CN1786152A - Engineering bacteria strain for producing recombination buman tPA and its preparation method - Google Patents

Abstract

The invention discloses a manufacturing recombination man tPA colibacillus strain and making method. It offers colibacillus strain BL21 (DE3)/rtPAm-1, CCTCC M205112. Its cell contains a recombinant plasmid pETrtPAm-1. The plasmid is cloned a recombination mutation man tPA gene. The gene has nucleotide sequence showed in SEQ ID NO.1, and contains T7 promoter sequence, ribosome binding sequence, and KozaK sequence. The BL21(DE3)/rtPAm-1 strain is induced to effectively express recombination mutation tPA albumen. The produced recombination tPA molecule has small molecular weight, strong activity, long half life, and high stability.

Description

A kind of engineering strain and preparation method who produces recombination buman tPA

Technical field

The present invention relates to biotechnology, be specifically related to a kind of colibacillus engineering strain, relate to the preparation method of engineering strain simultaneously.This bacterial strain can produce recombination mutation tPA albumen, can be used for treating clinically diseases such as Acute Myocardial Infarction, cerebral thrombosis, pulmonary infarction, medically has important use value.

Background technology

Buman tPA is that (tissue type plasminogen activator tPA), is a kind of serine protease for people's tissue-type plasminogen activator.It contains 527 amino acid, molecular weight 68KD, can become the activation of the Profibrinolysin of non-activity plasmin and bring into play the thrombus dissolving effect, be used for thromboembolism treatments such as Acute Myocardial Infarction, cerebral thrombosis apoplexy, pulmonary infarction clinically, be present good in the world thrombolytic drug, it is fast, safe to have a thrombolysis, more the back disability rate is low, to advantages such as people's no antigens.

TPA is mainly by the vascular endothelial cell expression-secretion, and concentration is very low in the blood plasma, is about 5ng/ml, about 5 minutes of transformation period, and therefore from blood plasma or tissue, directly extract tPA and can not be used for producing, can only adopt genetic engineering technique production.1987, the rtPA that the U.S. takes the lead in ratifying first genetic engineering technique production is used for the clinical treatment Acute Myocardial Infarction, commodity are called alteplase (Activase), and this product belongs to the original tPA of total length, belong to tPA first-generation product, significantly shortcoming is transformation period short (3-6 minute), active low, to the tPA inhibition PAI non-resistant of higher concentration in patient's blood plasma, thereby using dosage is very big, every pin contains tPA 150-200mg, and the price of every pin is also very expensive.

In order to solve the problem that original tPA exists, on the basis of tPA structure and functional study, the researchist further carries out aminoacid deletion and replaces Study on Variation tPA.TPA structurally can be divided into (finger plot structure territory, F district, 4-49aa), E district (somatomedin functional domain, 50-86aa), (kringle 1 in the K1 district, 87-175aa), (kringle 2 in the K2 district, 176-261aa) with (proteolytic enzyme district, P district, 262-527aa) five functional domains (naming ﹠ numbering of structural domain is according to gene pool Genbank login number GI137119 or document Pennica D.et al.Nature 301:214,1983) independently.Each ad hoc structure territory of t-PA and the relation between the various biologic activity of t-PA are illustrated, F district and K2 district all can specificity be incorporated into fibrin clot, K1 district and liver receptor are in conjunction with relevant, P contains serine protease in the district, be responsible for the fiber proenzyme is cut into cellulase, contain the binding site (aa296-299) of the active inhibition PAI of tPA on it.The disappearance in any one district or variation do not influence other regional structure and activity among the tPA, and this provides the foundation for the reorganization of tPA, transformation (being referred to as the tPA mutant).Countries such as the U.S., Germany, Japan, Britain all improve the performance of tPA by reorganization and variation in research, and have separately an intellecture property, as the reteplase of German Pola graceful (Boehringer Mannheim) company exploitation (Reteplase, rPA), nPA enzyme (Lanoteplase), the Japan of the TNK enzyme (Tenecteplase) of U.S.'s Genentech (Genentech) company exploitation, U.S.'s Bristol-mayer Si Pubo (Bristol-MyersSpuibb) company exploitation defends the Monteplase (Monteplase) etc. that material (Eisai) is built the exploitation of ripple institute.Reteplase (Reteplase) is a kind of Recomposed tPA product of producing with the intestinal bacteria system expression, form by two structural areas of K2P, disappearance F, E, three zones of K1, contain 355 amino acid (Kohnert U, et al.Protein Engineering 5:93,1992, the patent No.: EP 0 382 174 A1).Its character and natural tPA molecule have had bigger change, stability is improved, transformation period rises to 15-18 minute, but bigger decline is arranged with fibrinous avidity, it is combined into reversible the combination with fibrinous, binding ability is significantly less than natural tPA, and still contains the binding site of the active inhibition PAI of tPA in the reteplase structure, and in vitro tests shows that the PAI-1 of 5IU/ml just can make the reteplase inactivation of 5ng/ml.Its specific activity is 5 * 10 ⁵IU/mg, using dosage 20-40mg, preparation was preserved validity period 2 years at 2 ℃, and the dissolving rear stability is poor, requires clinically to use in 4 hours.The TNK enzyme is a kind of total length tPA that produces with Chinese hamster cell (CHO), contain 527 amino acid, variation takes place to replace in totally 6 amino acid three positions, and promptly the Thr103 in K1 district and Asn117 are replaced by Asn and Glu respectively, and the Lys296-His-Arg-Arg299 in P district is replaced by 4 Ala.Compare with original t-PA, the activity of the external anti-PAI of TNK enzyme improves 80 times, and the transformation period prolongs 4 times, and activity has only 82% of original tPA in vivo, and binding fiber albumen ability keeps 87% (Keyt.B.A.et al.PNAS, USA, 91:3670,1994).TNK enzyme using dosage is still bigger clinically, and every pin content reaches 50mg.Monteplase is a kind of total length tPA that is produced by body hamster kidney cell, contain 527 amino acid, the Cys84 in E district is replaced into Ser, its action effect is similar to t-PA, the ability of plasminogen activation is strong 14.9 times when not having than scleroproein when scleroproein exists, but itself and scleroproein combination rate are about 1/3 of original t-PA, and it is descended to some extent to the thrombus site specific.More than three kinds of tPA mutant all go on the market, also has a kind of tPA mutant nPA enzyme (Lanoteplase) that waits for U.S. food and drug administration (FDA) approval in addition, F, E district have been lacked, and modified the glycosylation site in K1 district, compare with original t-PA, its transformation period prolongs 37min, and has improved thrombolysis activity, but reduced fibrinous avidity, and the rate of intracranialing hemorrhage that causes is higher than t-PA.Although various tPA mutant improve the performance of tPA aspect different, s-generation Recomposed tPA still exists poor heat stability, the problem that specificity is not high, using dosage is big and production cost is high.

