CN105331617B - Gene for encoding human hepassocin, carrier and transgenic cell, application thereof as well as preparation method of human hepassocin - Google Patents

Abstract

The invention relates to the field of bioengineering and particularly discloses a gene for encoding human hepassocin. The gene has a nucleotide sequence as shown in SEQ ID No:1. In addition, the invention also discloses a carrier containing the gene, a transgenic cell as well as application thereof to the preparation of a medicine for preventing and/or treating liver injury. The invention also relates to a preparation method of the human hepassocin. The new gene for encoding the HPS (Hepassocin), provided by the invention, can efficiently express the HPS in a host cell, so that the formation of polymer precipitation is effectively avoided; in addition, the obtained HPS has good curative effect for the liver injury, for example, the obtained HPS can be used for effectively promoting the proliferation of hepatocyte, resisting the liver injury, and reducing the transaminase level of liver injury tissue and tissue hepatocyte necrosis.

Description

The gene of encoding human liver cytokines, carrier, transgenic cell and their application and people liver The preparation method of cytokines

Technical field

The present invention relates to bioengineering field, in particular it relates to the gene of a kind of encoding human liver cytokines, containing this gene Carrier and transgenic cell, and they in preparation for preventing and/or treating the application in the medicine of hepatic injury, further relate to one Plant in cell, the method especially producing people's liver cytokines with high level in mammalian cell.

Background technology

China is viral hepatitis district occurred frequently, calculates according to existing investigation, and there are 1.3 hundred million people's Hepatitis B carriers in China, 38000000 people carry hepatitis C virus, and the whole nation is annual occurs acute viral hepatitis about 1,200,000 example, dies from liver patient about 300,000 every year People.Wherein hepatitis gravis, acute hepatic failure etc. have onset urgency, poor prognosis, feature that case fatality rate is high, and clinic still lacks to be had The treatment means of effect." prevention hepatic necrosis, promote liver cell regeneration " remains main therapeutic strategy, and its prognosis is main Depend on that the hepatocellular degree of necrosis of disease damage and regeneration capacity, this principle problem in science below are the molecules that hepatic injury is repaired Mechanism.

The tail of some lower animals such as Gekko Swinhonis, the limb etc. of newt, the even all of internal organs of Stichopus japonicus can regenerate, but Higher mammal especially mammalian tissues regeneration capacity is very limited, but liver is but few in number to have powerful regeneration One of organ of ability.Liver at least carries the physiological function of more than 5000 kinds, is one of most important organ of human body.Although Normal liver tissue is in the hepatocyte of mitosis state and only accounts for 0.0012%-0.01%, but the multiplication capacity of liver and The ability adapting to metabolic alterations demand is not lost.Liver has the strongest injury repairing ability, be partly cut away when liver or When hepatocyte is by destroying (as poisoning, ischemia, anoxia, virus infect), remaining hepatocyte quickly enters division cycle, passes through Powerful regeneration capacity repairs rapidly the hepatic tissue of damage, and such as, muroid can be extensive at 8-10 days after carrying out 70% hepatectomy Multiple normal liver size.

Find and stop hepatic necrosis, promote the focus of the medicine always research of liver cell regeneration, and based on liver regeneration Feature, seek treatment the hope that the active substance always people of severe liver disease dream of from liver itself, especially special The growth factor acting on liver one of people's primary study target especially of the opposite sex.As far back as the seventies LaBrecque It is reported in mammal Liver Regeneration Deng i.e. and there is the cell active factor hepatocyte stimulation that a species specificity rush liver breeds Material (Hepatic Stimulatory Substance, HSS), research subsequently shows that this factor is widely present in mammal In the liver of young baby, embryonic liver and regenerating hepatic tissue.The application such as Chinese scholar old Cheng Wei eighties 4-6 month human foetus liver cell suspension (h-FLCS) treatment liver failure obtain good therapeutic effect, thus promoted this research at home carry out application.

The repair mechanism of hepatic injury is sufficiently complex, the ginseng jointly such as cytokine, somatomedin, immune system, metabolism network With wherein.After liver sustains damage, on the one hand showing as hepatocellular apoptosis or necrosis, body is after perception damages, accordingly The system such as metabolism, immunity can occur to change accordingly to adapt to news；On the other hand hepatocellular propagation is shown as, damage After, some immediate early gene (immediate early genes) such as c-fos, c-jun etc. are expressed and are raised rapidly, lure simultaneously Lead withered no (family name) cell, macrophage etc. and produce inflammatory cytokine including TNF-α, so activate produce NF-κ B, IL-6, STAT3 etc., cell is entered the G1 phase by the G0 phase；Additionally, TNF-α also stimulates generation TGF-α, other somatomedin ratio collaborative As HGF, EGF etc. promote cell to be entered the M phase by the G1 phase.During hepatocyte growth, cytokine, somatomedin play pass The effect of key, they produce cascade reaction by corresponding signal transduction pathway, interact, not only start but also maintain liver Regenerative process.It is currently known the material that hepatocyte can be promoted to divide a lot, mainly includes two big classes: cytokinin and auxiliary Mitotic growth factor class, the former includes that hepatocyte growth factor (Hepatocyte growth factor, HGF), liver are thin Born of the same parents generate element (hepatopoietin, HPO), epidermal growth factor (EGF), transforming growth factor (TGF-α) etc., and the latter wraps Include norepinephrine (Norepinephrine), estrogen (Estrogens), insulin (Insulinum) and pancreas hyperglycemia Element (Glucagon) etc..Such as in liver regeneration, HGF, by liver non-parenchymal cell synthesis secretion, acts on hepatic parenchymal cells Specific receptors c-Met on film and the synthesis of cell cultured supernatant DNA, the paracrine approach regulation and control that HGF and c-Met is formed are hepatocellular Growth and differentiation；There is Autocrine in HPO in liver, starts MAPK path by the specific receptors of surface of hepatocytes and promote Hepatocellular propagation；TGF-α provides mitogenesis stimulus object with the form of autocrine for hepatocyte, and EGF is then with endocrine Form promote hepatocellular division.

