CN1262659C - Human protein C expression method and its special primer and expression carrier - Google Patents

Abstract

The present invention discloses an expression method of human protein C, special primers thereof and an expression vector thereof. The special primers provided by the present invention for amplifying human protein C genes are SEQID No.: 1 and SEQID No.: 2 disclosed in the sequence table. The expression vector of the human protein C is a recombinant Pichia yeast expression vector pPIC9-hPC containing the human protein C genes. In the expression method of the human protein C, the recombinant Pichia yeast expression vector pPIC9-hPC is converted into Pichia yeast to obtain a positive clone, and then the positive clone is induced and cultured to express the human protein C. The present invention uses a gene cloning technology to successfully build the vector pPIC9-hPC for yeast expression. An electroporation converting method is used to obtain yeast transformants with exogenous genes, and the detection by an APTT method shows that the expressed human protein C can prolong the time of blood coagulation to perform a function of blood coagulation resistance. The method of the present invention has extensive industrial prospects in the production of the human protein C.

Description

A kind of expression method of human protein C and primer special thereof and expression vector

Technical field

The present invention relates to expression method and the primer special and the expression vector of a kind of human protein C in the medical biotechnology field.

Background technology

Anticoagulation system in the blood mainly comprises three individual system: antithrombin-III, protein C system and tissue factor pathway inhibitor, their fellowship anti-freezings.Protein C system is by PROTEIN C (protein C, PC), zymoplasm is regulated albumen (thrombomodulin, TM), Protein S (protein S, PS) and PROTEIN C inhibition (protein Cinhibitor, PCI) form, solidifying of blood played important regulatory role, and wherein PROTEIN C is the key component in this system.PROTEIN C is as a kind of important anticoagulant substances in the blood, play an important role for the function of regulating blood coagulation system and fibrinolytic system, low and the venous thrombosis of its blood plasma level, the sudden purpura of newborn infant, disseminated inravascular coagulation (disseminated intravascular coagulation, DIC), hepatic diseases etc. is relevant.

Nineteen sixty, Mammen and Seegers find PROTEIN C, called after " self-excitation thrombogen II at that time when handling thrombogen (prothrombin) mixture _a(autoprothrombin II _a) ".1970, Seegers purifying from ox blood obtained this material.1976, Stenflo isolated a kind of vitamin k-dependent protein (vitamin K-dependent protein) from Ox blood plasma, because of its 3rd peak that is positioned at the albumen wash-out is called PROTEIN C.Seegers confirms PROTEIN C and his the previous autoprothrombin II that finds _aBe same albumen.

PROTEIN C is the glycoprotein that a kind of vitamin K relies on, and belongs to serine protease, and is synthetic in liver cell.Its molecular weight is 62kDa, and optical extinction coefficient is 14.5, and iso-electric point is 4.4-4.5.The normal contents of PROTEIN C is about 2-6mg/L in the human plasma, and asexuality difference has the trend that increase is arranged with age growth.The PROTEIN C gene is positioned at No. 2 karyomit(e)s of people (2q13-2q14) with the form of single copy, and long 11.2kb comprises 3 ' the distolateral wing sequence of 5 ' the distolateral wing sequence of 6kb, 9 exons, 8 introns and 4kb, and wherein intron accounts for 83% of full sequence.The mRNA length overall of PROTEIN C is 1795nt, accounts for 0.02% of total mRNA.The cDNA of PROTEIN C is cloned, comprises 5 ' non-coding region (73bp), open reading frame (1383bp), 3 ' non-coding region (296bp) and polyA tail (38bp).