The expression system that is used to produce tPA at present mainly is animal cell expression system and escherichia expression system.Be used at first production for treating with the cell system of tPA be melanoma cell (Griffiths, JB.et al.Adv.Biochem.Eng.Biotechnol.1987,34:147-166).Subsequently a series of gone on the market and be about to the listing tPA derivative products in, also be to adopt the zooblast system to produce mostly, as the TNK enzyme (Tenecteplase) of U.S.'s Genentech (Genentech) company exploitation, the nPA enzyme (Lanoteplase) of U.S.'s Bristol-mayer Si Pubo (Bristol-MyersSpuibb) company exploitation, be to produce with Chinese hamster cell (CHO), Japan defends material (Eisai) and builds the Monteplase (Monteplase) of ripple institute exploitation and produce with body hamster kidney cell.Since the eukaryotic cell culture systems exist the cell speed of growth slow, produce the yielding poorly of tPA, medium component costliness, culture condition and require characteristics such as height, cause the tPA production cost very high, cause the tPA product price high, patient is difficult to bear economically.

Intestinal bacteria (Escherichia coli) expression system becomes the first-selected host cell of most protein expression study and production owing to its easy and simple to handle, low production cost.Adopt the escherichia expression system production for treating promptly to receive in early days at the tPA drug research that the researchist pays close attention to and achieving success (Harris TJ et al.Mol.Biol.Med.1986,3:279-292 with tPA; Datar RV.et al.Biotechnology.1993,11:349-357), at present in the tPA product of listing, reteplase (Reteplase by the exploitation of German Pola graceful (Boehringer Mannheim) company, rPA) promptly be to produce, but need cotransformation recombinant expression plasmid and helper plasmid to come express recombinant tPA by coli strain K12.The present invention utilizes e. coli bl21 (DE3) to obtain a kind of colibacillus engineering strain BL21 (DE3)/rtPAm-1, can efficiently express novel reorganization variation tPA albumen (rtPAm-1), and inheritance stability, and going down to posterity still keeps higher tPA output more than 30 times.

Summary of the invention

The object of the present invention is to provide a kind of coli strain of producing recombination buman tPA.The Recomposed tPA that this bacterial strain produces has little, active strong, the long half time of molecular weight, characteristics that stability is high, can be used for treating clinically diseases such as Acute Myocardial Infarction, cerebral thrombosis, pulmonary infarction, is worth medically having important use.

Another object of the present invention is to provide a kind of method for preparing the coli strain of producing recombination buman tPA, and method is easy, easy handling.

This colibacillus engineering strain called after BL21 (DE3)/rtPAm-1, bacterial strain has been submitted Chinese typical culture collection center preservation to, preservation address: China. Wuhan. Wuhan University, preservation date: on October 09th, 2005, deposit number is CCTCC M205112, classification name: Escherichia coli BL21 (DE3)/rtPAm-1.Contain recombinant plasmid pETrtPAm-1 in its cell, contain the described recombination mutation buman tPA of SEQ ID NO.1. gene order in the plasmid.Coli strain BL21 (DE3)/rtPAm-1 is prepared by following steps:

1. the structure of plasmid pETrtPAm-1

(1) amplifies the FE fragment and the P fragment of tPA gene respectively with PCR method.

(2) with restriction enzyme BamHI and KpnI cutting FE fragment, with restriction endonuclease KpnI and HindIII cutting P fragment, with restriction endonuclease BamHI and HindIII cutting pQE30 plasmid (the pQE30 plasmid is available from QIAGEN company), carrying out three fragments with the T4 ligase enzyme connects, construction recombination plasmid pQErtPAm-1, contain Recomposed tPA gene on it, by F, E, the P district of original tPA gene forming.

(3) two mutagenic primers of design adopt the PCR fixed-point mutation method on Recomposed tPA gene original tPA molecule R298, R299 coding place all to be sported the E coding.