But above-mentioned various somatomedin is all nonspecific to hepatocellular stimulation.As a example by HGF, study table Bright, widely, biological action is varied such as the propagation of negative regulation tumor cell, promotes non-for the target cell of HGF effect Liver cell regeneration is repaired with kidney, stomach, Nao Deng tissue injury, and positive regulation hemopoietic promotes embryo and multiple allelotaxis, promotes kidney The motions etc. such as cell.Liver regeneration has the strongest organ specificity, finds the stimulating factor of liver specificity for deep understanding The molecular mechanism of liver regeneration is significant, and may provide novel drugs for severe liver disease treatment.

2000, Hara etc. was obtained from rat liver by subtractive cDNA clone and xenopus leavis oocytes expression screening method Obtain a kind of new hepatocyte growth promoting factors Hepassocin (HPS, also referred to as people's liver cytokines).Next year, humanized HPS is also It is cloned.Sequence retrieval shows humanized HPS Yu HFREP1 (Hepatocyte-Derived Fibrinogen-Related Protein 1) very high homology.HFREP1 is FGL1 gene expression product, and FGL1 (Fibrinogen-like 1) is Yamamoto is equal to a kind of gene screened by subtractive hybridizing method for 1993, and its expression product HFREP1 belongs to fiber egg Bai Yuan family.Fibrinogen and Fibrinogen associated protein C end have 4 conservative cysteine residues, and HFREP1 is same The C-terminal homology of Fibrinogen β, γ comprises 4 cysteine, but lacks fibrin blood coagulation and form necessary blood Platelet binding site, cross-linking zone and thrombin sensitivity site, therefore Yamamoto speculates that this albumen is one and blood coagulation not phase The secretory protein closed.2002, Jun YAN etc. was found that the homologous protein MFREP-1 (mouse of Mus HPS in mice Fibrinogen-related protein-1), 2004, Jun YAN etc. obtained again people LFIRE-1 (liver Fibrinogen-related gene-1), and LFIRE-1 is the homologous genes of HPS and HFREP1, therefore Jun YAN again will Its named LFIRE-1/HFREP1 gene, LFIRE-1/HFREP1 gene and HFREP1 gene have identical reading frame (Open Reading Frame, ORF), the most somewhat different in 5 ' untranslated regions, and it was found that LFIRE-1/HFREP1 is one Planting the gene of liver specific expression, this gene down-regulated expression in hepatoma carcinoma cell the most also has suppression liver cancer cell growth Characteristic.

People's Hepassocin gene is positioned at chromosome 8p 22-p21.3, is made up of 8 exons, a length of 1.2kb of mRNA, ORF total length 936bp, encodes 312 amino acid whose polypeptide, and wherein 22 aminoacid of N-terminal are its signal peptide sequence.People HPS is many Peptide only has the different of 3 amino acid sites, i.e. Asn69 from HFREP1 albumen, and Ile72, Leu105, HFREP1 are then respectively Asp69, Val72, Pro105.Mus HPS cDNA is 1.4kb, 314 aminoacid of protein polypeptide total length, wherein 24 ammonia of N-terminal Base acid is its signal peptide sequence.People HPS is 81.4% with the homology of Mus HPS albumen before excision signal peptide, and excises signal It is 83.8% after peptide.People's HPS protein polypeptide monomer is 34kDa, is contained within 5 cysteine residues, and after adding reducing agent HPS promotees liver proliferation activity and disappears.Western blot result displays that HPS exists two kinds of natural form of 34kDa and 68kDa, therefore This speculates that HPS plays a role with the form of homodimer in vivo.

HPS mRNA has low expression in normal rat liver, and damages when hepatectomy or D-Gal do cause When hindering, HPS mrna expression raises rapidly, and maintains higher level at 24-72h or 10-40h, is gradually restored to normal water afterwards Flat.The Mus source HPS of purification by the experiment of [3H]-TdR incorporation methods prove in vitro can the Hepar Mus of strong impulse original cuiture real Cell plastid DNA synthesizes, and has the strongest mitogenic activity.The result of in situ hybridization shows, it is thin that HPS is expressed in liver parenchyma Born of the same parents.Result for the hybridization research of people's HPS mRNA tissue dot shows that HPS high expressed, in Adult Liver, secondary is expressed in tire liver, Bone marrow and pancreas have faint expression, other tissue is not the most expressed HPS.Additionally, the rush liver proliferation activity of HPS has the strongest Tissue specificity, HPS only has a rush proliferation activity to normal hepatocyte, and to other tissue and hepatoma carcinoma cell the most accordingly Activity；HPS also has certain species specificity simultaneously.HPS is the controlling gene of liver specificity transcription factor HNF-1, in vain Interleukin-6 also assists in its expression regulation by STAT path.The research of this laboratory shows that HPS the most also can promote hepatocyte Propagation, and there is clear and definite anti-liver injury effect.Injection external source HPS can promote that hepatomegaly Partial Resection rat hepatocytes is bred, and has Effect ground reduces hepatic injury rat blood serum transaminase level, promotes hepatic injury reparation, improves acute experimental liver failure survival of rats Rate.

Although HPS has preferable antihepatitis activity, but still suffers from more problem in its production application.There is biology The restructuring HPS of activity is expressed, such as escherichia coli, Pichia sp., Chinese hamster ovary cell in various cells, although Expression in escherichia coli can reach 44.1g/L, but its expression-form mostly is inactive inclusion bodies, and Expression in Chinese hamster ovary celI is only 10mg/L, cannot meet far away coml feasibility.Therefore, it is badly in need of the HPS that exploitation is new Production method to obtain the substantial amounts of highly purified restructuring HPS albumen with biologic activity.

Summary of the invention

The invention aims to overcome disadvantages described above, it is provided that the gene of the coding HPS of a kind of optimization, containing this gene Carrier and transgenic cell, and they in preparation for preventing and/or treating the application in the medicine of hepatic injury, further relate to The preparation method of a kind of HPS and a kind of pharmaceutical composition.The gene of the coding HPS that the present invention provides may be implemented in eukaryotic cell The restructuring HPS that under culture systems, efficient substantial amounts of expression is active.By the nucleotide sequence coded protein sequence optimized Identical with by the corresponding nucleotide sequence coded protein sequence being not optimised.

