CN1059237C - Method for producing recombined human liver cytokinin and its use for curing serious hepatic disease - Google Patents

Abstract

The present invention relates to a method for producing recombined human liver cytokinin (rHPO) and the application for curing serious hepatic diseases, which belongs to the field of a biologic product manufacturing method and the application. On the basis of obtaining hHPO cDNA and successfully building a high-level expression bacterial strain, a whole set of technologies of inducing expression of engineering bacteria, separation and cracking of inclusion bodies and purification and recoverability of protein is established to obtain a pure product of recombined human liver cytokinin. The recombined human liver cytokinin has the biologic characteristics that an isoelectric point is 7.2, and the molecular weight is 15280 daltons. The recombined human liver cytokinin can be used for the primary culture of liver cells and the proliferation of hepatoma carcinoma cells at the outside of a human body. The recombined human liver cytokinin can be used for promoting DNA synthesis of liver cells of a rat in vivo after 1/3 of the liver is cut and CCl4 is damaged, preventing necrosis of liver cells and obviously enhancing survival rate of the rat with liver function failure induced by CCl4. Experiments indicate that rHPO can be possibly the effective medicine for treating serious hepatic diseases in clinic.

Description

The production method of RHPO and treat the purposes of serious hepatopathy

The present invention relates to a kind of production method and purposes field of Bio-engineering Products, more particularly is the production method and the product therapeutic action clinically of a kind of RHPO (rHPO).

Liver is the internal organs that body has powerful regenerative power, the research of relevant its Regulation Mechanism has been carried out over one hundred year in the world, but the present known liver regeneration relative growth factor such as pHGF (HGF), transforminggrowthfactor-(TGF-α) etc., its effect all lacks liver specificity, be difficult to explain to have organ specific liver regeneration Regulation Mechanism, therefore, the focus that to seek novel liver regeneration regulatory factor be this field always.1975, LaBrecque etc. at first reported the short liver multiplicaiton factor that contains a kind of tool thermostability in the rat regenerating hepatic tissue.The mid-80, we also find the similar factor in people's tire hepatic tissue, and systematic study has been carried out in its biological activity, physico-chemical property, protein purification and clinical application.Because this type of factor applying clinical is treated serious hepatopathy and obtained better curative effect, thereby extremely payes attention to, the molecular cloning of this type of factor is devoted in the Duo Jia laboratory always both at home and abroad.Nineteen ninety-five, we utilize the functional screening method to be separated to the plain cDNA of human hepatocyte growth from people's tire liver cDNA library, finish its nucleotide sequencing and pass through to make up eucaryon, prokaryotic expression plasmid, its biologic activity is studied, confirm this gene expression product in vivo can cell cultured supernatant propagation, and apply for that in March, 1996 Chinese invention patent, application number are 96103203.0.But do not see as yet that so far relevant human liver cell generates the plain system process report that efficiently expresses and obtain RHPO both at home and abroad.In recent years, the domestic serious hepatopathy of extracting from sucking pig liver, new calves liver of hepatocyte proliferation activity factor in treatment of using clinically obtains satisfactory effect, but is subjected to restrictions such as material source, product purity, racial difference, and its application is subjected to bigger restriction.The production of RHPO has overcome these restrictions, is expected to the medicine that provides the serious hepatopathy of effective treatment for clinical.

The objective of the invention is to obtain the RHPO of biologically active and be used for treating clinically serious hepatopathy by prokaryotic expression, purifying.

It to the effect that generates plain cDNA and successfully making up on the basis of efficient expression strain obtaining human liver cell the present invention, separation and cracking, protein purification and the renaturation technology of a whole set of engineering bacteria abduction delivering, inclusion body have been set up, to obtain the pure product of RHPO, and the biology performance of RHPO is described, show that RHPO has the purposes for the treatment of serious hepatopathy clinically.