(4) expression plasmid pET-His (plasmid pET-His is available from gene Dynamic Test Chamber company limited) is transformed.Utilize the pET-His plasmid DNA to be template, design a pair of primer and carry out pcr amplification, then with BamHI and HindIII cutting amplified production, the 1.1kb variation tPA gene that again PCR is produced cuts with restriction endonuclease BamHI and HindIII, connect two fragments with the T4 ligase enzyme, obtain recombinant plasmid pETrtPAm-1, transformed into escherichia coli DH5 α obtains coli strain DH5 α rtPAm-1.

(5) identify recombinant plasmid pETrtPAm-1: with double digestion, PCR method recombinant plasmid is carried out preliminary evaluation, carry out finally determining through dna sequencing again.

2. cultivate coli strain DH5 α rtPAm-1, extract recombinant plasmid pETrtPAm-1.

3. cold CaCl ₂Legal system is equipped with e. coli bl21 (DE3) competent cell

4. with recombinant plasmid pETrtPAm-1 transformed into escherichia coli BL21 (DE3) competent cell, obtain coli strain BL21 (DE3)/rtPAm-1 through screening and evaluation.

Coli strain BL21 (the DE3)/rtPAm-1 of gained of the present invention has following characteristics:

1. the used coli strain BL21 of the present invention (DE3), available from gene Dynamic Test Chamber company limited, its genotype is F ^-OmpT hsdSB (rB-mB-) gal (λ cI 857 ind1 Sam7 nin5 lacUV5-T7genel) dcm (DE3).BL21 is the most widely used host bacteria source of marking protein, and its advantage is to lack lon and ompT proteolytic enzyme, is beneficial to exogenous gene expression protein matter.BL21 (DE3) is a λ DE3 lysogenic bacteria, have the T7 rna polymerase gene on the DE3, the T7 rna polymerase gene is controlled by the lacUV5 promotor, just have to a certain degree when not inducing and transcribe, express target protein, under the condition that has inductor isopropylthio-(IPTG) to exist, can produce the T7 RNA polymerase in a large number, thereby make target protein obtain high level expression.

2. penbritin (Amp) there is resistance, can on the LB-Amp flat board, grows, on flat board, be the typical intestinal bacteria bacterium colony of diameter 1～2mm.The rounded protuberance of bacterium colony, mean diameter are 1.6mm, and appearance is moistening smooth, white.The opticmicroscope hypothallus is a rod-short, the form uniformity.

3. under isopropylthio-(IPTG) inductive condition, can efficiently express novel recombinant human tPA (rtPAm-1).The rtPAm-1 molecular weight of albumen of expressing is about 38kD, forms the inclusion body of non-activity in born of the same parents, and expression level reaches more than 30% of cell protein total amount.

4. passage number is more than 30 times, inheritance stability, stable yield.

5. the Recomposed tPA albumen of its expression is different from existing other recombination buman tPA (as rt-PA, TNK enzyme, reteplase reteplase etc.).This recombination buman tPA is made up of F, E, the P-structure district of natural tPA, has removed K1 and two structural areas of K2 of natural tPA.In addition, compare, also contain the replacement sudden change of 3 amino acid codings, be respectively: S262G (TCC-GGT), R298E (AGG-GAG), R299E (AGG-GAG) (digitized representation is the amino acid position in the natural tPA molecule) with natural tPA molecule.

In sum, coli strain BL21 provided by the invention (DE3)/rtPAm-1 can produce rtPAm-1 albumen high-levelly, utilizes 10 liters of fermentor tanks to carry out pilot scale fermentation production, and the level of producing rtPAm-1 reaches 1100mg/L.The rtPAm-1 inclusion body detects with molten fine circle method through renaturation and purifying, has the effect that activates the former cellulolytic activity of fibrinolytic enzyme, and activity reaches 5-6 * 10 ⁵IU/mg reaches or is better than international like product level.This bacterial strain genetic stability height, continuous passage still keeps identical throughput more than 30 generations.Further animal experiment shows: the rtPAm-1 nontoxicity, have no side effect, the transformation period reaches 20-30 minute in the body, reaches international like product level.Colibacillus engineering strain BL21 provided by the invention (DE3)/rtPAm-1, throughput is strong, the output height, the genetic stability height, the novel recombination mutation tPA molecular weight of producing is little, stability is high, activity is strong, on every index, meet or exceed international like product level, possess production application and be worth.Appearance has repeatability within the present invention, and those of ordinary skill can obtain this bacterial strain and utilize this bacterial strain to produce recombination mutation tPA according to step provided by the present invention.

Description of drawings

Fig. 1 plasmid pETrtPAm-1 makes up synoptic diagram

1. the acquisition of Recomposed tPA gene (rtPA gene).

Design two pairs of primers, primer 1 and the primer 2 FE fragment that is used to increase, the primer 3 and 4 P fragment that is used to increase.With tPA cDNA is template, obtain FE district, the P district fragment of tPA gene by PCR method, two fragments are through the restriction enzyme cutting, carrying out three fragments with pQE30 plasmid that enzyme is cut is connected, recombinant plasmid pQErtPA in the middle of obtaining, on contain Recomposed tPA gene, by F, E, the P district of original tPA gene forming.

2. the acquisition of recombination mutation rtPAm-1 gene (rtPAm-1 gene).

Adopt the PCR fixed-point mutation method.Designing two mutagenic primer primers 5 and primer 6, is template with pQErtPA, carries out pcr amplification with primer 1 and 5, primer 4 and 6 respectively.And then, carry out the PCR second time with two kinds of PCR products mixing, template---primer extends two fragments each other, produces the variation tPA gene (rtPAm-1) of 1.1Kb.The position in the mutational site of round dot display design.