To achieve these goals, on the one hand, the invention provides the gene of a kind of encoding human liver cytokines, wherein, this base Because having the nucleotide sequence shown in SEQ ID No:1.

Second aspect, the invention provides a kind of recombinant vector, and wherein, this recombinant vector contains described in claim 1 Gene and expression vector.

The third aspect, the invention provides a kind of transgenic cell, and wherein, this transgenic cell contains claim 1 institute The gene stated.

Fourth aspect, the invention provides the gene of encoding human liver cytokines as above, recombinant vector as above, The transgenic cell as above application in preparation people's liver cytokines.

5th aspect, the invention provides the preparation method of a kind of people's liver cytokines, and wherein, the method includes: at encoding human Under conditions of the gene of liver cytokines can be expressed, cultivate transgenic cell as above, so that it expresses people's liver cytokines.

6th aspect, the invention provides the gene of encoding human liver cytokines as above, recombinant vector as above, Transgenic cell as above, preparation method as above are in preparing the medicine being used for preventing and/or treat hepatic injury Application.

The gene of the coding HPS of the optimization that the present invention provides, it is possible to high efficient expression HPS in host cell, is prevented effectively from The formation of polymer precipitation, and the HPS obtained has good curative effect, if effectively facilitating hepatocellular increasing to hepatic injury Grow, anti-liver injury, reduction hepatic injury tissue transaminase level and tissue hepatic necrosis.

Other features and advantages of the present invention will be described in detail in detailed description of the invention part subsequently.

Accompanying drawing explanation

Accompanying drawing is used to provide a further understanding of the present invention, and constitutes the part of description, with following tool Body embodiment is used for explaining the present invention together, but is not intended that limitation of the present invention.In the accompanying drawings:

Fig. 1 is the construction of eukaryotic expression vector figure of HPS.

Fig. 2 is transient transfection HPS gene HPS egg after 48 hours in Western-blot detection Chinese hamster ovary celI culture supernatant White collection of illustrative plates, wherein, wherein, swimming lane 1 represents that under reducing condition, HPS presents the single band of 34KDa, swimming lane in a reduction state 2 represent under non-reduced state, present the band of dimer 68KDa.

Fig. 3 is that transient transfection HPS gene is after 48 hours in SDS-PAGE detection Chinese hamster ovary celI culture supernatant after purification The collection of illustrative plates of HPS albumen, wherein, wherein, swimming lane M represents that molecular weight protein marker, swimming lane R represent that under reducing condition, HPS is also Presenting the single band of 34KDa under original state state, swimming lane represents under non-reduced state, presents the band of dimer 68KDa.

Fig. 4 be the expression product of the HPS gene using the coding of original series shown in SEQ ID No:3 purified after Western-blot collection of illustrative plates.

Fig. 5 is the propagation collection of illustrative plates that under variable concentrations, HPS stimulates L02 cell.

Fig. 6 is the collection of illustrative plates of the damage of HPS protection L02 cell resistance CCl4 induction under variable concentrations, and hepatocellular LDH releases High-volume as evaluation index.

Fig. 7 shows the HPS protective effect to liver in zoopery, wherein:

Fig. 7-1 is that (* P < 0.05, relative to right on the impact of the survival rate of the acute hepatic injury mice that CCl4 induces for HPS According to)；

Fig. 7-2 is the HPS impact on the transaminase level of the acute hepatic injury mice that CCl4 induces, and evaluation index is ALT (A) and the level of AST (B) (n=8, * P < 0.05 is relative to comparison；#P < 0.05 is relative to HSS；&P < 0.05 is relative to GSH)；

Fig. 7-3 is that (A is pathological picture to the HPS acute hepatic injury mice Histopathology Effect of inducing CCl4；B is HPS On the impact of degree of necrosis of the Liver Tissue of CCl4 induction, (n=5, * P < 0.05 is relative to PBS；#P < 0.05 relative to HSS；&P < 0.05 is relative to GSH)；

Fig. 7-4 is that (A is pathological picture for the impact of the acute hepatic injury mice hepatocellular apoptosis that CCl4 is induced by HPS；B is (n=5, * P < 0.05 is relative to PBS on the impact of CCl4 induced liver injury mouse liver cell apoptosis for HPS；#P < 0.05 relative to HSS；&P < 0.05 is relative to GSH)；

Fig. 7-5 be HPS on the impact of the expression of PCNA in CCl4 acute hepatic injury mice liver, and evaluate liver thin with this (n=5, * P < 0.05 is relative to PBS for the cultivation effect of born of the same parents；#P < 0.05 is relative to HSS；&P < 0.05 is relative to GSH).

Detailed description of the invention

Hereinafter the detailed description of the invention of the present invention is described in detail.It should be appreciated that described herein specifically Embodiment is merely to illustrate and explains the present invention, is not limited to the present invention.

On the one hand, the invention provides the gene of a kind of encoding human liver cytokines, wherein, this gene has SEQ ID No:1 Shown nucleotide sequence.

The amino of the nucleotide sequence coded people's liver cytokines (HPS, hepatocyte growth promoting factors) shown in SEQ ID No:1 Acid sequence is as shown in SEQ ID No:2.

Genes of SEQ ID No:3 compared to original coding HPS, genes of SEQ ID of the coding HPS that the present invention provides No:1, changes base 201, accounts for the 21% of base sum, and G+C content is 50.34%.

The gene of the encoding human liver cytokines according to the present invention, the two ends of this gene can also be added well known in the art various Sequence, such as, restriction enzyme site, promoter, enhancer, purification tag (such as, histidine-tagged) etc..Known, these sequences Row will not change the instinct of the nucleotide sequence coded people's liver cytokines shown in SEQ ID No:1, the most more will not change obtained The performance of HPS, and carry out after sequence as above adds, its effect brought also is that those skilled in the art can understand in advance Phase.

