CN1348003A - Hirudin gene hv 2-47k and its synthesis and expression - Google Patents

Abstract

Based on the amino acid sequence data of over twenty natural hirudin mutants and by using HV2 hirudin as prototype, novel hurudin gene hv2-47k capable of inserting pPIC9 vector is finally obtained through replacing the asparagine (N) with lysine (K), selecting methanol yeast Picha pastoris preferred genetic codon, synthesizing eight-section deoxynucleotide segment (primer) and recursive PCR splice. The gene hv2-47k is expressed in secretion mode in methanol yeast Pichia pastoris, and in cylinder fermentation, the novel hirudin expression amount in the fermented supernatant liquid is not lower than 30000 ATU/ml (after inducement for 50 hr) or 2.0 g/L yield.

Description

Hirudin gene hv2-47k and synthetic and expression thereof

The present invention relates to a kind of reconstituted drug, particularly hirudin gene and synthetic and expression thereof.

R-hirudin (Hirudin) is to separate a class polypeptide that obtains from Hirudo japonica (Hirudomedicinalis) sialisterium the earliest.Afterwards, the investigator separates the r-hirudin varient that obtains as many as 20 kinds from various bloodthirsty leech, they mostly are made up of more than 60 (64-69 is not waited) amino-acid residues, wherein maximum three kinds of varients (HirudinVariants) of being made up of 65 or 66 amino acid are used in research, be called HV1, HV2 and HV3, all natural hirudin C-hold all sulphatings (RobertHand, et al Transgenica-Topics in Clinical Biotechnology http://pharminfo.com/pubs/transgen/th-hir.html) of the 63rd tyrosine Tyr (Y).

R-hirudin all has very high avidity to people and other mammiferous zymoplasms, is the strongest zymoplasm natural inhibitor of finding so far.It is in conjunction with the center irreversible combination to take place by catalytic center and Fibrinogen with zymoplasm, makes the zymoplasm inactivation.R-hirudin of a part and the zymoplasm of a part form firm, non-covalent mixture, and therefore, the zymoplasm of inactivation is irreversible basically, and this just makes the hematoblastic activation process of fibrinogenic conversion and thrombin-mediated be blocked.

The N-end (1～39) of r-hirudin mainly contains hydrophobic amino acid, stablize by three intrachain disulfide bonds, and C-end (40～65) is rich in tart amino acid, and wherein the peptide section of 10 amino-acid residues compositions of C-end is specificity bind thrombin and the critical area that makes it inactivation.47 Methionins (K) residue to HV1 and HV3 discovers that K is to the active of r-hirudin and with the interaction of zymoplasm also very important (Mao, S.J.T., etal., Anal.Biochem.161:514-518,1987; Harvey, R.P., etal., PNAS (USA) 83:1084-1088,1986; Dodt, J., etal., FEBSLett.2 29:87-90,1988).The three-dimensional structure that 47 Methionins of HV2 may influence r-hirudin simultaneously by hydrogen bond and electrostatic interaction with and with the combination of zymoplasm, its thermostability is increased, biological activity improves 61% (Chinese biological chemistry and molecular biology journal such as Lu Yu peak, 1998; 14 (1): 32-37)

Limited in view of the natural hirudin source, in the later stage eighties, all begin one's study both at home and abroad and adopt genetic engineering technique to produce lepirudin 023 ludon, to satisfy the needs of pharmacology and clinical study test.The company that is engaged at present hirudin gene engineering research exploitation abroad is as many as 11 families.Wherein German Hoechst company holds primary Isoleucine (I) to become leucine (L) N of HV2, the 2 63 the tyrosine Tyr desulfurization r-hirudin Lepirudin (chemical name of producing: Leu1, Thr2-63-desulfohirudin, trade(brand)name REFLUDAN), its powder injection successively went on the market in the Europe and the U.S. in 1998, be used for the treatment of the thrombocytopenia (HIT, Heparin-induced thrombocytopenia) that heparin brings out.The 63rd the desulfurization r-hirudin that the Ciba-Geigy of Switzerland (NOVARTIS) company adopts recombinant technology to express in cereuisiae fermentum, called after Desirudin, trade(brand)name Revasc, be mainly used in the complication after bone surgery and other major operations, as dvt (DVT, Deep vein thrombo-sis) and pulmonary infarction (PE, Pulmonary embo lism), existing in Europe listing (Aventis-Refludan Prescribing Information http://www.Aventispharma-s.com/Pis/refludan-TXT.html).

