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CN1328380C - High-efficiency expression recombinant hirudin and producing method thereof - Google Patents

High-efficiency expression recombinant hirudin and producing method thereof Download PDF

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Publication number
CN1328380C
CN1328380C CNB011414154A CN01141415A CN1328380C CN 1328380 C CN1328380 C CN 1328380C CN B011414154 A CNB011414154 A CN B011414154A CN 01141415 A CN01141415 A CN 01141415A CN 1328380 C CN1328380 C CN 1328380C
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China
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hirudin
protein
acid
dna molecular
novel
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CN1420176A (en
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赵凯
吴祖泽
苑宾
张津辉
李满文
熊志红
蒋中华
董春娜
曹菊荣
于爱平
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SHANGHAI TASLY PHARMACEUTICAL CO Ltd
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Tianjin Tasly Pharmaceutical Co Ltd
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Abstract

The present invention relates to an anti-thrombus medicine of biological engineering and a production method thereof, particularly to novel hirudin and a production method thereof. A whole gene of variant HV2 is obtained by a method of whole artificial synthesis, and a section of proper front-leading sequences is added to the upper stream of a 5'terminal of the gene; a secretion type expression carrier pHV2 of pichia yeast is built, a host cell GS115 is converted by the carrier, and the carrier is integrated into the chromosome of the host cell. A high-efficiency expression strain is sieved. The production efficiency of a product of recombination gene, the expression level of the product and the biological activity are improved by the steps of optimized fermentation induction and separating purification, and a research of pharmacodynamics shows that the anti-coagulation performance and the anti-thrombus performance are higher than that in a literature reported. Thus, the present invention provides a material source for the further research of the large-scale production and the clinical application of a second type of hirudin.