Also needing to herein be intended that, the gene of described encoding human liver cytokines has the core shown in SEQ ID No:1 On the premise of nucleotide sequence, as long as this gene can encode the HPS protein with as above function, can be known in the art And expected scope interior interpolation any type other bases of sum purpose, these genes also should be claimed in the present invention In the range of.

Second aspect, present invention also offers a kind of recombinant vector, and wherein, described recombinant vector contains as above SEQ ID No:1 encoding gene and expression vector.

According to the present invention provide recombinant vector, described expression vector can be known in the art various can connect outside The expression vector of source gene, such as, described expression vector can be in carrier T, prokaryotic expression carrier and carrier for expression of eukaryon At least one.Wherein, the Main Function of carrier T and prokaryotic expression carrier can be preliminary identification functional gene and target protein Function, and the effect of carrier for expression of eukaryon can be to be transformed in cell in order to follow-up.Therefore, it is possible to meet above-mentioned application Various carrier T (such as, pMD18-T, pMD19-T etc.), prokaryotic expression carrier (such as, pET32a etc.) and carrier for expression of eukaryon (such as, pGN-M, pcDNA3.1 etc.) are used equally to the present invention.

The third aspect, the invention provides a kind of transgenic cell, and wherein, described transgenic cell contains as above Encoding gene.

Described transgenic cell can be prokaryotic cell or eukaryotic cell.The selection of these cells is people in the art Well known to member.And described transgenic cell can not be grown for complete organism.

Fourth aspect, present invention also offers the gene of encoding human liver cytokines as above, recombinates load as above The application in preparation people's liver cytokines of body, transgenic cell as above.

5th aspect, the invention provides the preparation method of a kind of people's liver cytokines, and wherein, the method includes: at encoding human Under conditions of the gene of liver cytokines can be expressed, cultivate transgenic cell as above, so that it expresses people's liver cytokines.

According to the present invention, essentially consist in the optimization of the gene of encoding human liver cytokines due to the inventive point of the present invention, and for The method carrying out this gene expressing can be implemented according to various methods well known in the art, such as, and CN200510000255.9 Patent application in disclosed method.The application is herein incorporated by the way of quoting in full.

6th aspect, present invention also offers the gene of encoding human liver cytokines as above, recombinates load as above Body, transgenic cell as above, the preparation method of people's liver cytokines as above are used for preventing and/or treating liver in preparation Application in the medicine of damage.

Application according to the present invention, described hepatic injury can include various hepatic injury well known in the art, such as, Acute Hepatic Damage and/or chronic hepatic injury.

Application according to the present invention, it is preferred that the medicine of described prevention and/or treatment hepatic injury includes promoting that hepatocyte increases Medicine, the medicine of anti-liver injury, the medicine reducing hepatic injury tissue transaminase level and the medicine of anti-tissue hepatic necrosis grown At least one in thing.

Herein it is understood that gene, as above containing encoding human liver cytokines as above in any of the above medicine At least one in people's liver cytokines of described recombinant vector, transgenic cell as above and coding.

As known from the above, the HPS that the present invention provides can be by promoting that hepatocellular propagation, anti-liver injury, reduction liver damage The mechanism such as injured tissue transaminase level, anti-tissue hepatic necrosis play prevention and/or the effect for the treatment of hepatic injury.Therefore, may be used The hepatic disease improved by the regulation of these mechanism is within the scope of the present invention.

It addition, the gene of the present invention, albumen all can be prepared as the form of pharmaceutical composition, described compositions can be prepared For different dosage forms, and it is added with the compositions such as corresponding excipient, pharmaceutically acceptable carrier.Concrete is chosen as ability Well known to field technique personnel, in this not go into detail.

Hereinafter will be described the present invention by embodiment.In following example,

The method using synthetic obtains the sequence and such as of the optimization as shown in SEQ ID No:1 that the present invention provides Original series shown in SEQ ID No:3.

Embodiment 1

The structure of the recombinant vector that the present embodiment provides for the present invention is described

(1) acquisition of HPS gene PCR product

Use forward primer P1 (the 5 '-GG shown in SEQ ID No:4GGTACCATGGCAAAGGTCTTCAGC-3 ') and Downstream primer P2 (5 '-AAGGAAAAAA shown in SEQ ID No:5GCGGCCGCAATCACGTTTGGGATAAAGTCG-3 ') right Sequence shown in SEQ ID No:1 carries out PCR amplification, obtains amplified production.Wherein, the nucleotides sequence of underscore part is classified as enzyme Cut site.Amplification system and condition are as follows:

Amplification system:

Component	Volume
		Template	1μl
10×LA Taq Buffer(Mg2+plus)	2.5μl
		dNTPs(10mM)	2μl
P1(10μM)	0.5μl
		P2(10μM)	0.5μl
LA Taq(5U/μl)	0.25μl
		ddH2O	18.25μl

Total system

25μl

Amplification condition:

94 DEG C of denaturations 5min, carry out 94 DEG C of degeneration 45s, 55 DEG C of annealing 45s and 72 DEG C of extend 1min 30 afterwards and follow Ring, after loop ends, 72 DEG C maintain 7min, last 4 DEG C of insulations.

(2) structure of expression vector

Pcr amplification product as obtained above is connected in carrier T by the method utilizing this area conventional, and order-checking is identified correct After, use double digestion (KpnI/NotI) that the carrier T and expression vector pGN-M that connect genes of interest are carried out enzyme action, double to be formed Cohesive end, is then connected the digestion products obtained by T4DNA ligase, thus obtains expression vector.

(3) qualification of expression vector

Expression vector obtained as described above is converted bacillus coli DH 5 alpha (DE3).Screened by ammonia benzyl resistance culture base, picking 5 single bacterium colonies are cultivated, and extract plasmid, carry out PCR and corresponding enzyme action is identified, select the correct clone of amplification to send to company and survey Sequence, sequencing result proves identical with HPS gene order (SEQ ID No:1), and the reading frame of expression vector is complete, and restructuring is described Vector construction success, recombinant vector structure is as shown in Figure 1.

