacteria that transform;

(5) separate the also staphylokinase of purifying expression.

Described glucokinase gene can be any known glucokinase gene.

In the present invention, we are separated to 9 strain hemolytic streptococcus aureuses from Burn Ward burn patient infective wound surface, be seeded in respectively on the agar plate that contains human plasma, select a strain fibrinolytic the highest as the pcr amplification template, obtain the cDNA fragment of staphylokinase.Find that through sequencing this cDNA is a kind of new dna sequence dna of glucokinase gene, and is different with known sequences.

The amplification of glucokinase gene can use the method for PCR to carry out, and can be used for the upstream primer of PCR according to the sequences Design of known glucokinase gene, and its sequence is:

5 ' CAC GAA TTC ATG TCA AGT TCA TTC GAC AAA 3 ': and downstream primer, its sequence is:

5’GGC?GAA?TTC?TTA?TTT?CTT?TTC?TAT?AAC?AAC?3’。Wherein GAA TTC is an EcoR I restriction enzyme site; ATG is an initiator codon; TTA is a terminator codon.Carry out the pcr gene amplification according to known method then.

Carrier pBV220 is a kind of known carrier (Zhang Zhiqing etc., viral journal, 6 (2): 111-116,1990).

On the pBV220 vector plasmid, the restriction enzyme site of adjacent SD sequence has eight, and they are successively in proper order: EcoR I, Sma I, BamH I, Sal I, Hinc II, Pst I, HindIII, Xmn I.Generally speaking, the translation skill of the distance meeting remarkably influenced target protein matter of SD sequence and initiator codon.When design of primers, we have designed the single restriction enzyme site of EcoR I at upstream and downstream primer two ends, and Sak cDNA inserts pBV220 EcoR I restriction enzyme site after EcoR I enzyme is cut like this, and is nearest with the SD sequence, can improve the translation skill of target protein.Glucokinase gene and carrier PBV220 with amplification cuts with EcoR I enzyme respectively then, connects then, is built into expression plasmid.

Described intestinal bacteria can be any known coli strains, preferred DH5 α bacterial strain.

The present invention also provides a kind of new glucokinase gene and encoded protein matter thereof.：ATG TCA AGT TCA TTC GAC AAA GGA AAA TATMet Ser Ser Ser Phe Asp Lys Gly Lys TyrAAA AAA GGC GAT GAC GCG AGT TAT TTT GAALys Lys Gly Asp Asp Als Ser Tyt Phe GluCCA ACA GGC CCG TAT TTG ATG GTA AAT GTGPro Thr Gly Pro Tyt Leu Met Val Asn ValACT GGA GTT GAT GGT AAA GGA AAT GAA TTGThr Gly Val Asp Gly Lys Gly Asn Glu LeuCTA TCC CCT CGT TAT GTC GAG TTT CCT ATTLeu Ser Pro Arg Tyr Val Glu Phe Pro IleAAA CCT GGG ACT ACA CTT ACA AAA GAA AAALys Pro Gly Thr Thr Leu Thr Lys Glu LysATT GAA TAC TAT GTC GAA TGG GCA TTA GATIle Glu Tyr Tyr Val Glu Trp Ala Leu AspGCG ACA GCA TAT AAA GAG TTT AGA GTA GTTAla Thr Ala Tyr Lys Glu Phe Arg Val ValGAA TTA GAT CCA AGC GCA AAG ATC GAA GTCGlu Leu Asp Pro Ser Ala Lys Ile Glu ValACT TAT TAT GAT AAG AAT AAG AAA AAA GAAThr Tyr Tyr Asp Lys Asn Lys Lys Lys GluGAA ACG AAG TCT TTC CCT ATA ACA GAA AAAGlu Thr Lys Ser Phe Pro Ile The Glu LysGGT TTT GTT GTC CCA GAT TTA TCA GAG CATGly Phe Val Val Pro Asp Leu Ser Glu HisATT AAA AAC CCT GGA TTC AAC TTA ATT ACAIle Lys Asn Pro Gly Phe Asn Leu Ile ThrAAG GTT GTT ATA CAA AAG AAA TAALys Val Val Tle Glu Lys Lys

The present invention also provides a kind of recombinant expression vector, and it is the pBV220 carrier that contains glucokinase gene.

Described coli strain can be any known coli strain that is used for expression alien gene, preferred DH5 α bacterial strain.

In expression in escherichia coli sak protein matter, its expression amount reaches the 60-70% of bacterial protein with method of the present invention, apparently higher than the expression amount of currently known methods.