Description

High-efficient conveying Hirudin And Production Method
The present invention relates to a kind of bionic medicine for treating thrombus thing and production method thereof, particularly a kind of novel r-hirudin and production method thereof.
R-hirudin is the small molecular protein that is produced by leech saliva, can form non-covalent stable compound with α-zymoplasm, thereby the fibrinogenic ability of completely destroy zymoplasm cracking, thereby makes it to become anti-freezing, medicine for treating thrombus thing.Natural hirudin has a series of analogues, and main r-hirudin analogue comprises: r-hirudin I type (HV1), r-hirudin II type (HV2) and r-hirudin III type (HV3).Because the content of r-hirudin is few in hirudinaria manillensis, a large amount of extraction r-hirudins are impossible from leech.Constantly perfect along with genetic engineering technique begins to utilize protokaryon and eukaryotic expression system to come the mass production lepirudin 023 ludon.Present HV1 product is official listing.But HV2 and HV3 product-free listing still.Because therefore the lepirudin 023 ludon large usage quantity for improving productive rate, reduces cost, and obtains the lepirudin 023 ludon of high expression level, good stability, become the problem that must solve.
The objective of the invention is to seek a kind of new r-hirudin 2 type genes, the step that simplifies the operation is raised the efficiency, and obtains the r-hirudin of novel texture.
The objective of the invention is to be achieved through the following technical solutions:
According to the natural HV2 gene order of bibliographical information, adopt total man worker's synthetic method to obtain the full gene of HV2.Have following two schemes to implement the first string simultaneously: total man worker's synthetic gene coded sequence is compared with natural hirudin 2 type dna encoding sequences, and variation has taken place the Nucleotide 121 and 156 respectively.The 121st of the dna encoding sequence that the embodiment of the invention is related is guanylic acid by the cytidylic acid variation, and the 156th is cytidylic acid by the thymidylic acid variation.Add one section leader sequence in its 5 ' end upstream simultaneously, have 12 Nucleotide, its sequence is CTCGAGAAAAGA.Second scheme: total man worker's synthetic gene coded sequence is compared with natural hirudin 2 type dna encoding sequences, divides to be listed in 121.The 121st of related dna encoding sequence is guanylic acid by the cytidylic acid variation, and the 156th is cytidylic acid by the thymidylic acid variation, and the 157th is guanylic acid by the adenylic acid (AMP) variation.Add one section leader sequence in its 5 ' end upstream simultaneously, have 12 Nucleotide, its sequence is CTCGAGAAAAGA.
By engineered method, to above two kinds of lepirudin 023 ludon dna sequence dnas, make up its pichia spp secreted expression carrier pHV2, recombinant expression vector transformed host cell GS115, and be integrated in the karyomit(e) of host cell.Screening efficiently expresses engineering cell strain, engineering cell strain is carried out fermentation culture, specifically expressing goal gene under the inductor effect.Expression product carries out separation and purification, the analysis of aminoacid sequence and the mensuration of biologic activity.At the gene structure characteristics of lepirudin 023 ludon and the growth characteristics of pichia spp, optimize the production technique of fermentation, separation, purifying.Only both can obtain purer product with the separation of two steps, purifying process.The aminoacid sequence of resulting novel r-hirudin is compared with the aminoacid sequence of the natural r-hirudin that obtains 2 types, variation has taken place in the 47th amino acids in first scheme, the 47th amino acids of the aminoacid sequence of the novel r-hirudin that the embodiment of the invention is related is a Methionin by the asparagine variation, variation has taken place in the 47th and the 53rd amino acids in alternative plan, and the 47th amino acids of the aminoacid sequence of the novel r-hirudin that the embodiment of the invention is related is that the 53rd of Methionin is aspartic acid by the asparagine variation by the asparagine variation.
Because the synthetic of hirudin gene of the present invention all contains one section suitable leader sequence at its upstream, so can obtain high level expression in yeast expression system.Its expression product secretion is in the extracellular, simplified purification procedures, improved the production efficiency of recombination product, product expression level and biologic activity, and the pharmacodynamic study of anti-freezing, anti-bolt shown, be higher than bibliographical information, this provides the material source for the scale operation of r-hirudin II type and the further investigation of clinical application.
Pichia spp involved in the present invention is by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preservation date is August 23 calendar year 2001, and classification called after pichia pastoris is numbered 0619.
Below be embodiments of the invention
Attached explanation:
1 efficiently expresses the r-hirudin dna sequence dna for the present invention (scheme one is related)
2 efficiently express amino acid sequence of hirudin for the present invention (scheme one is related)
3 efficiently express the r-hirudin dna sequence dna for the present invention (scheme two is related)
4 efficiently express amino acid sequence of hirudin for the present invention (scheme two is related)
One, contains the acquisition of two kinds of novel hirudin genes of leader sequence
Utilize total man worker's synthetic method, the synthetic novel r-hirudin that contains leader sequence, wherein a kind of is that 121 Nucleotide are guanylic acid in its structure gene, 156 is cytidylic acid, another kind of 156 is cytidylic acid for 121 Nucleotide in its structure gene are guanylic acid, and 157 is guanylic acid.And at the synthetic leader sequence that comprises 12 Nucleotide of XhoI restriction enzyme site in 5 ' end upstream of above structure gene, at its synthetic terminator and EcoRI restriction enzyme site of containing in 3 ' end downstream.XhoI and EcoRI double digestion, ℃ preservation of electrophoresis rate of recovery thing-20 are standby.
Two, make up the expression plasmid of yeast that efficiently expresses
With pBSK XhoI and EcoRI double digestion, electrophoresis reclaims, and two kinds of synthetic products are connected with pBSK, in Transformed E .coli DH5 α (preserve in this laboratory) recipient bacterium, obtain recombinant clone through screening, plasmid DNA is extracted in amplification back, apportion check order (the results are shown in 1).Recombinant plasmid pBSK-HV2 XhoI and EcoRI double digestion, electrophoresis reclaims small segment, with Pichia anomala expression plasmid pPIC9 (available from Invitrogen company) XhoI and EcoRI double digestion, reclaim segment with above-mentioned two after product reclaims and spend the night, obtain recombinant plasmid pHV2 with the connection of T4 ligase enzyme.Be converted into the positive amplification of E.coli TOP 10F (available from Invitrogen company) screening upgrading grain clone.
Three, obtain the Yeast engineering bacteria of high expression level r-hirudin 2 types
With recombinant plasmid pHV2 Sall single endonuclease digestion, to reclaim enzyme and cut product, method transformed competence colibacillus pichia spp GS115 bacterial strain (available from Invitrogen company) is changeed in electricity consumption.Filter out high expression level active bacterial strain.Hirudin activity can reach 1500ATU/ml in its supernatant after shake-flask culture was induced in 48 hours.Expression product is through being accredited as target protein (the results are shown in 2).
Four, scale operation r-hirudin
The Pichia yeast engineering that above-mentioned screening obtains utilizes type fast through being accredited as methyl alcohol, carry out large scale fermentation with the large vol fermentor tank, fermented liquid supernatant engineered protein expression amount reaches more than the 1.5g/L, fermented liquid target protein electrophoresis purity is more than 80%, the centrifugal supernatant of fermented liquid is through ultrafiltration, two step of ion-exchange purification procedures, purity of protein can reach 98.8%, total yield 50-60%, this r-hirudin that efficiently expresses has been simplified operation steps, and has reached the processing requirement of low-cost scale operation.
1：
5-CTC?GAG?AAA?AGA?ATT?ACT?TAC?ACT?GAT?TGT?ACA?GAA?TCG
GGT?CAA?AAT?TTG?TGC?CTC?TGC?GAG?GGA?AGC?AAT?GTT?TGC
GGT?AAA?GGC?AAT?AAG?TGC?ATA?TTG?GGT?TCT?AAT?GGA?AAG
GGC?AAC?CAA?TGT?GTC?ACT?GGC?GAA?GGT?ACA?CCG?AAG?CCT
GAA?AGC?CAT?AAC?AAC?GGC?GAT?TTC?GAA?GAA?ATT?CCA?GAA
GAA?TAT?TTA?CAA?TAA?GAA?TTC-3
2：
ITYTDCTESGQNLCLCEGSNVCGKGNKCILGSNGKGNQKVTGEG
TPKPESHNNGDFEEIPEEYLQ
3：
5-CTC?GAG?AAA?AGA?ATT?ACT?TAC?ACT?GAT?TGT?ACA?GAA?TCG
GGT?CAA?AAT?TTG?TGC?CTC?TGC?GAG?GGA?AGC?AAT?GTT?TGC
GGT?AAA?GGC?AAT?AAG?TGC?ATA?TTG?GGT?TCT?AAT?GGA?AAG
GGC?AAC?CAA?TGT?GTC?ACT?GGC?GAA?GGT?ACA?CCG?AAG?CCT
GAA?AGC?CAT?AAC?GAC?GGC?GAT?TTC?GAA?GAA?ATT?CCA?GAA
GAA?TAT?TTA?CAA?TAA?GAA?TTC-3
4：
ITYTDCTESGQNLCLCEGSNVCGKGNKCILGSNGKGNQKVTGEG
TPKPESHNDGDFEEIPEEYLQ