Embodiment 2

The HPS gene that the present embodiment provides for the present invention is described transient expression in Chinese hamster ovary celI

By Chinese hamster ovary celI by 5 × 10⁵The density of individual cell/ml is inoculated in 30ml shaking flask, and culture medium is that CD-DG44 cultivates Base (Gibco 12610-010), at 37 DEG C, 5%CO₂Overnight incubation in environment.Next day collects cell, uses Opti-MEM culture medium (Gibco31985-070) resuspended, adjust its density to 1.4 × 10⁷Individual cell/ml, the restructuring that Example 1 successfully constructs carries Body 40 μ g, with 1 × 10⁷Individual cell mixes, and adds in the electric revolving cup of 4mm diameter, turns with Bio-Rad electroporation electricity, and condition is: electricity Pressure 300V, electric capacity 950 μ F, resistance ∞, once, the cell after being turned by electricity adds continuation in CD-DG44 culture medium and cultivates electric shock.Turn Contaminate latter 48 hours and collect culture supernatant, detect protein expression by ELISA.By PBS solution by coated antibody (Mouse Anti-human Hepassocin antibody, R&D, cat#MAB1614) it is diluted to 2 μ g/ml, add in 96 hole elisa plates, Every hole adds 100 μ l, is placed in 4 DEG C, is coated overnight.Discard and be coated liquid, wash plate 1 with PBST (PBS solution containing 0.1%Tween-20) Secondary.Every hole adds 320 μ L confining liquids (PBS solution containing 1%BSA), and room temperature reaction 2h, PBST wash 2 times, with confining liquid by 1: 100 dilution proportion samples, add 100 μ l sample diluting liquids, 32 DEG C of reaction 1h in every hole.Plate is washed 5 times with PBST, each 1min, 1:100 dilution one anti-(Goat anti-humanHepassocin antibody, R&D, cat#AF1614) is pressed, often with confining liquid Hole adds the 100 anti-diluents of μ L mono-, 32 DEG C of reaction 1h.Plate is washed 5 times, each 1min with PBST.1:20000 dilution is pressed with confining liquid Two anti-(Donkey anti-Goat-HRP, Southernbiotech, cat#6420-05), every hole adds the 100 anti-dilutions of μ l bis- Liquid, 32 DEG C of reaction 45min.Plate is washed 5 times, each 1min with PBST.Add freshly prepared TMB working solution (TMB 2- Component Microwell Peroxidase Substrate Kit, KPL, Cat.No.50-76-03) 100 μ l, lucifuge 32 DEG C reaction 20min, add stop buffer.By the OD value at enzyme connection readout instrument detection 450nm.Result shows to optimize the expression of construct Level is 4 μ g/ml.

By Western-blot detect collected by cells and supernatant in the expression of HPS.Draw reset and add 5 on 75 μ l × Albumen loading buffer (60mM Tris-Cl pH6.8,2%SDS, 14.4mM 2 mercapto ethanol, 25% glycerol, 0.1% Bromophenol blue) in water-bath, boil 5min, SDS-PAGE electrophoresis, go to (4 DEG C, constant current 80mA, 1h) pvdf membrane, 5% skim milk Closing 3h, one anti-(FGL1 (G-15)-R, SC-55957-R) 4 DEG C overnight, TBS-T washes film 3 times (each 5min), two anti-(SC- 2004) incubated at room 45min, TBS-T washes 4 times (each 10min), ECL Color Appearance System (Thermo) testing goal albumen, aobvious Shadow.As in figure 2 it is shown, wherein, M is molecular weight protein marker to result, and swimming lane 1 represents that under reducing condition, HPS is in a reduction state Presenting the single band of 34KDa, swimming lane 2 represents under non-reduced state, presents the band of dimer 68KDa.

Embodiment 3

The cDNA producing the HPS optimized by gene order under serum-free condition stably expresses recombined human liver cytokines Chinese hamster ovary celI system

For the generation of the cell line described in this report, as described in example 1 above, use is inserted with sequence optimisation The pGN-M plasmid of HPS gene.According to embodiment 2, Chinese hamster ovary celI is transfected, receive cell after 48 hours, count, by 200 Cell is transferred to 96 orifice plates by the density of individual cells/well, add in the medium MTX (methotrexate, Sigma, Cat.No.: M8407) continue to cultivate to final concentration of 200nM, screen for the expression of people's liver cytokines by ELISA method, without expressing Or the cell of low expression goes out of use, based on ELISA result, the clone body being positive people's liver cytokines HPS is carried out time in 6 orifice plates Cultivate.Again when about 50% converges, the culture medium of each clone body is sampled and is analyzed expression by ELISA.Base In ELISA data, selected high-expression clone body is transferred in T25 flask, after 3-5 days, is transferred to T75cm²In flask, 3-5 After it, with 0.2 × 10⁶Cell is transferred in 125ml shaking flask by the density of individual living cells/ml, and shaking flask is placed on humidified incubator (37 DEG C and the CO of 5%₂On horizontal shaker in).At cell density more than 0.5 × 10⁶After individual living cells/ml, by 0.2 × 10⁶ The density of individual living cells/ml is seeded in the 125ml shaking flask more renewed carry out time cultivation, until setting up reproducible growth spy Levy (the most within 2 weeks).Collect culture supernatant and small part cell every day, monitor its growth and industry characteristics.ELISA detects knot Really show the recombined human liver cytokines selected in CD Opti-CHO culture medium (Invitrogen, Cat.No.:12681-011) Stable cell lines expression, up to 800mg/L, determines total cell count and survival rate by Trypan Blue, and result shows: training Support to cell viability when the 10th day still greater than 95%.The culture of selected clone is maintained in Selective agar medium.

Embodiment 4

The purification of people's liver cytokines that the present embodiment provides for the present invention is described

According to the recombined human liver cytokines in standard method purifying cells culture supernatant.The sun will picked out in embodiment 3 After sex clone amplification culture, upper tank fermentation, collects supernatant, by 2 step anion-exchange chromatography purification, purified product warp after 10 days SDS-PAGE detect, result as shown in Figure 3: wherein, M is molecular weight protein marker, swimming lane 1 be under reducing condition HPS and also Presenting the single band of 34KDa under original state state, swimming lane 2 is under non-reduced state, presents the band of dimer 68KDa.