Brief Description Of Drawings:

Fig. 1 .Puc-Sak construction of recombinant plasmid.

Fig. 2. the dna sequence dna that a kind of recombinant staphylokinase is new.

Fig. 3 .pBV-Sak construction of recombinant plasmid.

Fig. 4 .Sak expression product is identified.Wherein swimming is with 1: without the inductive engineering strain, swimming is with 2:

Engineering bacteria 1, swimming is with 3: engineering bacteria 2, swimming is with 4: molecular weight is 14.5kd's

Standard protein.

Fig. 5. to the scanning of Fig. 4 dual-wavelength scanner perspective.

Fig. 6 .r-Sak surveys the purity result with HPLC behind the gel column purifying.

Embodiment

By following example technical characterictic of the present invention is described in detail embodiment one: the screening of goal gene

Be separated to 9 strain streptococcus aureuses from certain hospital's Burn Ward, it is inoculated in respectively on the agarose plate that contains blood plasma.Make negative control with host cell.Agarose plate making method is: 1% agarose 20ml boils, put 56 ℃ standby, get the 5ml human plasma be stored in 56 ℃ 20 minutes, fall dull and stereotyped behind the two mixing.After the cooling bacterium to be measured and negative control bacterium are inoculated in this plate, cultivated liquid for 37 ℃, observe the dissolving circle of periphery of bacterial colonies.Negative control does not have, and in the 9 strains bacterium to be measured, has 3 strains that dissolving circle is arranged, choosing wherein the dissolving circle maximum as the pcr amplification template.Embodiment two: the structure of Sak engineering strain

1, design of primers and pcr amplification

Contain the primer of EcoR I restriction enzyme site and promotor according to the sequences Design upstream of known glucokinase gene, its sequence is:

5 ' CAC GAA TTC ATG TCA AGT TCA TTC GAC AAA 3 '; The primer that contains EcoR I restriction enzyme site and terminator with the downstream:

5’GGC?GAA?TTC?TTA?TTT?CTT?TTC?TAT?AAC?AAC?3’。Wherein GAA TTC is an EcoR I restriction enzyme site; ATG is an initiator codon; TTA is a terminator codon.

After the design of primers, look into the no dimer of 3 ' end through the DNA database.After primer is synthetic, with the highest staphylococcic bacterial lysate of fibrinolytic as template, loop parameter be 94 ℃ 60 seconds-45 ℃ 40 seconds-72 ℃ 60 seconds, through 30 circulations, obtain the Sak gene fragment.

2, cDNA clone and order-checking

The Sak pcr amplified fragment is cut through EcoR I enzyme, reclaim, with cut through EcoR I enzyme after vector plasmid Puc18 reorganization, transformed into escherichia coli filters out the positive strain that contains the Sak gene, called after Puc-Sak (Fig. 1), it is carried out determined dna sequence, and dna sequence dna is a kind of new sequence (Fig. 2).

3, the expression of cDNA in intestinal bacteria

The Puc-Sak plasmid is cut through enzyme, reclaims the Sak gene fragment.Prokaryotic expression carrier pBV220 is after enzyme is cut, with the Sak gene fragment reorganization of reclaiming, transformed into escherichia coli (Fig. 3).Same penbritin-LB plate screening, the single bacterium colony of picking, the preparation plasmid, enzyme is cut evaluation, contains Sak gene expression characteristics person, called after pBV-Sak.With pBV-Sak 30 ℃ be cultured to OD600 0.4-0.6 after, induced 4-6 hour for 42 ℃, centrifugal collection bacterium carries out SDS-gel electrophoresis (Fig. 4), with the scanning of dual-wavelength lamellar scanning instrument, Computer Processing records the 60-70% (Fig. 5) that target protein accounts for bacterial protein.

4, expression product is measured:

＜1〉existence is measured: the substratum supernatant after engineering bacteria is induced carries out the biological activity determination feminine gender, has got rid of expression product and has existed with secretor state; Engineering bacteria after inducing is observed through high power lens, seen that a small amount of inclusion body is arranged; Engineering bacteria ultrasonic disruption after inducing is centrifugal, centrifugally that cleer and peaceful precipitation is not carried out determination of activity and the SDS-gel electrophoresis determines that staphylokinase 95% is present in intercellular substance with solvable state, and 5% exists with the inclusion body form.