The present invention also successfully makes up on the basis of efficient expression strain at the hepatocyte forming agent cDNA that clones people voluntarily; Set up and optimized separation and cracking, protein purification and the renaturation technology of a whole set of engineering bacterium fermentation, inclusion body, it is characterized by: 1) to the fermentation of engineering bacteria low density, its best abduction delivering time is cell density OD ₆₀₀=0.3-0.4; 2) the inclusion body denaturing agent is 8M urea or uses 0.05mol/L NH ₄The 7mol/L Guanidinium hydrochloride that contains 1% mercaptoethanol of AC damping fluid preparation; 3) adopt renaturation solution (2mol/L urea, 0.2mmol/L Sleep-promoting factor B, 1mmol/L reduced glutathion, 100mmol/L Tris-HCl pH8.0) or dilution refolding solution (100mmol/L urea, 0.2mmol/L Sleep-promoting factor B, 1mmol/L reduced glutathion, 100mmol/L Tris-HCl pH8.0) to carry out renaturation; 4) column chromatography purification can adopt the column chromatography purification of recombinant protein or inclusion body to carry out column chromatography purification, its flow process such as Fig. 3 under the sex change condition; Wherein on the sp-FF post behind the sample sodium acetate buffer wash-out not in conjunction with phase, with 300mmol/LNacl stepwise elution rHPO at first peak; DEAE-Sepharose FF (Pharmacia) anion-exchange column uses 50mmol/L Tris-HCl (pH8.0) buffer solution elution not in conjunction with phase.With 350mmol/L NaCl stepwise elution, rHPO is at first peak; 5) column chromatography purification can adopt inclusion body to carry out column chromatography purification, its flow process such as Fig. 4 under the sex change condition; Wherein use 2mol/L Guanidinium hydrochloride wash-out on the AcA44 gel filtration chromatography behind the sample, rHPO is in second peak; Use 0.05mol/L NaCl (0.05mol/L NH on the MONO Q ion exchange chromatography behind the sample ₄The Ac preparation) rHPO is eluted, rHPO is in main peak; Obtain human liver cell by this method and generate plain pure product, purity reaches 99%, and its n terminal amino acid sequence (Fig. 2) is measured with consistent according to nucleotide sequence deduced amino acid (Fig. 1); It is 7.2 that human liver cell generates plain iso-electric point, and molecular weight is 15280 dalton; In the external propagation that can promote primary cultured hepatocyte and liver cancer cell, can promote CCL in vivo ₄It is synthetic to cause damage liver cell DNA, and can significantly improve CCL ₄Induce the acute liver failure rats survival rate, thereby the prompting RHPO can be treated serious hepatopathy by applying clinical.

Accompanying drawings specific implementation method of the present invention is as follows:

Fig. 1 is the rHPO aminoacid sequence of deriving according to nucleotide sequence

The people rHPO n terminal amino acid sequence of Fig. 2 for measuring

Fig. 3 is a process flow sheet of the present invention

Fig. 4 is the technical process of the embodiment of the invention two

Fig. 5 is the SDS-PAGE detected result of engineering bacteria abduction delivering level

Fig. 6 is rHPO purified product SDS-PAGE result

Be respectively from left to right among the figure: protein standard, full bacterium lysate, purifying HPO

Embodiment one:

One, the technical essential of present embodiment

1. fermentation using bacteria is the low density high expression level

2. the inclusion body denaturing agent is a urea

3. carry out column chromatography purification (two-step approach) behind the renaturing inclusion bodies again

4. carry out column chromatography purification (single stage method) under the inclusion body sex change condition

Two, embodiment experimental implementation step (Fig. 3)

1. this chamber of engineering strain reorganization pBV-rHPO-1/JM109 makes up.Picking list colony inoculation is in 5ml LB nutrient solution test tube (containing penbritin 100ug/ml) from the engineering bacteria plate of 4 ℃ of preservations, 30 ℃, 200 rev/mins shaking culture are after 12 hours, are inoculated in to shake in 5% ratio (to contain penbritin 100ug/ml) in the bottle 30 ℃, 200 rev/mins shaking culture can be used as seed liquor after 12 hours.