Embodiment 5

The present embodiment is used for people's liver cytokines lyophilized preparation formula and the preparation method that the present invention provides is described

After fully dissolving, aseptic filtration, subpackage, and lyophilization.

Comparative example 1

This comparative example is for illustrating the recombinant vector using existing HPS encoding gene to build

Method according to embodiment 1 carries out the structure of recombinant vector, and except for the difference that, genes of interest is original HPS coding Sequence, that is, original series as shown in SEQ ID No:3.

Comparative example 2

This comparative example is for illustrating to use expression and the purification of expression product of existing HPS gene

The expression of HPS gene and the purification of expression product, except for the difference that, purpose base is carried out according to the method for embodiment 2-4 Because original HPS coded sequence, that is, original series as shown in SEQ ID No:3.ELISA testing result finds, original The expression of HPS sequence is only the 1/3 of the expression of the preferred HPS sequence that the present invention provides, the i.e. expression of original series It is 1.3 μ g/ml, and the expression of the sequence of the optimization that the present invention provides is 4 μ g/ml.Western-blot testing result such as Fig. 4 Shown in, wherein, M is molecular weight protein marker, and swimming lane 1 represents that under reducing condition, HPS presents the list of 34KDa in a reduction state One band, swimming lane 2 represents under non-reduced state, presents the band of dimer 68KDa；Compared with Fig. 2, there is significant poly Body and degraded band.

By as above data it can be seen that the HPS encoding gene that the present invention provides can effectively improve the expression of HPS Amount, and also effectively prevent the generation of HPS polymer deposited phenomenon in expression product.Therefore, the present invention is the extensive of HPS Production provides the foundation.

Test case 1

The HPS that this test case provides for the present invention the is described proliferation to Human normal hepatocyte system L02

By L02 cell good for cultivation conditions, counting with after trypsinization, adjusting cell concentration is 5 × 10⁴/ ml, takes 100 μ l are inoculated in 96 well culture plates, and culture medium is the RPMI-1640 containing 10% hyclone, at 37 DEG C, and 5%CO₂Incubator Middle cultivation 12h, then, changes culture fluid, serum-concentration is reduced to 0.5%.With this understanding, add respectively in cultivating system Enter 0,20,50, people's liver cytokines HPS of obtaining in 100ng/ml variable concentrations embodiment 4.Dosing is placed on 37 DEG C, 5%CO₂Training Supporting and cultivate 16h in case, every hole adds 25 μMs of EdU (5-acetenyl-2' deoxyuridine, Guangzhou is sharp rich, R11053.2), Upper machine (High content screening system microscope, Operetta PerkinElmer) meter of taking pictures after 8h Number, calculates EdU incorporation efficiency.Result is as shown in Figure 5.

Result shows, HPS can promote L02 cell proliferation in vitro.

Test case 2

This test case is used for the protective effect of HPS CCl4 anti-to the L02 cell induced damage illustrating that the present invention provides

By every hole 2 × 10⁴Cell density L02 cell is seeded to 96 orifice plates.Serum-free is changed after cell attachment RPMI1640 culture medium Nature enemy 12h, be subsequently added in the embodiment 4 of variable concentrations obtain HPS (0,20,50,100ng/ Ml) stimulating 12h, often group arranges 5 multiple holes.Then add 8nM CCl4 and stimulate 12h, (green according to LDH citotoxicity detection kit Skies C0017) description detection LDH activity: cell processed after before predetermined detection time point 1h, to sample maximum enzyme Hole (cell control well being left intact) alive adds the LDH releasing agent of 10% culture volume, continues gently after mixing Cell culture incubator is hatched, after arriving stimulation time point, 96 orifice plates is centrifuged 5min in 400 × g, collect supernatant 120 μ l and be transferred to In 96 new orifice plate respective aperture, every hole add respectively 60 μ l LDH detection working solutions (lactic acid solution: 1 × INT solution: enzymatic solution= 1:1:1), mixing, room temperature lucifuge is hatched 30min in horizontal shaker jog, is measured absorbance at 490nm.Cytotoxicity or Mortality rate (%)=(sample absorbance-sample controls hole absorbance)/(cell maximum enzyme hole alive absorbance-sample controls hole is inhaled Luminosity) × 100.Result is as shown in Figure 6.

Result shows that HPS can reduce the release of the LDH of damaging cells, thus plays a protective role hepatic injury.

Test case 3

The HPS that this test case provides for the present invention is described is to CCl₄The impact of the survival rate of induced liver injury mice

Use the CCl of 50%₄With the dosage inducing mouse hepatic injury of 5.0ml/kg, test packet is divided into PBS negative control Dosage group (1.0mg/ in group, HSS positive controls, bifendate positive controls, HPS low dose group (0.2mg/kg), HPS Kg), HPS high dose group (5.0mg/kg) 6 groups, respectively injection CCl₄Front 12h, injection CCl₄Rear 24h, 48h, 72h are administered.Often Organize 21 mices.

Result is as shown in Fig. 7-1, and PBS group mice 12h after modeling starts death, and administration group mice is to 24h after modeling Beginning with death, after modeling between 24h to 48h, PBS group mouse survival rate drastically declines, and is only 14.3% during to 48h, and this Time HSS group mouse survival rate be 40%, HPS middle and high dosage group mouse survival rate is all more than 60%.Have compared to PBS group There is significant difference (P < 0.01).Continuous Observation 168h, after 144h, the mice of survival is the most dead, now PBS group mice Survival rate reduces to 9.5%, bifendate group and HSS group more than 20%, HPS middle and high dosage group is then respectively as follows: 47.6%, 38.1%.HPS middle and high dosage group mouse survival rate is higher than positive control drug bifendate and HSS group, compared with PBS group, and survival Rate significantly improves (P < 0.05), and wherein in HPS, the protective rate of dosage group is 38.1%.To each group of death mice mean survival time Statistical result (table 1) display, compared with PBS group, HSS group and bifendate group death mice mean survival time do not have difference (P>0.05), tri-equal significances of dosage group of HPS increase (P<0.05), and HPS dosage group is more than positive drug control group.Each group is all The statistical result showed of the mean survival time of mice, the HPS administration group mice mean survival time apparently higher than PBS group (P < 0.05), additionally, HPS basic, normal, high three dosage group mice mean survival times than HSS group add respectively 15.43h, 33.71h, 32.57h, HPS middle and high dosage group all dramatically increases (P < 0.05) compared with HSS group and bifendate group.