Through after the analysis-by-synthesis, I am based on the aminoacid sequence of HV2, simultaneously with reference to HV1, the sequence of HV3,47 the l-asparagine (N) of HV2 is substituted by Methionin (K), the genetic codon of selecting for use methanol yeast Pichia pastoris to be liked, 8 sections oligonucleotide chains of synthetic (primer) (chain length 31-46 base does not wait), adopt recursion PCR (Recursive PCR) (Bi Qun, Huang Yixiu, the recurrence PCR of Zhu's hirudin gene in holy heptan synthetic and expression in intestinal bacteria and secretion Peking University's journal (natural science edition) 33 (6): 737-742,1997), splice novel this gene of r-hirudin hv2-47K and it is inserted among the Pichia yeast secretion type expression carrier pPIC9 construction of expression vector PIC8.2.Transform the Pichia yeast with this carrier, obtain the engineering bacteria that efficiently expresses.To the effect that of the present invention: 1.hv2-47k gene synthetic

According to the HV2-47K aminoacid sequence of design, consider the expression vector that will use, determine after the analysis of machine Autocad that as calculated full length gene is 222bp.We synthesize 8 oligonucleotide fragment/primers altogether to obtain this gene.For guaranteeing that the HV2 gene produces minimum assorted band through recursion PCR, every primer is with about 15 base pairs of adjacent primer strand complementary cohesive end.Because the carrier of selecting Pichia yeast secretion type expression carrier pPIC9 to insert as gene, so 5 ＇-ends in HV2-47K structure gene are designed the XhoI restriction enzyme site, and hold the codon that inserts KEX2 protease recognition sequence (K-R) between first amino-acid residue at this site and HV2-47K N-, so that target protein can successfully cut signal peptide when secretion; 3 '-end is TAA terminator codon and EcoRI restriction enzyme site (Fig. 1).Like this after the gene splicing success, through XhoI with EcoRI is two cuts, just can be inserted into the pPIC9 expression vector, construction of expression vector PIC8.2 (Fig. 3).2.hv2-47k Gene Sequence Analysis

The pPIC9 plasmid vector is a kind of shuttle vectors of Invitrogen company design, its plasmid that can increase in bacterium, the goal gene of integrating in yeast, inserting.This carrier has three sequencing primers: two target gene 5s '-the end primer (5 '-AOX1,2-Factor Seq) and one 3 '-the end primer (3 '-AOX1).Therefore; the structure of screening, order-checking and expression vector once can be finished; because the hv2-47k goal gene has only 222bp long; be inserted between the multiple clone site XhoI-EcoRI of pPIC9; so we select 5 for use '-the AOX1 primer is to goal gene in the pPIC9 expression plasmid check order (measure in last sea base health (Genecore) company, sequencer address is seen Fig. 2).By the result of order-checking as seen, the base sequence of goal gene does not go wrong when recurrence PCR splice, its 5 '-the XhoI point of contact of end, the base sequence of KEX2 enzyme recognition sequence (K-R), 3 '-end TAA termination codon and EcoRI restriction enzyme site be all with original design identical.In addition, from 3 '-end EcoRI site extend back AVrII, NotI restriction enzyme site the multiple clone site (MCS) of visible pPIC9.3.hv2-47k expression of gene

Express novel r-hirudin HV2-47K with the GS115-HV2-47K Pichia Yeast engineering bacteria that methanol induction makes up, adopt antithrombin vigor assay excretory r-hirudin amount (being the ATU of anti-freezing unit) to determine its expression level.Because the hv2-47k gene is a genetic codon of selecting for use methanol yeast Pichia pastoris to be liked, when shake-flask culture, hv2-47k gene expression amount in Pichia zymic nutrient solution supernatant is not less than 2,000ATU/ml (inducing 50 hours); When the 30L fermentation cylinder for fermentation, expression amount is not less than 30, and 000ATU/ml (inducing 50 hours) is equivalent to the output of 2.0g/L.

Synthetic eight sections few nucleic acid (primer) of embodiment 1:hv2-47k gene all synthesize in Shanghai bio-engineering corporation (Sangon), reclaim through the PAGE electrophoresis, are packed as the 10D/ pipe, synthetic numbering: 33502～33509, number certainly: P1～P8, in September, 97).The synoptic diagram of eight sections primer splicings is as follows:

For avoiding the phase mutual interference between the primer, earlier P3, P4, P5 and P6 are mixed and carry out first round PCR reaction, obtain one section of the centre of hv2-47k gene, be primer with P2, P7 again, first round PCR product is that template is carried out second and taken turns PCR, obtain the part broad in the middle of hv2-47k gene, taking turns the PCR product with second at last is template, is that primer splices complete hv2-47k gene with P1, P8.