Fermentation with 2% concentration seed liquor is inoculated in the 500ml LB nutrient solution (contain penbritin 100ug/ml, pH7.2), 30 ℃, 200 rev/mins shaking culture 3-5 hour.Treat that its nectar degree reaches OD ₆₀₀During=0.3-0.4, elevated temperature to 42 ℃ abduction delivering is 5 hours rapidly.Centrifugal collection bacterium, electrophoresis are identified the target protein expression contents.The wet about 5g/L of bacterium harvest yield.5 liters of ferment tank conditions are air flow 2L/min, dissolved oxygen 50% (DO adjusting), and stirring velocity 300-600 rev/min, (contain penbritin 100ug/ml, pH7.2) charging capacity is 4 liters for each LB nutrient solution.Seed liquor concentration 2%, 30 ℃ of growth temperatures treat that its nectar degree reaches OD ₆₀₀During=0.3-0.4, elevated temperature to 42 ℃ abduction delivering 5 hours.Centrifugal collection bacterium, electrophoresis are identified target protein expression contents (Fig. 5).The wet about 5g/L of bacterium harvest yield.

3. bacteria cell cracking

4 ℃ 3, centrifugal 30 minutes of 000rpm, supernatant discarded.Weighing bacterium wet weight adds lysate (50mmol/L Tris-HCl pH8.0,1mmol/L EDTA, 1mg/ml) N,O-Diacetylmuramidase, 0 ℃ of effect 30 minutes by the concentration of 10g bacterium/100ml.Ice-bath ultrasonic 30-45 second then, intermittently 2 minutes, ultrasonic three times altogether.Ultrasonic finishing, 4 ℃ 8, centrifugal 30 minutes of 000rpm, supernatant discarded is collected inclusion body.With 100mmol/L Tris-HClpH8.0 washing once.

4. inclusion body washs and purifying

(1) with 0.5%Triton X-100 (100mmol/L Tris-HCl pH8.0 preparation) resuspended precipitation (20g/100ml), stirring at room 40 minutes.10,000rpm, centrifugal 30 minutes, supernatant discarded.Precipitation with 100mmol/L Tris-HCl pH8.0 washing is once weighed;

(2) with 2mol/L NaCl (100mmol/L Tri-HCl pH8.0 preparation) resuspended step (1) precipitation (20g/100ml), stirring at room 120 minutes, 10,000rpm, centrifugal 30 minutes, supernatant discarded.Precipitation with 100mmol/L Tris-HCl pH8.0 washing is once weighed;

(3) with 4mol/L urea (100mmol/L Tris-HCl pH8.0 preparation) resuspended step (2) precipitation (20g/100ml), stirring at room 40 minutes.10,000rpm, centrifugal 30 minutes, supernatant discarded.Precipitation with 100mmol/L Tris-HClpH8.0 washing is once weighed.

5. inclusion body cracking

Adopt the 8mol/L urea cracking inclusion body of purifying.8mol/L urea (100mmol/L Tris-HClpH8.0 preparation), the inclusion body (20-30g/100ml) of the resuspended purifying of 0.5-1% mercaptoethanol, stirred overnight at room temperature.10, centrifugal 30 minutes of 000rpm, supernatant is the inclusion body cracked solution, and it is limpid transparent, faint yellow that solution should be.Precipitating available 8mol/L urea extracts once again.

6. renaturing inclusion bodies

(1) 2mol/L urea renaturation step 5, the inclusion body lysate is used 100mmol/L Tris, and pH8.0 is diluted to 2mol/L with urea by 8mol/L, regulates total protein concentration simultaneously and is no more than 500ug/ml.And in this renaturation system, add final concentration 0.2mmol/L Sleep-promoting factor B and final concentration 1mmol/L reduced glutathion.4 ℃ renaturation 8-10 hour.Solution should limpid transparent nothing precipitation after the renaturation.Repeatedly dialyse with 100mmol/L Tris-HCl pH8.0 damping fluid (containing redox system) and to remove urea, each urea concentration reduces by 25%.

(2) the dilution refolding method dropwise adds (100mol/L urea, 0.2mmol/L Sleep-promoting factor B, 1mmol/L reduced glutathion, 100mmol/L Tris-HClpH8.0) in the renaturation solution with metaprotein liquid.After total protein concentration was no more than the abundant renaturation of 1mg/mg, centrifugal removal precipitation concentrated through Millipore concentrating instrument (molecular weight holds back 5000), fully dialyses through 100mmol/L Tris-HCl pH8.0 damping fluid again, removes residual urea.