＜2〉biological activity determination:

A. the preparation of typical curve: the 150mg agarose adds 23ml physiological saline, boils dissolving, is cooled to 56 ℃, add human thrombin 1.76u, human fibrinogen 2.5mg pours culture dish into after shaking up, and solidifies the back punching, the sample of standard substance 10u, 5u, 2.5u, 1.25u, 0.625u, 0.31u is added in the hand-hole, put in the box of preserving moisture, cultivate 16-24h, measure the dissolving loop diameter for 25-30 ℃, with the active logarithm of 1g, 1g dissolving loop diameter logarithm is made typical curve.

Add 1.76 when b. 0.7% of sample determination: 13ml agarose boils postcooling to 56 ℃ with zymoplasm, the 2.5mg human fibrinogen shakes up the back and falls dull and stereotypedly, and it is standby to coagulate the back punching.The centrifugal supernatant of engineering bacteria after the inducing of ultrasonication is surveyed protein concentration, supernatant is by 1000 times, 2000 times, 4000 times, 6000 times different gradient dilutions, getting 20 μ l adds in the hand-hole, cultivate 24h for 30 ℃, survey dissolving circle size, Sak compares with standard, obtains corresponding activity unit according to dissolving circle size on typical curve.The ratio that records is lived and is 7-10 ten thousand units/mg.

The purifying of embodiment three, recombinant staphylokinase

Engineering bacteria after inducing is broken through ultrasonic wave, centrifugal collection supernatant.

1, ion-exchange

The Q-Sepharose post is after the damping fluid balance, above-mentioned sample is added this ion column, use the buffer solution elution foreign protein, use salt gradient buffer solution elution r-Sak then, merging contains the active solution of respectively managing of Sak and concentrates with ammonium sulphate precipitation, again with the damping fluid dissolving, purity reaches more than 85% centrifugal back albumen precipitation.

2, gel-filtration:

The Sephacryl-100 gel column again with going into gel column in the damping fluid dissolved sample, is used buffer solution elution with the previous step ammonium sulphate precipitation after the damping fluid balance, purity is measured through HPLC and reached (Fig. 6) more than 98%.

Claims

1, a kind of method of utilizing genetic engineering technique to produce staphylokinase comprises:

(1) amplification glucokinase gene;

(3) with constructed expression plasmid transformed into escherichia coli;

(4) cultivate the staphylokinase that intestinal bacteria also separate, purifying is expressed that transforms.

2, in accordance with the method for claim 1, it is characterized in that the amplification of described glucokinase gene is to use the PCR of following primer to carry out:

5’CAC?GAA?TTC?ATG?TCA?AGT?TCA?TTC?GAC?AAA?3’；

5 ' GGC GAA TTC TTA TTT CTT TTC TAT AAC AAC 3 ', and the glucokinase gene of amplification be to use EcoR I enzyme to cut being connected of carrier pBV220 after connect.

3, in accordance with the method for claim 1, it is characterized in that described coli strain is a DH5 α bacterial strain.

4, a kind of glucokinase gene or its code degeneracy body with following sequence: ATG TCA AGT TCA TTC GAC AAA GGA AAA TATAAA AAA GGC GAT GAC GCG AGT TAT TTT GAACCA ACA GGC CCG TAT TTG ATG GTA AAT GTGACT GGA GTT GAT GGT AAA GGA AAT GAA TTGCTA TCC CCT CGT TAT GTC GAG TTT CCT ATTAAA CCT GGG ACT ACA CTT ACA AAA GAA AAAATT GAA TAC TAT GTC GAA TGG GCA TTA GATGCG ACA GCA TAT AAA GAG TTT AGA GTA GTTGAA TTA GAT CCA AGC GCA AAG ATC GAA GTCACT TAT TAT GAT AAG AAT AAG AAA AAA GAAGAA ACG AAG TCT TTC CCT ATA ACA GAA AAAGGT TTT GTT GTC CCA GAT TTA TCA GAG CATATT AAA AAC CCT GGA TTC AAC TTA ATT ACAAAG GTT GTT ATA CAA AAG AAA TAA

5, a kind of by the described sequence of claim 4 or its code degeneracy body encoded protein matter.

6, a kind of recombinant expression plasmid, it is the pBV220 carrier that contains the described sequence of claim 4 or its code degeneracy body.

According to the described recombinant expression plasmid of claim 6, it is characterized in that 7, described sequence links to each other by EcoRI single endonuclease digestion site with described carrier.

8, a kind of with claim 6 or the formed engineering bacteria of 7 described recombinant expression plasmid transformed into escherichia coli cells.

According to the described engineering bacteria of claim 8, it is characterized in that 9, described intestinal bacteria are DH5 α bacterial strain.

10, in accordance with the method for claim 1, it is characterized in that described glucokinase gene is glucokinase gene or its code degeneracy body with the described sequence of claim 4.