3. expression plasmid pET-His is transformed, remove the coding region of 6His on it.

Design a pair of primer according to the pET-His plasmid sequence, primer 7 and primer 8 are template with the pET-His plasmid DNA, carry out pcr amplification.With BamHI and HindIII cutting amplified production, obtain not having the pET plasmid of 6His coding region then.

4. the acquisition of plasmid pETrtPAm-1.

The recombination mutation tPA gene (rtPAm-1 gene) of the 1.1kb that PCR is produced connects the pETs plasmid of transforming with restriction endonuclease BamHI and HindIII cutting with the T4 ligase enzyme, obtains recombinant plasmid pETrtPAm-1.The enzyme of Fig. 2 recombinant expression plasmid pETrtPAm-1 is cut and is identified and PCR evaluation (0.7% agarose gel electrophoresis)

1. through the pET carrier of transformation.Removed the 6His coding region, its big or small 2.6kb.

2.DNA molecular weight sign (Marker)

3, the 4.pETrtPAm-1 plasmid is through the fragment of KpnI and the generation of HindIII double digestion.Wherein the band of 0.8kb is the P fragment in the rtPAm-1 gene, and the band of 2.9kb then is that the FE fragment of tPA adds pET plasmid fragment.

5,6. be the PCR product of template with pETrtPAm-1.1.1kb amplified band be the rtPAm-1 gene fragment.

Fig. 3. coli strain BL21 (DE3)/rtPAm-1 abduction delivering produces rtPAm-1 albumen (detection of 10%SDS-polyacrylamide gel electrophoresis)

1. cultivate through LB, 2YT liquid nutrient medium, rtPAm-1 albumen does not appear in the tropina of coli strain BL21 (the DE3)/rtPAm-1 before isopropylthio-(IPTG) is induced.

2. isopropylthio-(IPTG) is induced 6h, ultrasonic disruption coli strain BL21 (DE3)/rtPAm-1 cell, the albumen in the centrifugal supernatant that obtains.There is not rtPAm-1 albumen.

3. isopropylthio-(IPTG) is induced 6h, ultrasonic disruption coli strain BL21 (DE3)/rtPAm-1 cell, the albumen in the centrifugal precipitation that obtains.RtPAm-1 albumen occurs, the arrow place shows rtPAm-1 albumen.The rtPAm-1 albumen of expressing accounts for more than 30% of total cell protein, and its size is about 38kD.

4. will induce the rtPAm-1 inclusion body sex change of acquisition to be dissolved in 8M urea, and, utilize molecular sieve chromatography G50 to carry out the rtPAm-1 albumen of purifying again with phosphate buffered saline buffer (PBS) the dialyzed overnight renaturation of pH8.0.Through above-mentioned processing, the proteic purity of rtPAm-1 reaches more than 95%.

5. protein molecule quantitative character (Marker)

Fig. 4. detect the effect of the proteic plasminogen activation cellulolytic activity of rtPAm-1 with molten fine circle method

1. negative control: physiological sodium chloride solution, point sample 2 μ l.Molten fine circle does not appear.

2. standard control: standard buman tPA sample, point sample 10 units (10IU).Molten fine circle appears.

3. experimental group: through the rtPAm-1 albumen of renaturation and purifying, point sample 5 μ l.Molten fine circle appears.

The result shows that the rtPAm-1 albumen of coli strain BL21 (DE3)/rtPAm-1 expression of the present invention has the effect of plasminogen activation cellulolytic activity.

Fig. 5. the tropina (10%SDS-polyacrylamide gel electrophoresis) in 30 generations of coli strain BL21 (DE3)/rtPAm-1 continuous passage

M: protein Marker

1-7: respectively be starting strain, the 5th, 10,15,20,25,30 generation bacterial strain tropina.The rtPAm-1 albumen that the arrow indication starting strain and the bacterial strain that goes down to posterity are expressed.

The result shows that after going down to posterity through 30 generations, coli strain BL21 (DE3)/rtPAm-1 produces the proteic ability of rtPAm-1 and do not change.

Embodiment

The present invention is described in further detail below in conjunction with embodiment.Experimental technique among the embodiment, condition is carried out routinely, with reference to as Sa nurse Brooker etc., experimental technique described in the molecular cloning experiment guide (second edition,, Science Press in 1992).

The structure of embodiment 1. plasmid pETrtPAm-1

Fig. 1 has shown the structure principle and the process of recombination mutation tPA gene.Natural sophisticated buman tPA contains 527 amino acid, form (F, E, K1, K2 and P) by 5 independent structures districts, the P district contains inhibition PAI land and the enzymic activity district of tPA, K2 contains PAI zone of action and scleroproein land, K1 is relevant with liver removing tPA, F district (referring to the type structural area) is relevant with protein interaction, and when K1K2 lacked, the F district still can interact with scleroproein, Profibrinolysin.The recombination mutation rtPAm-1 gene that the present invention is designed is to consider stability, prolong half-life and the increase of the increase tPA resistance to inhibition PAI.When the design gene is rebuild and is made a variation, carry out in two steps, at first construction goes out to contain the tPA gene (disappearance K1 and K2) of F, E, P, and then the replacement of encoding at PAI action site introducing amino acid suddenlys change, obtain as the gene order as shown in the SEDID NO.1., at last this gene is connected in the expression plasmid, is built into plasmid pETrtPAm-1.Embodiment is as follows:

