CN1109508A - Full DNA sequence of recombinative glucokinase - Google Patents

Abstract

The invention provides a recombinant staphylokinase DNA sequence, excluding signal peptidase, and number 33 basic group of the DNA is A, the number 275 basic group is T, the kinase DNA assembly expression vector to host bacteria, through transcription in the host bacteria, recombinant staphylokinase protein is translated to cure Myocardial Infarction, cerebral thrombosis and venous thrombosis disease such as deep venous thrombosis.

Description

Full DNA sequence of recombinative glucokinase

The present invention relates to a kind of recombinant DNA sequence, particularly relate to a kind of full DNA sequence of recombinant staphylokinase.

The external in recent years researchist as countries such as Belgium, Japan, Germany has done a large amount of research work at the preparation and the medical use this respect of recombinant staphylokinase, and enters the clinical experimental study stage.As " nucleic acids research " (Nucleic Acids Research) the 22nd phases 11 of nineteen eighty-three volume, be entitled as " Nucleotide sequence of the staphylokinase gene from Staphylococcus aureus " and reported a kind of nucleotide sequence of determining staphylokinase (Sak), its peptide sequence of inference has 163aa, signal peptide has 27aa, is positioned at NH2 and does not hold.

The object of the present invention is to provide a kind ofly not contain signal peptide, and the 33rd bit base of DNA is A that the 275th bit base is the full DNA sequence of the recombinant staphylokinase of T.

The full DNA sequence of recombinant staphylokinase of the present invention is:

TCA AGI TCA TTC GAC AAA GGA AAA

Ser Ser Ser Phe Asp Lys Gly Lys

TAT AAA AAA GGC GAT GAC GCG AGT

Tyr Lys Lys Gly Asp Asp Als Ser

TAT TTT GAA CCA ACA GGC CCG TAT

Tyt Phe Glu Pro Thr Gly Pro Tyr

TTG ATG GTA AAT GTG ACT GGA GTT

Leu Met Val Asn Val Thr Gly Val

GAT GGT AAA GGA AAT GAA TTG CTA

Asp Gly Lys Gly Asn Glu Leu Leu

TCC CCT CAT TAT GTC GAG TTT CCT

Ser Pro His Tyr Val Glu Phe Pro

ATT AAA CCT GGG ACT ACA CTT ACA

Ile Lys Pro Gly Thr Thr Leu Thr

AAA GAA AAA ATT GAA TAC TAT GTC

Lys Glu Lys Ile Glu Tyr Tyr Val

GAA TGG GCA TTA GAT GCG ACA GCA

Glu Trp Ala Leu Asp Ala Thr Ala

TAT AAA GAG TTT AGA GTA GTT GAA

Tyr Lys Glu Phe Arg Val Val Glu

TTA GAT CCA AGC GCA AAG ATC GAA

Leu Asp Pro Ser Ala Lys Ile Glu

GTC ACT TAT TTT GAT AAG AAT AAG

Val Thr Tyr Phe Asp Lys Asn Lys

AAA AAA GAA GAA ACG AAG TCT TTC

Lys Lys Glu Glu Thr Lys Ser Phe

CCT ATA ACA GAA AAA GGT TTT GTT

Pro Ile Thr Glu Lys Gly Phe Val

GTC CCA GAT TTA TCA GAG CAT ATT

Val Pro Asp Leu Ser Glu His Ile

AAA AAC CCT GGA TTC AAC TTA ATT

Lys Asn Pro Gly Phe Asn Leu Ile

ACA AAG GTT GTT ATA GAA AAG AAA

Thr Lys Val Val Ile Glu Lys Lys

The full DNA sequence assembling expression vector of recombinant staphylokinase of the present invention is transformed into the host bacterium, by transcribing, translates into recombinant staphylokinase protein in the host bacterium.Can be used for treating thrombotic diseases such as myocardial infarction, cerebral thrombosis, degree of depth phlebothrombosis.