PROTEIN C in the human plasma exists with two kinds of forms: duplex molecule and single chain molecule account for 80-90% and 10-20% respectively.Double-stranded PROTEIN C is made up of light chain and heavy chain, and interchain is connected by disulfide linkage.(γ-Gla) structural domain contains 9 γ-Gla residue to the Gla of PROTEIN C light chain N end, is to be processed by L-glutamic acid carboxylation in the new polypeptide chain.This γ-Gla residue is PROTEIN C and Ca ²⁺High-affinity bonded position combines Ca ²⁺γ-Gla residue can be further interact with electronegative membrane phospholipid and make PROTEIN C present its biological activity.In this external PROTEIN C reactivation process, its Gla structural domain can directly combine with TM, so 9 Glas play crucial effects to the anticoagulating active of keeping PROTEIN C.There is a high-affinity Ca who has nothing to do with γ-Gla in two Urogastrons (EGF) structural domain on the light chain with coming from the EGF precursor ²⁺Combining site is that zymoplasm-thrombomodulin mixture activated protein c is necessary.There is a serine protease spline structure territory in PROTEIN C heavy chain C end, is the active region of PROTEIN C.The PROTEIN C precursor is subjected at the peptide bond between Arg157 and Asp158 (from the aminoterminal counting of ripe single chain protein C) rupturing after the effect of trypsin-like endopeptidase, then, remove Arg157 and Lys156 by a kind of hydroxyl PEPB B sample proteolytic enzyme from the C-end, disulfide linkage connects light chain and heavy chain forms double-stranded PROTEIN C.

PROTEIN C has powerful anti-freezing, short fibrinolytic and potential anti-inflammatory action, demonstrates important clinical application value.As far back as later 1980s, the PROTEIN C that people just will extract from blood plasma is applied to the protein C deficiency patient.In recent years, PROTEIN C infectious diseases particularly the therapeutic action in the severe infections receive much attention.According to statistics in 1996, the entire United States PROTEIN C year aggregate demand up to 100 kilograms, and increase year by year.Up to now, the used PROTEIN C of clinical treatment mostly is the PROTEIN C enriching agent that extracts from human plasma.But the content of PROTEIN C in human plasma is low, the transformation period short, extracts native protein C from concentrate of coagulation factors or human plasma, cost height not only, complex process, and have problems such as purity of protein, stability and security, haematogenous PROTEIN C product can not satisfy clinical needs far away.From the eighties so far, states such as the U.S., Canada, Japan carry out the r-aPC always (recombinated human protein C, research and development rhPC) have obtained expressing in mammalian cell.

The 26S Proteasome Structure and Function complexity of PROTEIN C, with the posttranslational modification process, the PROTEIN C that gives expression to high level and biologically active from cell is comparatively difficult in the translation that experience is complicated in its biosynthetic process.Prokaryotic expression system is not because of carrying out the required modification of PROTEIN C, and its expressed proteins C lacks biologic activity.People had carried out expression in HEK293, CHO, BHK, and have low, active not high and the problems such as posttranslational modification when in heterologous host, expressing of expression amount at mammalian cell expression, as the expression product glycosylation and carboxylated not exclusively, the single stranded form proportion is too high.When PROTEIN C was expressed in mammalian cell, there is some difference for its glycosylation, translation post-treatment and native protein C, and complex manufacturing, cost height also are very important problems in addition.Because the intrinsic advantage of mammary tissue in recent years, has become main target organ, the especially large usage quantity of producing pharmaceutical protein, the product that need carry out the protein translation post-treatment in animal body.Galactophore biological reactor becomes the important means of producing pharmaceutical protein C.At present, study at transgenic mice and transgenic pig.(Ann N Y Acad Sci.1992 such as Velander, 665:391) with WAP be promotor, change the cDNA of human protein C over to the CD-1 mouse, obtain 6 transgenic mices after testing, the PROTEIN C expression level is 1.0-3.0 μ g/ml in its milk, and the peak period reaches 8-12 days.The expressed PROTEIN C of transgenic mice has higher anticoagulating active, and the ratio of strand and two strands is at 5-30%, and is approximate with native protein C.But do not analyze the glycosylation and the aminoacid sequence of recombinant protein c in this research.In the research of transgenic pig, the expression amount of PROTEIN C reaches 0.1-1mg/ml in the milk, keeps stable in its lactation period of 26 days.With mouse WAP is promotor, and its regulating and controlling sequence can be controlled human protein C and efficiently express in pig mammary gland, per hour reaches 380 μ g/ml, and the PROTEIN C in its anticoagulating active and blood plasma source is close.30-60% in the secreted recombinant protein c of pig is γ-carboxylated fully, the mammary gland that shows transgenic pig can carry out sufficient γ-carboxylated to PROTEIN C, but owing to can not effectively cut in milk, transgenic pig excretory single chain protein C accounts for higher ratio.PROTEIN C must be cut propetide Lys ^-2-Arg ^-1Be connected light chain and heavy interchain Lys with removing ¹⁵⁶-Arg ¹⁵⁷After, could form sophisticated molecule, the mammary gland of transgenic mice and transgenic pig all can not effectively cut.PROTEIN C for energy render transgenic animal direct secretion double chain form, Drews etc. select paired basic aminoacids nickase (paired basic amino acid cleavingenzyme for use, PACE) have another name called people's amyloid protein precursor processive enzyme of furin, with PROTEIN C coexpression in the transgenic animal body, human protein C is cut in correct position, thereby gives expression to sophisticated double chain form.