1.tPA the segmental pcr amplification of gene FE fragment and P

At first synthetic two pairs of primers are used for two segmental amplifications

Primer 1:GAATTC GGATCCATGGGAAGATCTTACCAAGTGATC (the underscore place is BamHI cutting sequence GGATCC, after connect codon ATG, and contain Kozak sequence C CATGG)

Primer 2: GTCGAC GGTACCCGTGGCCCTGGTATC (containing KpnI cutting sequence GGTACC)

Primer 3:GTCGAC GGTACCTGCGGCCTGAGACAG (containing KpnI cutting sequence GGTACC)

Primer 4:AGATCT AAGCTTCTCGAGTCACGGTCGCATGTTGTC (containing HindIII cutting sequence A AGCTT)

With tPAcDNA is amplification template, add primer 1 and primer 2 in one set of reaction tubes, the FE fragment that is used to increase, another group adds primer 3 and 4, the P fragment is used to increase, two groups of PCR reaction conditionss are identical: 94 ℃ of 5min, 94 ℃ of 1min afterwards, 55 ℃ of 1min, 72 ℃ of 1min totally 33 circulations, last 72 ℃ of 10min, pcr amplification product are through the phenol extracting, and 1% low melting-point agarose gel electrophoresis is reclaimed.

2. the structure of recombinant plasmid pQErtPA

With restriction endonuclease BamHI and KpnI cutting FE fragment, with KpnI and HindIII cutting P fragment, with BamH I and KpnI cut vector plasmid pQE30 (the pQE30 plasmid is available from QIAGEN company), utilizing the T4 ligase enzyme to carry out three fragments connects, make up the pQErtPA clone, transformed into escherichia coli DH5 α competent cell, positive bacterium colony is obtained in screening on penbritin (Amp)-LB flat board.

3. on the tPA gene, the R298 of original tPA molecule, R299 coding is all sported the E coding

Adopt the PCR fixed-point mutation method to suddenly change.At first synthetic two mutagenic primers (representative sudden change place of underscore place):

Primer 5:CTCTCCGGGCGA CTCCTCGTGCTTGGCAAAGATGGC

Primer 6:GCCATCTTTGCCAAGCAC GAGGAGTCGCCCGGAGAG

With the pQErtPA plasmid is template, uses primer 1 and 5 respectively, and primer 4 and 6 carries out pcr amplification, and the PCR reaction conditions is: 94 ℃ of 5min, 94 ℃ of 1min afterwards, 52 ℃ of 1min, 72 ℃ of 1min totally 33 circulations, last 72 ℃ of 10min.Pcr amplification product is through the phenol extracting, and 1% low melting-point agarose gel electrophoresis is reclaimed.Two kinds of PCR products that will reclaim then mix, and carry out the PCR second time, and the PCR reaction conditions is: 94 ℃ of 10min, 94 ℃ of 1min afterwards, 53 ℃ of 1min, 72 ℃ of 1min totally 33 circulations, last 72 ℃ of 10min.Template---primer extends two fragments each other, produces the variation tPA gene (rtPAm-1) of 1.1Kb.

4. expression plasmid pET-His is transformed

Expression plasmid pET-His is available from gene Dynamic Test Chamber company limited.Original pET-His plasmid can give expression to continuous 6 Histidines so that protein purification, but does not meet biological products pharmacy requirement, increases operation and significantly improves production cost and cut fusogenic peptide Duan Zehui after purifying again.Therefore the main purpose of this step is the coding region of removing 6 Histidines on the pET-His plasmid.Shown in Figure 1, respectively design a primer according to the sequence of the left and right sides, 6His coding region of pET plasmid:

Primer 7:CG CTC CAG GGATCC GCA GAA TTC (forward)

Primer 8:CA TGA GCG GGA TCC TTC TTA AAG TTA AAC (oppositely)

Utilizing above-mentioned primer, is that template is carried out PCR with the pET-His plasmid, and the PCR reaction conditions is: 94 ℃ of 10min, 94 ℃ of 1min afterwards, 53 ℃ of 1min, 72 ℃ of 3min totally 33 circulations, last 72 ℃ of 10min.PCR can obtain a linear DNA amplified production, and an end contains restriction enzyme site BamH I, and the other end contains restriction enzyme site BamHI and HindIII (Fig. 1).

5. the structure of recombinant expression plasmid pETrtPAm-1:

The linear DNA amplified production that previous step PCR obtains cuts with BamHI and HindIII, and reclaims with 0.7% low melting-point agarose gel electrophoresis, and the rtPAm-1 gene that PCR obtains in going on foot the 3rd is used behind BamHI and the HindIII double digestion and reclaimed.Connect two fragments with the T4 ligase enzyme, obtain recombinant plasmid pETrtPAm-1, transformed into escherichia coli DH5 α competent cell (bacillus coli DH 5 alpha is preserved by this laboratory), obtain positive bacterium colony with penbritin (Amp)-LB plate screening, through identifying (step as follows), called after DH5 α rtPAm-1.