Table 1 people's liver cytokines HPS increases the mean survival time of the acute hepatic injury mice of 50%CCl4 induction

* P ＜ 0.05, * * P ＜ 0.01, * * * P ＜ 0.001, vs PBS

It can thus be seen that the HPS that the present invention provides can improve CCl₄The survival rate of induced liver injury mice.

Test case 4

The HPS that this test case provides for the present invention is described is to CCl₄The impact of the transaminase level of induced liver injury mice

Use 1%CCl₄Inducing mouse hepatic injury, test packet is divided into PBS negative control group, HSS positive controls, connection Dosage group (1.0mg/kg) in benzene dibasic acid esters positive controls, GSH positive controls, HPS low dose group (0.2mg/kg), HPS, HPS high dose group (5.0mg/kg) 7 groups, respectively at injection CCl₄Front 12h, injection CCl₄Rear 24h, 48h, 72h are administered.

As shown in Fig. 7-2, ALT, AST level of respectively organizing is at CCl₄The most first raise after stimulation, reach maximum at 24h, subsequently Being gradually lowered, finally recover to normal level, after modeling, 12h, PBS group ALT is about 1800U/L, positive drug group and HPS administration Group ALT all at below 1000U/L, substantially less than PBS group (P < 0.05)；After modeling, 24h, PBS group ALT level is about 42000U/L, and the ALT level of positive drug group and HPS administration group is all substantially less than PBS group (P < 0.05), in HPS administration group The ALT level of dosage group is substantially less than positive drug HSS group and GSH group (P < 0.05), but with bifendate group no significant difference, 36h, the ALT of administration group are also preferably below PBS control group.The level of AST each time point there is also significant difference (P < 0.05)。

It can be said that bright, the HPS that the present invention provides can reduce CCl₄The transaminase level of induced liver injury mice.

Test case 5

The HPS that this test case provides for the present invention is described is to CCl₄The shadow of the hepatic pathology damage of induced liver injury mice Ring

HPS is evaluated to 1%CCl by HE staining₄The acute hepatic injury mice hepatic tissue pathology degree of injury of induction Impact, test packet is divided into PBS negative control group, HSS positive controls, GSH positive controls, HPS low dose group (0.2mg/ Kg), dosage group (1.0mg/kg), HPS high dose group (5.0mg/kg) 6 groups in HPS, result as shown in Fig. 7-3A, normal condition Under, hepatocyte is polyhedron shape close-packed arrays, and nucleus is big and round, and Cytoplasm is uniform；1%CCl₄After damage, 6h hepatocyte is swollen Swollen, acidophile degeneration, cellularity starts to change, and after modeling, 12h has obvious inflammatory cell infiltration, and nucleus is solid simultaneously Contracting, and deeply contaminate, cellularity destroys, and lobules of liver limiting plate causes irregular destruction because of inflammation, hepatic necrosis, header District's inflammatory cell infiltration, has necrotic zone to occur, after modeling, large area necrocytosis occurs in 24h, and administration group is compared to feminine gender Matched group, at each time point pathology damage lesser extent, shows necrosis of liver tissue range statistics result (Fig. 7-3B), is making 6h after mould, PBS group hepatic necrosis areal concentration (necrotic zone area/Statistical Fields area) is 0.1283, and positive drug group and HPS administration group hepatic necrosis degree is significantly lower than PBS group (P < 0.05), along with each group of liver tissue injury of the extension of trauma time Degree all increases the weight of, and reaches maximum necrosis area to 24h, and now PBS group necrosis of liver tissue density is up to 0.5662, and HSS group, GSH group, HPS middle and high dosage group necrosis of liver tissue density is respectively as follows: 0.3765,0.3104,0.2688,0.2834, hence it is evident that be less than PBS group (P < 0.05).Respectively group liver tissue injury degree is gradually lowered, and to 36h, administration group necrosis density is respectively as follows: 0.2461,0.2698,0.2597,02065,0.1706, hence it is evident that less than PBS group (P < 0.05), during 48h, HSS group and HPS are middle and high Dosage group is less than PBS group.Additionally, after modeling 6h, 12h, 24h, 48h, HPS middle and high dosage group necrosis of liver tissue degree is the brightest Aobvious less than positive drug HSS group, at 36h, 48h significantly lower than GSH group (P < 0.05).

Result above shows that HPS can alleviate CCl₄The degree of necrosis of liver injury model hepatic tissue, the wherein middle and high dosage of HPS The protected effect of group is preferable, in two dosage group murine liver tissue necrotic zone density of 24h less than 0.3, is better than GSH group and HSS Group.

Test case 6

The HPS that this test case provides for the present invention is described reduces the acute hepatic injury mice hepatocellular apoptosis of CCl4 induction