When using prokaryotic expression system expression higher eucaryote gene, its expression product does not often have biological activity, and separation and purification is comparatively difficult.Mammalian cell expression system has overcome the shortcoming of procaryotic cell expression system basically, but cultivates difficulty, and the cycle is long, cost is high, and expression level is undesirable.Compare with above two kinds of expression systems, the development of yeast expression system more and more is subjected to people's favor.Yeast cell be a kind of application prospect good, develop heterologous protein expression system rapidly, has the processing characteristics behind the similar mammalian cell protein translation, expression product does not mix the pyrogen material, especially during the secretion expression, the albumen of the less secretion of cell own is easy to the purifying of target protein; In addition, yeast cell has the characteristics of similar prokaryotic expression systems such as be easy to cultivate and fermentation costs is cheap again.Increasing foreign protein then obtains to efficiently express in yeast cell, and the especially development of pichia pastoris yeast expression system has promoted the utilization and the development of yeast expression system especially greatly.At present, do not see the correlative study that PROTEIN C is expressed in yeast.

The innovation and creation content

The primer special that the purpose of this invention is to provide a kind of human protein C gene of amplification.

Amplification provided by the present invention human protein C gene's primer special is the SEQ ID № in the sequence table: 1 and SEQID №: 2.

SEQ ID №: 1 by 34 based compositions; SEQ ID №: 2 by 28 based compositions; Wherein, SEQ ID №: 1 from 5 ' end the 4th to the 9th bit base be the restriction enzyme site of XhoI; SEQ ID №: 2 from 5 ' end the 3rd to the 8th bit base be the restriction enzyme site of EcoRI.

Second purpose of the present invention provides a kind of expression vector of human protein C, and its expression product can be secreted into outside the born of the same parents, is beneficial to the extraction of human protein C, is fit to suitability for industrialized production.

The expression vector of human protein C provided by the present invention is the restructured Pichia pastoris in expression carrier pPIC9-hPC that contains the human protein C gene.

Restructured Pichia pastoris in expression carrier pPIC9-hPC obtains by following step:

(1) uses reverse transcriptional PCR (RT-PCR), from people's tire liver total rna, angle and get human protein C cDNA;

(2) the human protein C cDNA that obtains in the step (1) is connected on the Yeast expression carrier pPIC9, obtains restructured Pichia pastoris in expression carrier pPIC9-hPC.

Wherein, the middle RT of step (1) is with Oligo (dT) ₁₅Be primer; The pcr amplification primer can be the SEQ ID № in the sequence table: 1 and SEQ ID №: 2.

The 3rd purpose of the present invention provides a kind of method of expressing human PROTEIN C.