6. the evaluation of plasmid pETrtPAm-1:

Adopt alkaline lysis, with reference to Sa Samu Brooker etc., molecular cloning experiment guide (second edition, 1992, Science Press), from positive bacterium colony DH5 α rtPAm-1 culture, extract plasmid pETrtPAm-1, with KpnI and HindIII double digestion, 0.7% agarose gel electrophoresis inspection, the result produces 0.8Kb and 2.9Kb two bands (Fig. 2), conform to the expection size, wherein the band of 0.8kb is represented the P fragment in the rtPAm-1 gene, and the band of 2.9kb then is that the FE fragment adds pET plasmid fragment.Further identifying with PCR, is template with the recombinant plasmid, carries out PCR with primer 1 and primer 4, can obtain size and be the amplified band of 1.1kb (Fig. 2), conforms to the expection size.

The order-checking of rtPAm-1 gene is undertaken by handsome (invitrogen) Bioisystech Co., Ltd on the plasmid.Sequencing primer is the t7 rna polymerase sequence universal primer that utilizes on the pET plasmid, sequencing result shows that the rtPAm-1 gene order and the desired design of acquisition is in full accord, rtPAm-1 full length gene 1083nt (comprising termination codon TGA), 360 the amino acid whose albumen of encoding.With the plasmid called after pETrPAm that produces.

The preparation of embodiment 2 engineering strain BL21 (DE3)/rtPAm-1

1. the extraction of recombinant plasmid pETrtPAm-1

DH5 α rtPAm-1 is inoculated into the LB substratum, and 37 ℃ of incubated overnight adopt alkaline lysis method of extracting plasmid pETrtPAm-1.

2.BL21 (DE3) preparation of competent cell

Use cold CaCl ₂Legal system is equipped with BL21 (DE3) competent cell.BL21 (DE3) bacterial classification inoculation in the 20mlLB liquid nutrient medium, was cultivated 3-5 hour for 37 ℃, culture ice bath 10min, the 4000rpm centrifugal collecting cell adds precooling 0.1M CaCl ₂Re-suspended cell, ice bath 30min, the 4000rpm centrifugal collecting cell adds 500 μ l0.1M CaCl ₂(by initial incubation volume 20ml) suspension cell places 4 ℃.

3. recombinant plasmid pETrtPAm-1 transforms BL21 (DE3)

The pETrtPAm-1 plasmid is mixed with BL21 (DE3) competent cell, 42 ℃ of heat shock 90s, ice bath 1-2min adds the LB liquid nutrient medium, cultivates 45min for 37 ℃, and coating penbritin (Amp)-LB flat board is cultivated 12-16h, positive bacterium colony can be occurred for 37 ℃.Identify through embodiment 3, can produce the proteic bacterial strain called after of rtPAm-1 BL21 (DE3)/rtPAm-1.

Embodiment 3 coli strain BL21 (DE3)/rtPAm-1 induce through isopropylthio-(IPTG) and produce rtPAm-1 albumen

From the rapid penbritin of previous step (Amp)-LB flat board, select single bacterium colony, being inoculated into 5ml contains in the LB liquid nutrient medium of 100 μ g/ml penbritins (Amp), cultivate 14h for 37 ℃, press 1: 40 transferred species then in the 2YT substratum, cultivate 2～3h for 37 ℃, adding isopropylthio-(IPTG) is 1mM to final concentration, 37 ℃ of abduction delivering 6h.Centrifugal collection thalline, be suspended in buffer A (20mMTris, 0.5M NaCl, pH8.0), the ultrasonic disruption cell, centrifugal collecting precipitation carries out the SDS-polyacrylamide gel electrophoresis, the analysing protein expression.

Fig. 3 is the result of 10%SDS-polyacrylamide gel electrophoresis, and after inducing through isopropylthio-(IPTG), coli strain efficiently expresses out rtPAm-1.The expressing protein molecular weight is about 38kD, conforms to the expection size.Expressing protein is the inclusion body form, after cytoclasis, all appears in the precipitation, and expression amount accounts for more than 30% of total protein of cell.

The fermentative production level of coli strain BL21 (DE3)/rtPAm-1 in 410 liters of fermentor tanks of embodiment

1. inclined-plane seed culture.LB inclined-plane solid medium contains penbritin (Amp) 100 μ g/ml, cultivates 16 hours for 37 ℃.

2. shake-flask seed is cultivated: the 2YT liquid nutrient medium, contain penbritin (Amp) 100 μ g/ml, every bottle of 100ml substratum, inoculation one ring, 37 ℃ shaking culture 12-14 hour.

3. fermentation culture, used substratum is a fermention medium: peptone 20g/L, yeast extract 8g/L, NaCl2g/L, MgSO ₄0.2g/L, bubble enemy 20 μ l/L, pure water preparation, 10 liters of culture volumes.Regulate pH value to 7.0 before the sterilization.Steam sterilizing, cylinder was pressed 0.1mpa (120 ℃) 20 minutes.

The control condition of fermentation culture and parameter:

10 liters of culture volumes, pH6.8-7.0

Inoculum size 5%-10%

Stirrer rotating speed 40Hz

Temperature controlling range 35-37 ℃

Tank pressure 0.05-0.07MPa

Air flow 3-5 liter/min (1: 0.3-1: 0.5)

By above-mentioned condition and parameter, be cultured to that bacterium amount 6-8 grams per liter, pH value rise to 7.2, DO drops to 20% when following, adds isopropylthio-(IPTG) to final concentration 1mM, 35-37 ℃ is continued cultivation, induced protein expression 6-8 hour.Fermentation whole process is 10-12 hour.