3 PCR reactions of whole splicing experience can be finished, and PCR process and condition are as follows: PCR reaction (PCR-1) for the first time: 20ul reaction system: 0.3ul ... P40.3ul ... P52ul ... P32ul ... P61ul ... Mix-d NTP (every kind of dNTP contains 5mM) 2ul ... 10xTaq-plus damping fluid 1ul ... it is 20ul to cumulative volume that Taq-plus enzyme (cushioning, be diluted to 1u/ul with lxTaq-plus) adds ultrapure water 11.4ul.PCR reaction (PCR-2) for the second time: 20ul reaction system: 4ul ... PCR-1 reaction product 2ul ... P22ul ... P71ul ... Mix-d NTP (every kind of dNTP contains 5mM) 2ul ... 10xTaq-plus damping fluid 1ul ... Taq-plus enzyme (cushioning, be diluted to 1u/ul) with lxTaq-plus

Adding ultrapure water 8.0ul is 20ul to cumulative volume.PCR reaction (PCR-3) for the third time: 100ul reaction system: 5ul ... PCR-2 reaction product 4ul ... P14ul ... P83ul ... Mixed-dNTP (every kind of dNTP contains 5mM) 10ul ... Taq-plus damping fluid 1ul ... Taq-plus enzyme (proenzyme dress liquid, the suitable 5u of 1ul)

Adding ultrapure water 73ul is 100ul to cumulative volume.The reaction parameter setting of PCR: PCR-1, PCR-2 all take turns at 94 ℃, 60S/58 ℃, 70S/72 ℃, 70S circulation 30, finish rear electrophoresis at every turn and inspect, and determine that specificity is good, and when having only a band, just negate answers product to make the template of next round PCR.Since our design draw the complimentary piece segment length, whenever do not produce assorted band when taking turns PCR, thereby last round of PCR product can use as template directly.PCR-3 takes turns at 94 ℃, 60S/58 ℃, 70S/72 ℃, 70S circulation 30, and 72 ℃ were extended 6 minutes.Each step PCR product of whole recurrence PCR in 1.2% agarose gel electrophoresis result as shown in Figure 4.The structure of embodiment 2:hv2-47k expression vector PIC8.2 adopts the post absorption method to collect the target gene fragment that amplifies the PCR-3 product, cuts with XhoI-EcoRI is two, and glue reclaims and connects usefulness; Get the pPIC9 plasmid, cut with XhoI-EcoRI is two equally, glue reclaims big fragment and connects and use carrier.Set up following ligation system: 7ul ... hv2-47k gene 1ul after the recovery ... pPIC9 carrier 1ul after the recovery ... 10XT4 dna ligase 0.5ul ... T4 dna ligase (about 3U) 0.5ul ... dH2010ul ... total reaction volume is the condition of ligation: 22 ℃, 60 minutes, 65 ℃, 10 minutes.Ligation liquid is transformed E coli TOP10F ' competent cell born of the same parents with the Calcium Chloride Method preparation, and method is referring to J.Sambrook etal, Molecular Cloning a laboratory Mannual, and 2nded 1989.Bacterium after the conversion is layered on the LB+Amp plate, choosing positive colony from the LB+Amp flat board cultivates, extract plasmid, with XhoI, the screening of EcoRI double digestion method, the energy enzyme cuts out about 220 (216) bp band persons and is the PIC9 plasmid that is inserted in the hv2-47k gene, with 5 '-(Invitrogen Kit provides the AOX1 primer, or the sequence that provides by Kit is synthetic) to objective gene sequencing, the result shows: recurrence PCR splices the sequence requirement that the hv2-47k gene conforms with design fully, and is special with expression plasmid called after PIC8.2.Structure gene nucleotide sequence such as Fig. 2 of hv2-47k, building process as shown in Figure 3.Embodiment 3: the GS115-HV2-47K Pichia Yeast engineering bacteria 1 of structure). and Ecoli Top10F ' bacterium that preparation conversion Pichia zymic expression vector PIC8.2 plasmid will contain the PIC8.2 plasmid is in the LB+Amp+Kan liquid culture, with alkali extraction process large quantity extracting plasmid.The PIC8.2 plasmid linearization is made to transform the usefulness of Pichia yeast host bacterium GS115 with SalI.2) the .Pichia zymic transforms with linearizing PIC8.2 expression vector dna transformed competence colibacillus Pichia yeast GS115His ^--Mut ⁺Host bacterium (competence adopts the Pichia Easy CompTransformation Kit preparation of Invitrogen company), press Pichia Expression Kit-ProteinExpression, Catalog No:K1710-01) described in step transform and screen.Because of host GS115 is His ^-(histidine defect type), can not the MD plate on growth (1.34%YNB, 4 * 10 ^-5%Biotin, 2%Dextrose 1.5%Agar), has only the successful transformant of conversion to grow.3) screening of .GS115-HV2-47K Pichia Yeast engineering bacteria is chosen single bacterium colony from the MD plate, be inoculated into and contain 10mlYPD substratum (1%Yeast extract, 2%Peptone, 2%Dextose), cultivated 30 hours for 30 ℃, under the condition of aseptic technique, it is that 50ml shakes in the bottle that the collection thalline forwards the volume that contains 10mlMM inducing culture liquid to, be not less than 300 rev/mins of cultivations down, and, getting a sample and detect hirudin activity every 10 hours.Adding anhydrous methanol after each sampling is 0.5-1.0% to final concentration.The about 60-70 of inducing culture hour.High expression level amount (when hirudin activity is the highest) occurred between 55-65 hour.Constant relatively for guaranteeing the nutrient solution volume, take out how many samples at every turn, just add the MM nutrient solution of respective volume, other adds about 0.5ml distilled water and replenishes the water that evaporates.Finally obtain the Pichia Yeast engineering bacteria that to express HV2-47K, called after GS115-HV2-47KPichia yeast.Curve is as shown in Figure 5 over time for the expression amount of HV2-47K.