A kind of method of expressing human PROTEIN C is that restructured Pichia pastoris in expression carrier pPIC9-hPC is transformed in the pichia spp, obtains positive colony, the described positive colony of inducing culture, expressing human PROTEIN C.

In order to obtain better effect, it is the methanol induction cultivation positive colony of 0.25-1% with final concentration also that pichia spp is preferably the GS115 bacterial strain, and wherein the final concentration of methyl alcohol is preferably 0.5%.

The present invention adopts gene clone technology, has successfully made up Yeast expression carrier pPIC9-hPC; Utilize the electroporation method for transformation, obtain having the yeast transformant of foreign gene, the human protein C of expression shows through partial thromboplastin time (APTT) method mensuration can prolong blood coagulation time, has played anticoagulant effect.Method of the present invention has wide industrial prospect in the production of human protein C.

Description of drawings

Fig. 1 is the RT-PCR amplification of human protein C

Fig. 2 is double digestion and the PCR qualification result of recombinant expression vector pPIC9-hPC

Fig. 3 is the sequencing result of recombinant expression vector pPIC9-hPC

Fig. 4 identifies the result of positive colony for bacterium colony PCR

Fig. 5 is for expressing the SDS-PAGE electrophorogram after supernatant concentrates

Fig. 6 concentrates back Western Blot detection figure for expressing supernatant

Embodiment

Embodiment 1, human protein C cDNA expression and the activity identification in the pichia spp cell

(1) human protein C gene's clone

1. design of primers:

According to human protein C mRNA sequence (http://www.ncbi.nlm.nih.gov/Genbank/), design upstream and downstream primer P1, P2 and restriction endonuclease sites, wherein add the XhoI restriction enzyme site at upstream primer, downstream primer adds the EcoRI restriction enzyme site: P1:5 '-GCA CTCGAGAAAAGAGCCAACTCCTTCCTGGAGG-3 ' (base sequence of line is the restriction enzyme site of XhoI) (SEQ ID № in the sequence table: 1); P2:5 '-CG GAATTCAGGGAGGGTCGCTAAGGTGC-3 ' (base sequence of line is the restriction enzyme site of EcoRI) (SEQ ID № in the sequence table: 2).

2. the preparation of human protein C cDNA

Get liquid nitrogen cryopreservation fresh fetal liver cells 0.75g, add Trizol reagent 7.5ml rapidly, be ground to liquid clarification after, chloroform/primary isoamyl alcohol 12000rpm 10min is centrifugal to add equal-volume, draws supernatant, with the dehydrated alcohol post precipitation, be dissolved in the water of 50 μ l DEPC processing ,-20 ℃ of preservations are standby.

Use reverse transcriptional PCR (RT-PCR), angle from people's tire liver total rna and get human protein C cDNA, concrete steps are as follows: get people's tire liver total rna 3 μ g, with Oligo (dT) ₁₅Be synthetic cDNA first chain of primer.Reaction system is 5 * reaction buffer, 4 μ l; 40U/ μ l RNA enzyme inhibitors 0.5 μ l; 500 μ g/ml Oligo (dT) ₁₅Primer 1 μ l; 2.5mmol/L dNTP 4 μ l; 10U/ μ l AMV ThermoScript II 1 μ l; Add the nuclease free deionized water to cumulative volume 20 μ l.Room temperature is placed 10min, and in 42 ℃ of water-bath 1h, 99 ℃ of insulation 3min are with the deactivation ThermoScript II.