Fermentation can obtain the inclusion body of Recomposed tPA.Continuously ferment with 10 liters of automatic fermenters and to produce 10 batches, thalline output (centrifugation weight in wet base) is stabilized in 8.0-10.0g/L, and tPA crystal (behind the purifying, weight in wet base) is stabilized in 900-1200mg/L, and mean yield reaches 1100mg/L.The tPA crystal checks that through the 10%SDS-polyacrylamide gel electrophoresis tPA protein content is about 90%.

Proteinic renaturation of embodiment 5 rtPAm-1 and purifying

1. will be through isopropylthio-(IPTG) inductive coli strain BL21 (DE3)/centrifugal collection of rtPAm-1, be suspended in buffer A (20mM Tris, 0.5M NaCl, pH8.0).

2. ultrasonic disruption cell, centrifugal collecting precipitation, wash 3 times after, be dissolved in 8M urea, under 4 ℃ of conditions, with phosphate buffered saline buffer (PBS) the dialyzed overnight renaturation of pH8.0.

3. utilize molecular sieve chromatography G50-Sephadex (available from peace agate West Asia company) to carry out purifying, pillar 10mMTris-Cl (pH7.5) balance, upper column quantity is a column volume 20%, use 10mMTris-Cl, 0.15-0.5MNaCl (pH7.5) gradient elution, monitor collection with nucleic acid protein detector (wavelength 280nm), tPA directly flows out (band black tea look) at first from post, and the back is the following albumen (colourless) of molecular weight 30KD.

Fig. 3 swimming lane 4 shows that through the rtPAm-1 albumen behind sex change dissolving, renaturation and the purifying behind G50-Sephadex sieve chromatography purifying, the proteic purity of rtPAm-1 can reach more than 95%.

Proteic active detection of the rtPAm-1 that embodiment 6 coli strain BL21 (DE3)/rtPAm-1 expresses

Adopt the solusphere method to carry out, step is as follows:

1.tPA standard substance: available from Beijing pharmaceutical biological product calibrating institute of the Ministry of Health, every bottle contains tPA30000IU, and with the dissolving of 3ml physiological sodium chloride solution, making tPA concentration is 10IU/ul.

2. human thrombin: be made into 100IU/ml with physiological sodium chloride solution, in-20 ℃ of preservations.

3. Profibrinolysin:, be made into 0.5mg/ml with physiological sodium chloride solution available from Beijing pharmaceutical biological product calibrating institute.

4. human fibrinogen: 30mg/ props up, Nat'l Pharmaceutical ﹠ Biological Products Control Institute's preparation standard reagent.Configuration before the experiment, earlier at 37 ℃ of water-bath preheating 15min, physiological sodium chloride solution is preheating simultaneously also before the configuration, and every adds the 5ml physiological sodium chloride solution then, and insulation 30min is left standstill in 37 ℃ of water-baths dissolves it fully, is configured to 6mg/ml solution.

5. fibrin plate preparation: take by weighing the 125mg agarose, add the 23ml physiological sodium chloride solution, boil dissolving, 60 ℃ of water-bath balances, add 280ul scleroproein (0.5mg/ml), add 14ul zymoplasm (100IU/ml), do not stop to shake up, muddy back is fallen dull and stereotyped, horizontal positioned, and room temperature is solidified.

6. active the detection: punch on fibrin plate with punch tool, diameter 2mm, in tPA sample and standard substance point hand-hole, 37 ℃ 8 hours, measurement transparent circle diameter.Transparent circle diameter according to standard tPA and rtPAm-1 sample calculates the sample activity.

The results are shown in Figure 4, can produce molten fine circle by plasminogen activation through the rtPAm-1 albumen behind sex change, renaturation and the purifying, through with the conversion of standard tPA, activity reaches 5-6 * 10 ⁵IU/mg reaches the level of international like product.

The inheritance stability Journal of Sex Research of embodiment 7 coli strain BL21 (DE3)/rtPAm-1

Streak inoculation bacterial strain on penbritin (Amp)-LB solid plate substratum is cultivated 24h for 37 ℃, and picking list bacterium colony continues streak culture, and every biography once is designated as a generation.In going down to posterity every 5 generation picking list colony inoculation 5ml LB liquid nutrient mediums (containing penbritin 100 μ g/ml), cultivate 14h for 37 ℃, press 1: 40 transferred species then in the 2YT substratum, cultivate 2～3h for 37 ℃, adding isopropylthio-(IPTG) is 1mM to final concentration, 37 ℃ of abduction delivering 6h.Centrifugal collection thalline, (20mMTris, 0.5MNaCl pH8.0), carry out SDS-polyacrylamide gel electrophoresis analysing protein expression to be suspended in buffer A.

Fig. 5 shows, after going down to posterity through 30 times, coli strain BL21 (DE3)/rtPAm-1 still can express rtPAm-1 albumen in stability and high efficiency ground after isopropylthio-(IPTG) is induced, show that coli strain BL21 (DE3)/rtPAm-1 has good genetic stability.

The proteic animal experiment of embodiment 8 rtPAm-1

1.rtPAm-1 the transformation period in the proteic body

Get three of rabbit, dosage by 100,000 unit/kilograms is injected from ear vein, injection finishes back 5 minutes blood-sample withdrawals for the first time, be added in the tubule of antithrombotics, centrifugal 1500 rev/mins of sedimentation cells, get 5 μ l supernatants and survey the tPA activity, adopted a blood sample and survey the tPA activity, the mean time of the active decline 50% of calculating tPA in later per 5 minutes.Through measuring rtPAm-1 at the intravital transformation period 20-30 of rabbit minute.