The r-hirudin hv2-47k gene base sequence and the aminoacid sequence of description of drawings: Fig. 1 design

5 '-hold to be restriction enzyme XhoI recognition sequence CTCGAG, and KEX2 protease recognition sequence amino acid correspondence

Codon AAAAGA; 3 '-hold sequencing result into terminator codon TAA and limiting enzyme EcoRI recognition sequence GAATTC Fig. 2 recurrence PCR method splicing hv2-47k

This figure has only intercepted the key component of former sequencer address, and wherein the fragment between XhoI point of contact and the EcoRI point of contact promptly

It is the agarose gel electrophoresis of three step of the process synoptic diagram Fig. 4 recurrence PCR method pcr amplification product of hv2-47k gene fragment Fig. 3 construction of expression vector PIC8.2

PCR-1: first round PCR product

PCR-2: second takes turns the PCR product

PCR-3: third round PCR product promptly obtains final hv2-47k gene

100bp Ladder ⁺Be dna molecular amount standard map 5 r-hirudin HV2-47k expression amount curve over time in Pichia pastoris

Wen Zhongyong capitalization roman HV2-47k represents the r-hirudin albumen that engineering bacteria is expressed, and represents hirudin gene with small letter italic hv2-47k.

Claims (4)

1. the novel r-hirudin HV2-47K of genetically engineered drug is characterized by, and this medicine is a genetic code of selecting for use methanol yeast Pichia pastoris to be liked, and the gene hv2-47k of synthetic expresses in the Pichia yeast and gets;

2. novel hirudin gene hv2-47k according to claim 1 and amino acid sequence coded thereof are as follows: CTC GAG AAA AGA ATT ACT TAC ACT GAT TGT ACA GAA TCG

Ile Thr Tyr Thr Asp Cys Thr Glu SerGGT CAA AAT TTG TGC CTC TGC GAG GGA AGC AAT GTT TGCGly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val CysGGT AAA GGC AAT AAG TGC ATA TTG GGT TCT AAT GGA AAGGly Lys Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly LysGGC AAC CAA TGT GTC ACT GGC GAA GGT ACA CCG AAG CCTGly Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys ProGAA AGC CAT AAC AAC GGC GAT TTC GAA GAA ATT CCA GAAGlu Ser His Asn Asn Gly Asp Phe Glu Glu Ile Pro GluGAA TAT TTA CAA TAA GAA TTCGlu Tyr Leu Gln

3. novel hirudin gene hv2-47k according to claim 1 can insert different expression vectors, selects eucaryon or procaryotic cell expression host to express, and does not insert the pPIC9 carrier and be not only limited to, and is transformed into and expresses in the Pichia yeast;

4. novel hirudin gene sequence according to claim 2 and aminoacid sequence thereof, the genetic code of every seed amino acid can be selected the different degenerate codons of the primary structure that does not influence novel r-hirudin HV2-47K for use, and is not limited only to the codon that the Pichia yeast is liked.

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