With the cDNA that is obtained is template, with Pyrobest archaeal dna polymerase amplification human protein C cDNA.The PCR reaction system is a template for getting above-mentioned RT reactant 7.5 μ l; 10 * reaction buffer, 5 μ l; Upstream and downstream primer P1 and each 25pmol of P2; 2.5mmol/L dNTP 4 μ l; Pyrobest archaeal dna polymerase 2.5U; Add deionized water to cumulative volume 50 μ l.The PCR reaction conditions is 94 ℃ of pre-sex change 4min, 94 ℃ of 30sec, and 58 ℃ of 45sec, 72 ℃ of 1min, 30 circulations continue to extend 5min for back 72 ℃.The dna fragmentation purification kit that the dna fragmentation that pcr amplification produces is used Promega company carries out purifying, is undertaken by operation instructions.Concrete steps are as follows:

A. with 1% sepharose that contains EB (selecting the TAE preparation for use) electrophoretic separation PCR reaction product, the result shows the dna fragmentation that obtains 1260bp as shown in Figure 1, and a is DL2000DNA Marker among the figure, and b is RT-PCR product (encoding sequence that does not contain signal peptide).

B. cut the gel piece that contains target DNA fragment, the about 300ng of size, gel piece put into the Ep pipe of 1.5ml, and 70 ℃ are incubated to gel and melt fully;

C. add 1ml purifying resin, abundant mixing 20sec, but can not use the wortex device mixing;

D. 3ml disposable syringe piston is extracted, microtrabeculae links to each other with syringe;

E. resin gel/DNA the mixture for preparing is moved into the syringe bucket, insert piston, push away sample gently and go into microtrabeculae;

F. move into 2ml 80% Virahol in syringe, wash microtrabeculae once with Virahol;

G. microtrabeculae is placed the 1.5mlEp pipe, the centrifugal 2min of 10000g room temperature is with dry resin;

H. move the new aseptic Ep pipe of microtrabeculae to, add 50ul deionized water or TE Buffer in microtrabeculae, after room temperature was placed 1min, the centrifugal 20sec of 10000g was with the eluted dna fragment.DNA behind the wash-out is stored in 4 ℃ or-20 ℃.

3. the preparation of expression of recombinant yeast plasmid pPIC9-hPC

Behind the human protein C cDNA purifying that is obtained, application limitations restriction endonuclease XhoI, EcoRI carry out the double digestion reaction.Yeast secreted expression carrier pPIC9 also carries out the double digestion reaction through two kinds of same restriction enzymes.Double digestion reaction cumulative volume is 20 μ l, pPIC94 μ g wherein, and XhoI is 10U, EcoR I is 20U, 10 * H damping fluid, 2 μ l.Reaction conditions is 37 ℃ of water-baths 3 hours.After enzyme is cut back sample purifying, standby in-20 ℃.In 10 μ l reaction systems, press an amount of target gene fragment of target gene fragment and 3: 1 mol ratio of carrier adding and carrier DNA, 10 * ligase enzyme damping fluid, 1 μ l and T ₄Dna ligase 2U.In 4-6 ℃ of water-bath, spend the night.

Conventional preparation bacillus coli DH 5 alpha competence.Get 10 μ l and connect product transformed into escherichia coli DH5 α competence, converted product is coated amicillin resistance LB flat board, the random choose white colony, after 37 ℃ of activation 12h are above, extract plasmid DNA, with P1, P2 is that primer carries out pcr amplification and XhoI, the EcoRI double digestion is identified, the result shows to obtain positive bacteria clone (bacillus coli DH 5 alpha that contains recombinant plasmid pPIC9-hPC) as shown in Figure 2; Among the figure, a is the double digestion negative control, and b identifies that for pPIC9-hPC PCR c is pPIC9-hPC XhoI and EcoRI double digestion, and d is DL2000marker.Select positive colony, the evaluation of checking order, the result shows that the cDNA sequence of the human protein C of being cloned is correct as shown in Figure 3.

(2) human protein C gene's expression

1. the preparation of yeast transformant

The linearizing of Yeast expression carrier: Yeast expression carrier pPIC9-hPC, is beneficial to and is integrated in the yeast genes group with the linearizing expression vector through the StuI single endonuclease digestion.Single endonuclease digestion reaction cumulative volume is 30 μ l, recombinant plasmid pPIC9-hPC5 μ g wherein, StuI 0.5U, 10 * M Buffer, 3 μ l.Behind 37 ℃ of water-bath 3h, add sample-loading buffer with termination reaction.Getting 5 μ l samples identifies in 1% agarose gel electrophoresis.After the purified recovery ,-20 ℃ of preservations are standby.