7. the column chromatography purification of recombinant protein

(1) strong cation exchange chromatography binding molecule sieve chromatography method is selected the fast flow velocity exchange column of third sulfo group-agarose strong cation (SP-FF Pharmacia) for use.With pH5.5 50mmol/L sodium acetate buffer balance SP-FF post (2.620cm).Sample on after renaturing inclusion bodies liquid is fully dialysed in sodium acetate buffer.Each sample 10ml (1mg/ml) that goes up.The sodium acetate buffer wash-out is not in conjunction with phase.With 300mmol/L NaCl stepwise elution, 280nm detects the eluted protein peak, and SDS-PAGE identifies each peak albumen distribution situation, and rHPO is at first peak.Above first peak sample fully dialysed with 50mmol/L Tris-HCl pH8.0 be splined on the 50mmol/L Tris-HCl pH8.0 equilibrated quick post of Sephacryl S-100 (802.6cm) after removing NaCl, 280nm detects the eluted protein peak, and SDS-PAGE identifies each peak albumen distribution situation.RhHPO is in main peak.

(2) anion-exchange chromatography binding molecule sieve chromatography method is selected DEAE-Sepharose FF (Pharmacia) anion-exchange column for use.Post is washed balance with 50mmol/L Tris-HCl pH8.0 stream earlier.Renaturing inclusion bodies liquid is directly gone up sample, last sample concentration 1mg/ml, volume 15ml.50mmol/L Tris-HClpH8.0 buffer solution elution is not in conjunction with phase.With 350mmol/L NaCl stepwise elution, 280nm detects the eluted protein peak, and SDS-PAGE identifies each peak albumen distribution situation, and rHPO-I is at first peak.

Above first peak sample fully dialysed with 50mmol/L Tris-HCl pH8.0 be splined on the 50mmol/L Tris-HCl pH8.0 equilibrated quick post of Sephacryl S-100 (802.6cm) after removing NaCl, 280nm detects the eluted protein peak, and SDS-PAGE identifies each peak albumen distribution situation.RHPO is in the end in the main peak.

(3) molecular sieve chromatography of inclusion body lysate under denatured state

The quick post of Sephacryl S-100 (802.6cm).The urea-denatured inclusion body sample of 8mol/L directly is splined on the post after 8mol/L urea (100mM Tris-HCl pH8.0) balance.280nm detects the eluted protein peak, and SDS-PAGE identifies each peak albumen distribution situation, and rHPO is the peak in the end.

Embodiment two

One, the technical essential of present embodiment

1. fermentation using bacteria is the low density high expression level

2. the inclusion body denaturing agent is a Guanidinium hydrochloride

3. inclusion body carries out column chromatography purification under the sex change condition, carries out the second step column chromatography purification after the target protein renaturation again

Two, present embodiment experimental implementation step (Fig. 4)

1. fermentation using bacteria is identical with embodiment 1

2. bacteria cell cracking is identical with embodiment 1

3. the inclusion body washing is identical with embodiment 1 with purifying

4. the inclusion body 0.05mol/L NH after the inclusion body cracking is washed ₄The 7mol/L Guanidinium hydrochloride cracking that contains 1% mercaptoethanol of Ac damping fluid preparation, 8, centrifugal 10 minutes of 000rpm reclaims the cracking supernatant, and this supernatant is used for following purifying.

5.AcA44 gel filtration chromatography 2mol/L Guanidinium hydrochloride (0.05mol/L NH ₄The Ac preparation) equilibrated AcA44 gel-filtration column (1.6 * 80cm).With this gel column on the step 4 cracking supernatant, each applied sample amount is 3ml.Use 2mol/L Guanidinium hydrochloride wash-out then, 280nm detects the eluted protein peak.SDS-PAGE checks the albumen in each peak, and rHPO is in second peak.