Describe the present invention's preparation and mensuration in detail below in conjunction with embodiment.The preparation of the full DNA sequence of recombinant staphylokinase of the present invention is:

1) screening of gold-coloured staphylococci: at first streptococcus aureus (S.aurens) is inserted the LB substratum of 50ml, cultivated 8 hours under 37 ℃ of temperature, rule on the flat board of LB-agar 1.5% 37 ℃ of following overnight incubation of temperature then.Picking list bacterium colony increases in the LB of 5ml nutrient solution, 37 ℃ of following overnight incubation of temperature, centrifugal collection supernatant liquor.Measure the activity of staphylokinase (Sak) in the supernatant liquor with the chromophoric substrate method, obtain the bacterial classification of two the highest single bacterium colonies of Sak activity altogether, be numbered S-1 respectively, S-29 as the streptococcus aureus that will screen (S.aurens), wherein the fibrinolytic of S-29 is higher, so be alternative.

2) has the extraction of the streptococcus aureus S-29 chromosomal DNA of fibrinolytic: streptococcus aureus S-29 is seeded in the LB substratum of 250ml with the ratio of 1:100, in 37 ℃ of temperature, constant-temperature shaking culture is 18 hours under the condition that rotating speed is 250 rev/mins, add each 250 μ g of DNAaseI and RNAase again, room temperature reaction 30 minutes, add solid 1.46gNacl then, waited to dissolve rearmounted ice bath 1 hour, under 4 ℃ of temperature, 11000 rev/mins of conditions of rotating speed centrifugal 10 minutes, collect supernatant liquor.In supernatant liquor, add 25g polyoxyethylene glycol 8000(PEG8000), ice bath 1 hour once more after waiting to dissolve.Under 4 ℃ of temperature, 11000 rev/mins of conditions of rotating speed centrifugal 10 minutes then, collecting precipitation.SM solution (Tris-Hcl of 5.8gNacl, 2gMgSO417H2O, 50ml concentration 1mmol/l, pH7.5,2% gelatin 5ml, adding distil water are to the 1000ml) dissolution precipitation that adds 4ml in precipitation gets crude extract.Add the equal-volume chloroform in crude extract, mixing under 4 ℃ of temperature, 3000 rev/mins of conditions of rotating speed centrifugal 15 minutes, is collected water.Centrifugal 2 hours again with 2500 rev/mins rotating speed, collecting precipitation.With the SM solution dissolution precipitation of 100ml, temperature is placed down for 4 ℃ and is spent the night.Adding Proteinase K (prote-inase k) to this Proteinase K final concentration then is 50 μ/μ g, add simultaneously 10% SDS to its final concentration be 0.5%, in the reaction hour down of 56 ℃ of temperature, be cooled to room temperature.Use phenol, phenol-chloroform, each extracting of chloroform respectively once.Add the sodium-acetate that 1/10 volumetric concentration is 3mol/l (PH7.0), the dehydrated alcohol that adds 2.5 times of volumes again precipitates.(concentration is that Tris-hcl and the concentration of 10mmol/l is the EDTA of 1mmol/l, and PH8.0) dissolution precipitation obtains phage DNA, and golden Portugal bacterium phage DNA collection of illustrative plates is preserved standby down in-20 ℃ as shown in Figure 1 to use TE solution at last.

3) amplification of recombinant staphylokinase gene: (S-phi-C DNA) is template with phage, by primer 1, primer 2, adopts PCR method amplification glucokinase gene, and the PCR reaction solution is:

Distilled water 33 μ l μ

10 * PCR damping fluid, 5 μ l

dNTP（2.5mmol/l） 4μl

Primer 1(100pmol) 1 μ l

Primer 2 (100pmol) 1 μ l

Phage S-Phi-CDNA(0.22 μ g/ μ l) 5 μ l

Mixing, 95 ℃ of following thermally denatures of temperature 5 minutes, ice bath immediately, adding 1 μ l concentration is the Taq polysaccharase of 2.5u/ml, the 50ml whiteruss.