Prepare yeast competent cell: a. as follows and inoculate single bacterium colony pichia spp GS115, in 30 ℃, 220-250rpm thermal agitation overnight incubation in 5ml YPD nutrient solution; B. get the 0.1-0.5ml yeast GS115 nutrient solution that spends the night and be inoculated in the new YPD nutrient solution of 500ml, continue 30 ℃ of 220-250rpm thermal agitations and be cultured to OD ₆₀₀=1.3-1.5; C. in 4 ℃ of centrifugal 5min of 1500g, abandon behind the supernatant with the resuspended precipitation of the pre-cold sterilization deionized water of 500ml; D. in 4 ℃ of centrifugal 5min of 1500g, precipitation is resuspended in the 250ml precooling deionized water; E. once more in 4 ℃ of centrifugal 5min of 1500g, with the aseptic sorbyl alcohol washing of the precooling of 20ml 1mol/L; F. in 4 ℃ of centrifugal 5min of 1500g, precipitation is resuspended in the 1ml 1mol/L precooling sorbyl alcohol, is approximately 1.5ml to volume, promptly can be used for transforming.

With electric shocking method foreign gene is imported pichia spp: a. selects the single restriction enzyme site in 5AOX1 zone that external source genophore pPIC9-hPC is carried out linearizing, adds EDTA solution and stops enzyme reaction; B. carry out sterilising treatment through behind phenol/chloroform extracting/ethanol sedimentation, last linearizing DNA is dissolved in (about 5-10 μ gDNA) in the 10 μ l aseptic deionized waters; C. the recombinant plasmid for preparing and 200 μ l are suspended in the pichia spp cytomixis in the sorbyl alcohol, forward in the aseptic electric shock cup of 0.2cm precooling, place 5min on ice; D. at 1500V voltage, under 25 μ F electric capacity and the 200 Ω resistance parameters, electric shock transforms pichia spp GS115; E. the sorbyl alcohol that adds 1ml 1mo/L precooling after electric shock is finished immediately was transferred in the aseptic 1.5ml centrifuge tube after soft the mixing, got 100 μ l immediately and coated on the MDS flat board that contains the 1mo/L sorbyl alcohol, cultivated 3d for 30 ℃ and occurred until the clone.

The yeast transformant that is obtained adopts bacterium colony PCR to identify positive colony: a. chooses single bacterium colony arbitrarily from the MDS flat board, each bacterium colony is placed on the sterilized water of 20 μ l with its big or small 1/4-1/2 of toothpick picking; B. handle 15min on ice, then change 37 ℃ of water-baths over to, place 15min, at last in 95 ℃ of thermal treatment 10min; C. get the bacterium liquid after the cold and hot processing of 10 μ l, operate by following PCR method again:

The PCR reaction system is: 10 * reaction buffer, 2.5 μ l; 25mmol/LMgCl ₂1.5 μ l; 2.5mmol/LdNTP 2 μ l; Primer P1, P2 are respectively 25pmol; Taq archaeal dna polymerase 5U; Recombinant plasmid 2 μ g; Add deionized water to 25 μ l.The PCR reaction conditions is 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec, and 58 ℃ of 45sec, 72 ℃ of 1min, 30 circulations continue to extend 5min for back 72 ℃.After reaction finishes, get 5 μ l samples and analyze pcr amplification product with 1% agarose gel electrophoresis, the result as shown in Figure 4, show to obtain among the positive colony figure that a.b.c.d.e.f.g.j.k is different clones, the positive contrast of h, i is DL2000Marker, 1 negative contrast.