6.rHPO renaturation with the rHPO sample that step 5 obtains, transfer to protein concentration with the 2mol/L Guanidinium hydrochloride and be lower than 500ug/ml, use 0.05mol/L NH then ₄Ac does 10 times of dilutions, and adds reduced glutathion and Sleep-promoting factor B in the sample solution after dilution, makes its final concentration be respectively 1mol/L and 0.2mmol/L.Sample after the dilution is put in 4 ℃ the chromatography cabinet and is spent the night.This day, with this dilute sample to 0.05mol/L NH ₄Ac fully dialyses.Then that dialyzed sample is centrifugal, supernatant is the rHPO sample after the renaturation.

7.MONO the rHPO sample flow of Q ion exchange chromatography after with renaturation is through 0.05mol/L NH ₄Ac equilibrated Q Sepharose Fast Flow anion-exchange column (1.6 * 10cm), not conjugated protein with same buffer solution elution.Use 0.05mol/L NaCl (0.05mol/L NH4Ac preparation) that rHPO is eluted again.Identify that through SDS-PAGE rHPO is in main peak.RHPO purified product SDS-PAGE the results are shown in Figure 6.The human liver cell of measuring generates plain n terminal amino acid sequence and sees Fig. 2.Iso-electric point is 7.2, and molecular weight is 15280 dalton.Embodiment three

One, the technical essential of present embodiment

1.rHPO short primary cultured hepatocyte and HTC liver cancer cell DNA are synthetic

2.rHPO hepatocyte DNA is synthetic in the body

Two, present embodiment experimental implementation step

1. hepatocyte proliferation activity mensuration is the active detection of external biological target cell with primary hepatocyte and HTC liver cancer cell in the body.The method of Seglaen is adopted in hepatocellular separation.Wistar rat overnight fasting is freely drunk water.The capable operation of 9:00-11:00 morning isolating hepatocytes.Anaesthetize with 0.4% Sodital intraperitoneal administration, 75% alcohol-pickled whole body flowed 5 minutes with no calcium PBS jar by hepatic vein, used 0.06% collagenase liquid of 37 ℃ of prior preheatings instead, speed jar stream with 10ml/min, simultaneously liver is separated with surrounding tissue, move to plate and continue a jar stream, peel off Glisson's capsule, shake off the liver parenchyma part gently, after removing blood vessel, fat, cross 200 order copper mesh and filtered through gauze cell, with PBS flushing three times.Adjust cell concn 6 * 10 ⁶/ ml gets 100ul and inoculates 96 well culture plates, trains nutrient solutions in 37 ℃, 5%CO to contain 1640 of 5% foetal calf serum ₂Cultivate 12h under the condition, add the rHPO (concentration 4-10ug/ml) of purifying.With Regular Insulin, the positive contrast of people's tire liver hepatic stimulator substance, the full bacterium lysate of JM109 transfection PBV220 empty plasmid (4-10ug/ml) is contrast simultaneously.The every hole of cultured continuously 24h adds 1.85 * 10 ⁴Bq ³H-TdR.Tryptic digestion behind the 3h, collecting cell also carries out liquid flashing counting.Stimulating activity is represented with stimulation index.The blank group of stimulation index=experimental group cpm/ cpm.Get HTC cell suspension (4 * 10 ⁵/ ml) 100ul is inoculated in 96 porocyte culture plates, with the RPMI-1640 that contains 5% calf serum at 37 ℃, 5%CO ₂After cultivating 6h under the condition, add testing sample (every sample four holes), continue to cultivate 20h, every hole adds 1.85 * 10 ⁴Bq ³H-TdR, behind the 3h, collecting cell is in filter paper, and carries out liquid flashing counting, and is active in the cpm value representation.The result shows that purification rHPO synthesizes at external former primary cultures of rat liver cell and the HTC liver cancer cell DNA of promoting.