Test kit PCR reaction conditions: each circulation is included in temperature and reacted 52 seconds down for 92 ℃; Reacted 60 seconds down for 55 ℃ in temperature; Reacted 70 seconds down for 72 ℃ in temperature; Circulate altogether 30 times.Under 72 ℃ of temperature, reacted again 15 minutes at last.Get 5 μ l electrophoresis detection, the dna segment about 400bp appears in electrophoresis detection (1% gel) PCR reaction product, and the PCR product collection of illustrative plates of SAK as shown in Figure 2.

The clone of glucokinase gene of the present invention and sequencing thereof are that above-mentioned dna segment is cut with restriction enzyme EcoR I and BamH1 enzyme respectively with carrier M13mp19, two segments after enzyme is cut connect with ligase enzyme T4DNA ligase again, the connector coated plate, choose positive colony, called after pDAGI, and carry out sequencing.(measure test kit available from Promega dna sequence analysis test kit, connect test kit and will show operation.Order-checking is undertaken by Ri Benjin swamp medical college).

Order-checking instrument: dna sequencing instrument (A.L.F.DNA SegnncerTM)

Sequential analysis:

Computer software A.L.F.Manager V2.5 Experinental

COnditions

Voltage 1500V

Electric current 38mA

Power 34W

Sample room was every 2 seconds

360 minutes time

40 ℃ of temperature

Probe power (laser intensity) 3mW

Clone's used carrier puc19 (ECoRI-Sakgene-BamH1-S

akgene)

The clone uses primer forward primer Puc18 primer-40

Reverse primer puc18 Reverse primer

Sequencing result as shown in Figure 3.

Claims (1)

1, a kind of full DNA sequence of recombinant staphylokinase, its sequence is:

TCA AGT TCA TTC GAC AAA GGA AAA

Ser Ser Ser Phe Asp Lys Gly Lys

TAT AAA AAA GGC GAT GAC GCG AGT

Tyr Lys Lys Gly Asp Asp Als Ser

TAT TTT GAA CCA ACA GGC CCG TAT

Tyt Phe Glu Pro Thr Gly Pro Tyr

TTG ATG GTA AAT GTG ACT GGA GTT

Leu Met Val Asn Val Thr Gly Val

GAT GGT AAA GGA AAT GAA TTG CTA

Asp Gly Lys Gly Asn Glu Leu Leu

TCC CCT CAT TAT GTC GAG TTT CCT

Ser Pro His Tyr Val Glu Phe Pro

ATT AAA CCT GGG ACT ACA CTT ACA

Ile Lys Pro Gly Thr Thr Leu Thr

AAA GAA AAA ATT GAA TAC TAT GTC

Lys Glu Lys Ile Glu Tyr Tyr Val

GAA TGG GCA TTA GAT GCG ACA GCA

Glu Trp Ala Leu Asp Ala Thr Ala

TAT AAA GAG TTT AGA GTA GTT GAA

Tyr Lys Glu Phe Arg Val Val Glu

TTA GAT CCA AGC GCA AAG ATC GAA

Leu Asp Pro Ser Ala Lys Ile Glu

GTC ACT TAT TTT GAT AAG AAT AAG

Val Thr Tyr Phe Asp Lys Asn Lys

AAA AAA GAA GAA ACG AAG TCT TTC

Lys Lys Glu Glu Thr Lys Ser Phe

CCT ATA ACA GAA AAA GGT TTT GTT

Pro Ile Thr Glu Lys Gly Phe Val

GTC CCA GAT TTA TCA GAG CAT ATT

Val Pro Asp Leu Ser Glu His Ile

AAA AAC CCT GGA TTC AAC TTA ATT

Lys Asn Pro Gly Phe Asn Leu ILe

ACA AAG GTT GTT ATA GAA AAG AAA

Thr Lys Val Val Ile Glu Lys Lys

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