Claims (7)

1, a kind of dna molecular of novel coding r-hirudin, it is characterized in that this dna molecular compares with the natural r-hirudin 2 type dna moleculars that derive from leech, the 121st Nucleotide of this dna molecular is that guanylic acid, the 156th Nucleotide are cytidylic acid by the thymidylic acid variation by the cytidylic acid variation, and the 157th Nucleotide is guanylic acid by the adenylic acid (AMP) variation; Its leader sequence is CTCGAGAAAAGA.
2, a kind of recombinant expression vector is characterized in that this recombinant expression vector contains the dna molecular of the described novel r-hirudin of claim 1, and this recombinant vectors is the pichia spp secreted expression carrier.
3, a kind of reconstitution cell is characterized in that the host cell of this reconstitution cell is transformed by the described recombinant vectors of claim 2; Wherein the described dna molecular of claim 1 is integrated in the reconstitution cell karyomit(e), and keeps above-mentioned molecule in going down to posterity, and keeps its genetic stability.
4, reconstitution cell according to claim 3 is characterized in that host cell is yeast cell GS115.
5, a kind of protein of novel r-hirudin is characterized in that this protein is coded by the described dna molecular of claim 1.
6, the protein of novel r-hirudin according to claim 5, it is characterized in that this protein compares with the natural hirudin 2 type protein that derive from leech, the 47th asparagine variation is aspartic acid for Methionin and the 53rd asparagine variation.
7, a kind of method of protein that produces as novel r-hirudin as described in claim 5 or 6 is characterized in that this method comprises the steps:
(1) synthetic r-hirudin dna molecular as claimed in claim 1;
(2) as the structure of expression vector as described in the claim 2;
(3) as the structure of claim 3 or 4 described reconstitution cells:
(4) culture expression of the reconstitution cell that obtained of step (3);
(5) separation and purification of the expression product that obtained of step (4);
(6) analysis and the bioactive mensuration of the aminoacid sequence of the purified product that obtained of step (5).
CNB011414154A 2001-09-24 2001-09-24 High-efficiency expression recombinant hirudin and producing method thereof Expired - Fee Related CN1328380C (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102373216B (en) * 2011-10-28 2013-06-05 元昊 Construction of recombinant eukaryotic hansenula engineering bacterial strains containing medical hirudin genes and production process of recombinant hirudin

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1073978A (en) * 1991-12-07 1993-07-07 赫彻斯特股份公司 Has the new synthetic isohirudins that improves stability
CN1088836A (en) * 1992-12-30 1994-07-06 中国科学院生物物理研究所 Lepirudin 023 ludon and complex thereof are used for preparation prevention and treatment thrombotic disease medicine
CN1113389A (en) * 1993-08-04 1995-12-13 西巴-盖尔基股份公司 High molecular weight desulphatohirudin
CN1348003A (en) * 2000-10-10 2002-05-08 梅伯皋 Hirudin gene hv 2-47k and its synthesis and expression

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1073978A (en) * 1991-12-07 1993-07-07 赫彻斯特股份公司 Has the new synthetic isohirudins that improves stability
CN1088836A (en) * 1992-12-30 1994-07-06 中国科学院生物物理研究所 Lepirudin 023 ludon and complex thereof are used for preparation prevention and treatment thrombotic disease medicine
CN1113389A (en) * 1993-08-04 1995-12-13 西巴-盖尔基股份公司 High molecular weight desulphatohirudin
CN1348003A (en) * 2000-10-10 2002-05-08 梅伯皋 Hirudin gene hv 2-47k and its synthesis and expression

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