We use TUNEL method to have rated the shadow of the acute hepatic injury mice hepatocellular apoptosis that 1%CCl4 is induced by HPS Ring.TUNEL method is commonly used for the research of Apoptosis Mechanism.During apoptosis, chromosomal DNA can be at some nucleic acid being activated Rupture under the effect of restriction endonuclease, produce 3 '-OH ends, under the effect of deoxyribonucleotide transferring enzyme (TdT), by fluorescein The dUTP of labelling is tagged to DNA 3 '-OH end, and the horseradish peroxidase being connected with fluorescein (HRP) can be with diaminobenzidine (DAB) produce the strongest color reaction (in dark-brown), and normal cell or the cell bred seldom can occur DNA to break Split, seldom can be colored, such that it is able to carry out apoptotic detection.Result is as shown in Fig. 7-4A, after injection CCl46h, and liver group Knitting interior existing apoptosis, to 24h, the ratio that apoptotic cell accounts for cell under the whole visual field is maximum, wherein PBS group hepatocyte Apoptosis is most, and administration group apoptosis is relatively fewer, and HPS middle and high dosage group TUNEL positive cell region is less than GSH and HSS Group.Positive rate statistical result (Fig. 7-4B) display of TUNEL, after injection CCl46h, PBS group apoptosis rate density is 0.0989, and in GSH group and HPS, dosage component is not: 0.03620,0.02293, all significantly lower than PBS group (P < 0.05), arrive 24h, each group density of apoptotic cells all increased, and PBS group density of apoptotic cells is 0.7481, and administration group apoptosis is the most relatively Low, HPS middle and high dosage group density of apoptotic cells is respectively as follows: 0.1916,0.2028, hence it is evident that less than PBS group (P < 0.05), 36h Time, in addition to HSS group, other each administration group apoptotic cells are all considerably less than PBS group (P < 0.05), along with hepatocellular increasing Growing, apoptotic cell ratio is gradually lowered, and is still above administration group to PBS group during 48h.Additionally, after modeling observe each time Between point, in HPS dosage group apoptosis rate all be substantially less than HSS group, HPS high dose group is the lowest at 12h, 24h, 36h, 48h In HSS group, HPS middle and high dosage group is substantially less than GSH group (P < 0.05) at 24h, 36h.

Result above shows, HPS can reduce the acute hepatic injury mice hepatocellular apoptosis of CCl4 induction.Damage the heaviest feelings Under condition, HPS middle and high dosage group apoptosis rate is about 20%, hence it is evident that less than PBS group, is better than GSH group and HSS group.

Test case 7

The HPS that this test case provides for the present invention is described improves the expression of PCNA in CCl4 acute hepatic injury mice liver

Proliferating cell nuclear antigen (Proliferating Cell Nuclear Antigen, PCNA) synthesizes with cell DNA Closely related, cell proliferation startup plays an important role.It is a kind of to exist only in proliferative cell the interim molecule expressed Amount is the protein of 36kDa, synthesizes, express being gradually increased in the G1 phase, reach maximum to the S phase in nucleus, and the G2/M phase contains Amount reduces, and this is consistent with DNA synthesis.In detection cell, the expression of PCNA can be as a finger of cell proliferation detection Mark.

Using the expression of SABC detection PCNA herein, research HPS is to the effect of hepatic tissue cell regeneration after damage.As Shown in Fig. 7-5, after CCl4 stimulates, PCNA positive cell increases, and 24h-48h administration group has more PCNA positive cell.PCNA Positive rate statistical result showed, 6h, PBS group PCNA positive rate 7.2% after modeling, GSH group, HSS group, the basic, normal, high dosage of HPS Group is respectively 7.2%, 19.1%, 9.2%, 29.2%, 8.5%, and wherein, HSS group and HPS 1.0mg/kg group are apparently higher than PBS Group (P < 0.05).Along with the extension of trauma time, each group PCNA positive rate is gradually increased, and to 24h, PBS group positive rate is 24.2%, and the middle and high dosage component of HSS group, HPS does not reach 57.5%, 67.6%, 50.0%, hence it is evident that higher than negative control PBS Group (P < 0.05).During 36h, each group PCNA positive cell remains at higher level, and HPS middle and high dosage group is apparently higher than PBS Group (P < 0.05), reduces to 11.0% to 48h, PBS group positive rate, and HSS group, HPS middle and high dosage group is still more than 25%, bright Aobvious higher than PBS group (P < 0.05).At each time point of damage, in HPS, dosage group PCNA positive cell ratio is all significantly greater than GSH group, HPS high dose group is then to be noticeably greater than GSH group (P<0.05) at 24h, 36h, 48h, with HSS group no significant difference (P> 0.05)。

Result above shows, HPS can promote hepatocellular propagation after damage, and it promotees cultivation effect and is better than GSH group, with HSS Group no significant difference, wherein at the time point that PCNA positive cell is most, HPS 1.0mg/kg and 5.0mg/kg can be by PCNA sun Property rate brings up to more than 50%.

The preferred embodiment of the present invention described in detail above, but, the present invention is not limited in above-mentioned embodiment Detail, in the technology concept of the present invention, technical scheme can be carried out multiple simple variant, this A little simple variant belong to protection scope of the present invention.

It is further to note that each the concrete technical characteristic described in above-mentioned detailed description of the invention, at not lance In the case of shield, can be combined by any suitable means.In order to avoid unnecessary repetition, the present invention to various can The compound mode of energy illustrates the most separately.

Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as it is without prejudice to this The thought of invention, it should be considered as content disclosed in this invention equally.

Claims (10)

1. the gene of an encoding human liver cytokines, it is characterised in that the nucleotide sequence of this gene is as shown in SEQ ID No:1.

2. a recombinant vector, it is characterised in that this recombinant vector contains the gene described in claim 1 and expression vector.

3. a transgenic cell, it is characterised in that this transgenic cell contains the gene described in claim 1.

Transgenic cell the most according to claim 3, wherein, described transgenic cell is eukaryotic cell.

5. the gene of encoding human liver cytokines described in claim 1, the recombinant vector described in claim 2 or claim 3 or 4 The application in preparation people's liver cytokines of the described transgenic cell.

6. the preparation method of people's liver cytokines, it is characterised in that the method includes: the gene in encoding human liver cytokines can table Under conditions of reaching, cultivate the transgenic cell described in claim 3 or 4, so that it expresses people's liver cytokines.

7. the gene of encoding human liver cytokines described in claim 1, the recombinant vector described in claim 2, claim 3 or 4 Preparation method described in described transgenic cell or claim 6 is used for preventing and/or treating the medicine of hepatic injury in preparation In application.

Application the most according to claim 7, wherein, described hepatic injury includes acute liver damage and/or chronic hepatic injury.

9. according to the application described in claim 7 or 8, wherein, the medicine of described prevention and/or treatment hepatic injury is anti-liver injury Medicine.

Application the most according to claim 9, wherein, the medicine of described anti-liver injury includes the medicine promoting hepatocyte growth At least one in the medicine that thing, the medicine reducing hepatic injury tissue transaminase level and anti-hepatic tissue cell are downright bad.