2. secretion property foreign protein abduction delivering in pichia spp GS115

A. picking positive colony pichia spp is inoculated in the 2ml BMGY nutrient solution, is cultured to OD in 30 ℃ of thermal agitations ₆₀₀=2-6 (approximately needing 16-18h), the cell of this moment is in logarithmic phase;

B. the centrifugal 5min of 1500-3000g under the room temperature, abandon supernatant after, with the resuspended yeast cell of BMMY, to OD ₆₀₀About=1.0 (approximately 10-20ml);

C. nutrient solution is gone to the triangular flask of 50ml, seal bottleneck, under 30 ℃, 220-300rpm condition, cultivate, induce the expression of foreign protein with three layers of gauze;

D. adding methyl alcohol to final concentration 0.5% (v/v) every day expresses with successive induction.Get 7ml yeast culture liquid, get supernatant after centrifugal, be used to detect proteic expression.

3. the evaluation of positive colony expression product

(1) sample collection

The positive colony that screens is carried out methanol induction express, 24h collects the expression supernatant respectively at interval, in 4 ℃, the centrifugal 5min of 3000rpm, get supernatant and store in-70 ℃ standby.

(2) sample concentration is handled

Sample in 4 ℃, the centrifugal 5min of 3000rpm, moves into the supernatant liquor of collecting in the dialysis tubing after room temperature is melted, be immersed in 70%PEG8000,4 ℃ of lower magnetic forces stir 4-6h, and 30ml left and right sides liquid is done thoroughly, again dialysis tubing is moved among the TE pH 8.0, continue dialysed overnight.Collect the sample in the dialysis tubing, store-70 ℃ standby.

(3) SDS-PAGE protein electrophoresis

Get 15 μ l samples and carry out SDS-PAGE according to a conventional method and detect the protein expression situation, the result shows that human protein C expresses, among the figure as shown in Figure 5, a is low molecular weight protein Marker:218KD, 118KD, 81.9KD, 48.7KD, 31.9KD, 25.5KD, 17.5KD; B.c.d.e.f.g.h is different clones; I is the pure product of human protein C.

(4) Western Blot detects the yeast expression supernatant

Get 15 μ l samples earlier through the SDS-PAGE protein isolate, use Western blotting behind the commentaries on classics film and detect the protein expression situation.Western Blot method is with reference to " molecular cloning ".Utilizing the electrophoresis of Liuyi Instruments Plant, Beijing and electricity to transfer from one department to another system carries out.Resist with the anti-human protein C of rabbit (available from Sigma company) respectively is first antibody more, and second antibody is the mouse-anti rabbit igg (available from Huamei Bio-Engrg Co.) of AP mark, the colour developing of BCIP/NBT substrate.Concrete steps are as follows:

A. sealing: confining liquid 5%BSA submergence nitrocellulose filter, 4 ℃ of sealings are spent the night;

B. wash film: wash nitrocellulose filter 3 times with lavation buffer solution, each 3-5min;

C. resist reaction with the anti-human protein C of rabbit: how anti-antibody diluent is by dilution in 1: 300 more, the submergence nitrocellulose filter, and shaking table is jolting gently, room temperature 2-4h;

D. wash film: discard to resist washings rinsing film 3 times, each 3-5min more;

E. react with ELIAS secondary antibody: antibody diluent diluted ELIAS secondary antibody by 1: 300, the submergence nitrocellulose filter, and shaking table is jolting gently, room temperature 2-4h;

F. wash film: discard ELIAS secondary antibody, washings rinsing film 3 times, each 3-5min;

G. colour developing: by 1: 1: 50 dilution NBT/BCIP, film is dipped in wherein, jog under the room temperature, the careful observation, treat that protein band reaches requirement (about 3-5min) after, use the distilled water flushing nitrocellulose filter, termination reaction, taking pictures gives over to permanent experimental record.The result shows to detect human protein C as shown in Figure 6, the negative contrast of a among the figure, and b.c.d.e.f.g is different clone, h is the pure product of human protein C.