2.rHPO synthetic rat 1/3 liver that adopts of hepatocyte DNA divides the excision model to carry out in the body.Set up mouse 1/3 lobectomy of liver model by the method that Higgins and Anderson introduce.Wistar big white mouse row 1/3 hepalobectomy, postoperative be abdominal injection rHPO (0.1mg/mI) 1ml immediately, establishes physiological saline and lotus empty plasmid recipient bacterium lysate (0.1mg/ml) control group simultaneously; Press 100uci/kg body weight abdominal injection 3H-TdR behind the 20h, behind the 2h, put to death rat and get hepatic tissue in the liver fixed position, carry out DNA extraction and 3H-TdR mixes counting by the method for Morley (6) introduction, the result calculates with cpm/ug DNA.The result shows that rHPO can promote hepatocyte growth in vivo.Example four

One, the technical essential of present embodiment

RHPO anti-liver injury activity

Two, present embodiment experimental implementation step

1. external CCl ₄The situ perfusion method separation Balb/c mouse liver cell that Seglaen introduces is pressed in the foundation of liver injury model.Isolated cells is with containing 10% calf serum, 10 ^-9Mol/L Regular Insulin and contain penicillin, the RPMI-1640 of Streptomycin sulphate suspends, by every hole 100ul (cell count 3 * 10 ⁴) be inoculated in 96 porocyte culture plates, 37 ℃, 5%CO ₂Cultivate under the condition after 10 hours, change and contain 5mM CCl ₄, the RPMI-1640 of 10% calf serum and different concns rHPO carries out LDH release experiment and cell survival state observation respectively at different time after changing liquid.

2.LDH release experiment is different time collecting cell culture supernatant after changing liquid, measures by Cytox96Non-Radioactive Cytotoxicity Assay operational guidance.

LDH release rate=reality discharges the release number of number-discharge naturally number/maximum release number-naturally

3. the cell survival situation adopts the MTS method to estimate the liver cell survival condition.。Every group 4 hole, totally three experiments detect each hole 490nm OD value with enzyme connection detector.

4. the foundation of liver injury model in the body

At first carry out preliminary experiment, according to definite 4ml/kg such as case fatality rate, pathological change (1 volume CCl ₄: be best lethal dose 1 volume soya-bean oil), 2ml/kg is the best dosage of causing injury.Get 40 mouse disposable every abdominal injection CCl respectively ₄(4ml/kg), random packet behind the 4h, 20 every group, per 12 hours abdominal injection rHPO 40ug are organized in treatment, establish the physiological saline control group simultaneously, write down the animal dead situation every day, and survival surpasses 96h and counts surviving animals.Carry out 3 batches of experiments altogether, to observe rHPO to CCl ₄The treatment effect of poisoning liver failure animal.

Get 18 mouse, press 2ml/Kg abdominal injection CCl ₄, be divided into 3 groups at random after 4 hours, 1 group: intravenous injection rHPO 10ug; 2 groups: intravenous injection rHPO 40ug; 3 groups: intravenous injection physiological saline; Injection in per 12 hours was once poisoned back 48 hours, carried out the DNA proliferation assay, got periphery determination of serum ALT, LDH level simultaneously; Get hepatic tissue and carry out pathological examination.

5.DNA carrying out hepatocyte growth with the 5 FU 5 fluorouracil labelling method, proliferation assay detects.Main process: injected for the last time behind the rHPO 12 hours, press 1.0ml/Kg abdominal injection marking fluid, live after 2 hours and kill animal, getting hepatic tissue in the liver fixed position fixes in formaldehyde solution, the routine paraffin wax section, the dimethylbenzene dewaxing, behind the serial dehydration of alcohol, section is with PBS liquid washing three times, anti-5 FU 5 fluorouracil monoclonal antibody incubated at room 60 minutes, PBS washs secondary, and section is with the mouse-anti rabbit igg incubated at room of peroxidase labelling 30 minutes, wash three times after, colour developing 10min, dehydration, transparent, mounting, microscopically are chosen 5 visuals field at random, the counting positive cell.

6. pathological examination is got hepatic tissue in the right lobe of liver fixed position and is put 10% formaldehyde solution, routine paraffin wax section, pathologic finding.