2. abnormal toxicity test:

Undertaken by tail vein injection and two test group of abdominal injection, by the dosage injection of 50,000 unit/kilograms, 20 of each sample injections are that contrast is rented with 20 injection stroke-physiological saline solution in addition, observe continuously 7 days.The mouse of two test group of result is all strong depositing in 7 days, no abnormal reaction, body weight gain is movable normal with feed.

SEQUENCE?LISTING

＜110〉Wuhan University Wuhan terrestrial life Engineering Co., Ltd

＜120〉a kind of coli strain and preparation method who produces recombination buman tPA

＜130〉a kind of coli strain and preparation method who produces recombination buman tPA

<160>1

<170>PatentIn?version?3.1

<210>1

<211>1083

<212>DNA

<213>Artificial

<400>1

atgggaagat?cttaccaagt?gatctgcaga?gatgaaaaaa?cgcagatgat?ataccagcaa 60

catcagtcat?ggctgcgccc?tgtgctcaga?agcaaccggg?tggaatattg?ctggtgcaac 120

agtggcaggg?cacagtgcca?ctcagtgcct?gtcaaaagtt?gcagcgagcc?aaggtgtttc 180

aacgggggca?cctgccagca?ggccctgtac?ttctcagatt?tcgtgtgcca?gtgccccgaa 240

ggatttgctg?ggaagtgctg?tgaaatagat?accagggcca?cgggtacctg?cggcctgaga 300

cagtacagcc?agcctcagtt?tcgcatcaaa?ggagggctct?tcgccgacat?cgcctcccac 360

ccctggcagg?ctgccatctt?tgccaagcac?gaggagtcgc?ccggagagcg?gttcctgtgc 420

gggggcatac?tcatcagctc?ctgctggatt?ctctctgccg?cccactgctt?ccaggagagg 480

tttccgcccc?accacctgac?ggtgatcttg?ggcagaacat?accgggtggt?ccctggcgag 540

gaggagcaga?aatttgaagt?cgaaaaatac?attgtccata?aggaattcga?tgatgacact 600

tacgacaatg?acattgcgct?gctgcagctg?aaatcggatt?cgtcccgctg?tgcccaggag 660

agcagcgtgg?tccgcactgt?gtgccttccc?ccggcggacc?tgcagctgcc?ggactggacg 720

gagtgtgagc?tctccggcta?cggcaagcat?gaggccttgt?ctcctttcta?ttcggagcgg 780

ctgaaggagg?ctcatgtcag?actgtaccca?tccagccgct?gcacatcaca?acatttactt 840

aacagaacag?tcaccgacaa?catgctgtgt?gctggagaca?ctcggagcgg?cgggccccag 900

gcaaacttgc?acgacgcctg?ccagggcgat?tcgggaggcc?ccctggtgtg?tctgaacgat 960

ggccgcatga?ctttggtggg?catcatcagc?tggggcctgg?gctgtggaca?gaaggatgtc 1020

ccgggtgtgt?acaccaaggt?taccaactac?ctagactgga?ttcgtgacaa?catgcgaccg 1080

tga 1083

Claims (4)

1. a coli strain of producing recombination buman tPA is characterized in that coli strain Escherichia coil.BL21 (DE3)/rtPAm-1, and its deposit number is CCTCC M205112.

2. a kind of coli strain BL21 according to claim 1 (DE3)/rtPAm-1 is characterized in that: contain recombinant plasmid pETrtPAm-1 in the Bacillus coli cells.

3. the recombinant plasmid pETrtPAm-1 among coli strain BL21 according to claim 2 (DE3)/rtPAm-1 is characterized in that: contain a kind of recombination mutation buman tPA gene, it has the nucleotide sequence shown in the SEQ ID NO.1.

4. preparation method who is used to realize the described a kind of colibacillus engineering strain of claim 1, it may further comprise the steps:

A, amplify the FE fragment and the P fragment of tPA gene respectively with PCR method;

B, usefulness restriction enzyme BamHI and KpnI cutting FE fragment, with restriction endonuclease KpnI and HindIII cutting P fragment, with restriction endonuclease BamHI and HindIII cutting pQE30 plasmid, carrying out three fragments with the T4 ligase enzyme connects, construction recombination plasmid pQErtPAm-1, contain Recomposed tPA gene on it, by F, E, the P district of original tPA gene forming;

C, two mutagenic primers of design adopt the PCR fixed-point mutation method on Recomposed tPA gene original tPA molecule R298, R299 coding place all to be sported the E coding;

D, expression plasmid pET-His is transformed, utilize the pET-His plasmid DNA to be template, design a pair of primer and carry out pcr amplification, then with BamHI and HindIII cutting amplified production, the 1.1kb variation tPA gene that again PCR is produced connects two fragments with restriction endonuclease BamHI and HindIII cutting with the T4 ligase enzyme, obtains recombinant plasmid pETrtPAm-1, transformed into escherichia coli DH5 α obtains coli strain DH5 α rtPAm-1;

E, evaluation recombinant plasmid pETrtPAm-1: recombinant plasmid is identified with double digestion, PCR method;

F, cultivation coli strain DH5 α rtPAm-1 extract recombinant plasmid pETrtPAm-1;

G, cold CaCl ₂Legal system is equipped with e. coli bl21 (DE3) competent cell;

H, with recombinant plasmid pETrtPAm-1 transformed into escherichia coli BL21 (DE3) competent cell, through screening with identify to obtain coli strain BL21 (DE3)/rtPAm-1.

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