4.APTT the anticoagulating active of method working sample

The method of the human protein C determination of activity test kit of producing with reference to American Diagnostic Inc.

With PROTEIN C defective blood plasma is matrix plasma, and white bole is surperficial contact activation agent, at Ca ²⁺Participate in being subjected to sample product PCT relevant down with the anticoagulating active of PROTEIN C.Measure the setting time of different dilution PROTEIN C contrast blood plasma, production standard curve, the setting time of working sample simultaneously.Concrete steps are as follows:

A. PROTEIN C is contrasted blood plasma by 1/2,1/4,1/8,1/16 doubling dilution after, get 0.1ml respectively, with 0.1ml PROTEIN C defective blood plasma and Protac ^TM0.05ml adding volume is in the 4ml transparent plastics small test tube, mixes back 37 ℃ of incubation 3min;

B. add APTT suspension 0.1ml rapidly, in 37 ℃ of incubation 2min;

C. add 0.025mol/LCaCl ₂Solution 0.1ml starts stopwatch immediately, in water-bath with 1-2 time/second the frequency mixing;

D. observe clotting of plasma situation.When solidifying appears in blood plasma, the immediate record PCT.

Per-cent activity with the PROTEIN C of reference standard is an X-axis, and the average blood plasma setting time is Y, and drawing best-fitting straight line by these points is typical curve.The result who searches typical curve be multiply by 2 to proofread and correct extent of dilution, promptly obtain the per-cent activity of sample.

With the positive contrast of reference blood plasma, and sample 0.05ml to be checked is got in the 8.0 negative contrasts of TE pH of buffer at every turn.Each sample replicate measurement three times.The result is as shown in table 1, shows that the yeast expression supernatant of preparation in the step 3 has anticoagulating active.

The anticoagulating active of the yeast expression supernatant of preparation in table 1. step 3

Sample	Clotting time (sec)			Mean+SD	The active % of blood plasma hPC
						1	2	3
	Reference blood plasma yeast expression supernatant negative control	146 110 40	140 105 42						143 108 40	143±3 107.7±2.5 40.1±1.2	100 50 0

Sequence table

<160>2

<210>1

<211>34

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

gcactcgaga aaagagccaa ctccttcctg gagg 34

<210>2

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

cggaattcag ggagggtcgc taaggtgc 28

Claims (8)

1, a kind of expression vector of human protein C is the restructured Pichia pastoris in expression carrier pPIC9-hPC that contains the human protein C gene.

2, restructured Pichia pastoris in expression carrier pPIC9-hPC according to claim 1 is characterized in that: described restructured Pichia pastoris in expression carrier pPIC9-hPC obtains by following step:

(1) uses reverse transcriptional PCR RT-PCR, from people's tire liver total rna, angle and get human protein C cDNA;

(2) the human protein C cDNA that obtains in the step (1) is connected on the Yeast expression carrier pPIC9, obtains restructured Pichia pastoris in expression carrier pPIC9-hPC.

3, restructured Pichia pastoris in expression carrier pPIC9-hPC according to claim 2 is characterized in that: the primer of described RT is Oligo (dT) 15.

4, according to claim 2 or 3 described restructured Pichia pastoris in expression carrier pPIC9-hPC, it is characterized in that: described pcr amplification primer is the SEQ ID № in the sequence table: 1 and SEQ ID №: 2.

5, a kind of method of expressing human PROTEIN C is that restructured Pichia pastoris in expression carrier pPIC9-hPC is transformed in the pichia spp, obtains positive colony, the described positive colony of inducing culture, expressing human PROTEIN C.

6, method according to claim 5 is characterized in that: described pichia spp is the GS115 bacterial strain.

7, according to claim 5 or 6 described methods, it is characterized in that: with final concentration is that the methanol induction of 0.25-1% is cultivated described positive colony.

8, method according to claim 7 is characterized in that: the final concentration of described methyl alcohol is 0.5%.