The result for observe rHPO external whether to CCl ₄The damage liver cell has provide protection, and we add 5mMol/L CCl in the primary cultured hepatocyte system ₄Liver injury situation in the analogue body, and selection LDH discharges and the liver cell survival condition is that index is observed.As shown, rHPO discharges normal liver cell LDH does not have obviously influence, but has obvious inhibition CCl ₄Induce the release of the liver cell LDH of damage, this each time point that acts on all exists and has dosage effect positive correlation.For than objective evaluation CCl ₄Cause and decrease the hepatocellular survival condition of back cultivation, detect it with the MTS method.Containing 5Mol/L CCl ₄Under the condition, cell survivaling number obviously descends the peak behind the cultivation 24h, but rHPO group survival number average is higher than control group (p＜0.05).

With 4ml/Kg dosage CCl ₄, successful inducing mouse generation liver failure, physiological saline group survival rate is 10%, rHPO treatment group is 30%, two group of significant difference (p＜0.01); With 2ml/Kg dosage CCl ₄Experimental hepatitis takes place in inducing mouse, and treat with rHPO (10ug, 40ug) and physiological saline, damage back 48h, two treated animal peripheral blood ALT, LDH level all obviously raise, but rHPO treatment group level reduces than control group, wherein 40ug group LDH level reduces by 34%, and the ALT level reduces by 38.3%; Pathological examination shows the extensive swelling of not treatment group liver cell, sex change under the light microscopic, visible many places necrosis around the central vein, 10ug treatment group does not relatively have significant difference with not treatment group, but 40ug treatment group sex change and necrosis all more not treatment group obviously alleviate; Liver cell DNA synthesized situation after immunohistochemical methods detection CCl4 induced liver injury.Show that rHPO-I has the effect of the DNA of promotion synthetic, wherein 10ug group echo cell contrasts increases by 1.7 times, and the 40ug group increases by 4.7 times.Show that rHPO has the prevention hepatic necrosis, promote the effect of liver cell regeneration.

In a word, the invention discloses the method for a kind of rHPO of production, comprise the separation and the series of processes such as cracking, protein purification and renaturation of engineering bacteria abduction delivering, inclusion body, and physico-chemical property, the biological activity assay method of producing product is provided.

Purified product of the present invention can be used for immune animal with the preparation corresponding antibodies as a kind of antigen, also can be used as a kind of reagent, is used for the detection of solid tissue, liquid sample; This product also is expected applying clinical and treats serious hepatopathy (as acute hepatic failure).

Claims (1)

1. the production method of a RHPO (rHPO, original name liver proliferating agent) is characterized by:

After the fermentation of a:HPO engineering bacteria low density, through the inclusion body sex change, adopt renaturation solution, its component is: 2mol/L urea, 0.2mmol/L Sleep-promoting factor B, 1mmol/L reduced glutathion, 100mmol/L Tris HCl, pH8.0 or dilution refolding solution, its component is: 100mmol/L urea, 0.2mmol/L Sleep-promoting factor B, 1mmol/L reduced glutathion, 100mmol/L Tris HCl, pH8.0 renaturation; B: the column chromatography purification of recombinant protein adopts SP FF cationic exchange coloum, the sodium acetate buffer wash-out is not in conjunction with phase behind the last sample, with 300mmol/L NaCl stepwise elution, rHPO is at first peak: DEAE Sepharose FF (Pharmacia) anion-exchange column is with 50mmol/L Tris HCl, the pH8.0 buffer solution elution is not in conjunction with phase, use the 350mmol/LNaCl stepwise elution, rHPO is at first peak;

C: column chromatography is under the inclusion body sex change condition: use 2mol/L Guanidinium hydrochloride wash-out on the AcA44 gel-filtration column behind the sample, rHPO is in second peak; Use 0.05mol/L NH behind the sample on the MONO Q ion exchange chromatography ₄The 0.05mol/LNaCl of Ac preparation elutes rHPO, and rHPO is in main peak;

D: the rHPO that is produced has following biological property: have accompanying drawing 1 aminoacid sequence, iso-electric point is 7.2, and molecular weight is 15280 dalton; In the external propagation that can promote primary cultured hepatocyte and liver cancer cell, can promote 1/3 liver to divide excision back and CCL in vivo ₄It is synthetic to cause damage back rat hepatocytes DNA, and can stop hepatic necrosis, significantly improves CCL ₄Induce the acute liver failure rats survival rate.

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