genetic recombinant expression and characterization of human augmenter of liver regeneration

ORIGINAL ARTICLEGenetic Recombinant Expression and Characterization of Human Augmenter of Liver RegenerationChun-Fang Gao ÆFei Guo Zhou ÆHao Wang ÆYing-Feng Huang ÆQiang Ji ÆJie ChenReceived:17March 2008/Accepted:3June 2008/Published online:9July 2008ÓSpringer Science+Business Media,LLC 2008Abstract Aims To establish a highly effective prokary-otic recombinant expression system for human augmenter of liver regeneration (hALR)and to characterize the recombinant hALR both in vitro and in vivo.Methods ALR cDNA was synthesized and inserted into expression vector pET28a +,the recombinant plasmid was transformed into BL21,and expression of hALR was induced by IPTG.Recombinant hALR (rhALR)was purified by sequential detergent wash,enterokinase (EK)digestion,gel-filtration,and chelating chromatography.The rhALR was identified by SDS-PAGE,immunoblotting,MALDI-TOF-MS,and N-terminal sequencer.Cell proliferative effect of rhALR on human hepatocytes was analyzed by MTT.The pro-tective effect of rhALR on liver function was observed on CCl 4-induced intoxicated mice.Results Recombinant expression plasmid of ALR [pET28(a +)-hALR]was con-firmed by restriction enzyme digestion and DNAsequencing.The expressed rhALR constituted 30%of total bacterial protein.Molecular weight was 15,029for mono-mer and 30,136for dimer by mass determination.N-terminal was M-R-T-Q-Q,exactly the same as antici-pated for hALR.The purified protein migrating at about 15KD showed excellent antigenicity in immunoblotting.The rhALR also showed a strong stimulative effect on hepa-tocyte proliferation.ALT and AST levels,liver histological structure,as well as the survival rate of CCl 4-intoxicated mice were significantly improved when rALR was administrated at 40l g/kg or 200l g/kg.Conclusions The rhALR is successfully expressed highly effectively with anticipated MW,N-terminal,and antigenicity.It could play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and improving liver function in CCl 4-intoxicated mice.Keywords Gene ÁAugmenter of liver regeneration (ALR)ÁRecombinant ÁHepatocytes ÁLiver functionAbbreviationsALR Augmenter of liver regeneration IPTG Isopropyl-b -galactopyranoside pET Plasmid for expression by T7RNA polymerase MW Molecular weightIntroductionLiver possesses the capacity to restore its tissue mass and attain optimal volume in response to physical,infectious,and toxic injury.The extraordinary ability of the liver to regenerate is regarded to be the effect of cross-talk among growth factors,cytokines,matrix components,and many other factors [1].Among the growth factors promotingChun-Fang Gao,Fei Guo Zhou:the authors contributed equally to this work.C.-F.Gao (&)ÁQ.JiDepartment of Laboratory Medicine,Eastern Hepatobiliary Hospital,Second Military Medical University,225Changhai Road,Shanghai 200438,Chinae-mail:gaocf1115@;wanggaob@ F.G.ZhouDepartment of Hepatic Surgery,Eastern Hepatobiliary Hospital,Second Military Medical University,Shanghai 200438,China H.WangDepartment of Laboratory Medicine,Changzheng Hospital,Second Military Medical University,Shanghai 200003,China Y.-F.Huang ÁJ.ChenLiver Fibrosis Research Center,Changzheng Hospital,Second Military Medical University,200003Shanghai,ChinaDig Dis Sci (2009)54:530–537DOI 10.1007/s10620-008-0372-1hepatocyte growth,augmenter of liver regeneration(ALR) is identified in regenerating rat and canine livers following partial hepatectomy and in the hyperplastic livers of weanling rats,but not in resting adult livers[2].Though initially recognized as a liver-specific,but not species-specific growth factor,ALR has been proved to distribute in many organs and tissues besides liver[3].Quantitative mRNA analysis,immunoblotting,and immunohistological studies revealed increased ALR expression in cirrhosis, hepatocellular carcinoma(HCC),and cholangiocellular carcinoma(CCC),indicating an important role for ALR in hepatocellular regeneration in liver cirrhosis,hepatocarci-nogenesis,and therefore of potential value in the clinical diagnosis of liver cirrhosis and cancer[4].Recently,ALR was identified as aflavin-dependent sulfhydryl oxidase with cytochrome c reductase activity and immune regula-tor,belonging to the new Erv/Alr family[5–7].It could control mitochondrial gene expression and the lytic activ-ity of liver-resident natural killer(NK)cells through regulating interferon-c(IFN c)[8,9].This secondary he-patotrophic protein,also known as hepatopoietin(HPO), can be obtained by gradient purification from hepatic stimulator substance(HSS)or genetic recombinant expression[10–12].However,none of these studies has characterized the rhALR both in vivo and vitron in detail. In this study,a highly effective prokaryotic recombinant human ALR(rhALR)expression system was established, and the biochemical features as well as functional char-acters of the rhALR were analyzed so as to provide experimental bases for developing it into a new potential product as hepatic growth factor for rescuing acute liver failure.Materials and MethodsConstruction and Recombinant Expression of rALRA378-bp coding region for rhALR was optimized for the prokaryotic expression system and avoiding unfavorable features,such as high GC content and undesirable folding domains,in reference to published data and the NCBI website(/molbio/)(sequence patented in China pending,No200710107893.X)[13].This dsDNA was synthesized by Genecore Company,Shanghai, China.The coding fragment with Eco RI,Xho I(underlined) adaptor,and enterokinase(EK)site(bold)was then obtained by PCR.The primers were:50TTG GAA TTC GAC GAC GAC GAC AAG ATG CGT ACC CAG CAG AAA30(upstream)and50TTG CTC GAG TTA GTC GCA AGA ACC GTC30(downstream).The Eco RI-and Xho I-treated fragment was ligated to linearized pET28a(+) vector.The recombinant plasmid was transformed into DH5a and identified with restriction enzyme digestion and DNA sequencing.The correct recombinant plasmid was transformed into BL21(DE3).Cells were grown to an optical density of0.5at600nm and were induced to express hALR by the addition of isopropyl-b-D-thiogalactoside (IPTG)(Amesco,America)to afinal concentration of1mM and incubation for4h.For gel-electrophoretic analysis of successful protein induction,bacteria from0.5-ml aliquots of the induced and uninduced cultures were pelleted and resuspended in100l l of protein sample buffer.Purification and Preparation of rALRBacterial cells were collected by centrifugation(10min at 4,000g),washed once with ice cold PBS and resuspended in PBS,and then frozen at-80°C.The preparation of the inclusion body was performed as described before[14]. Briefly,bacteria were thawed at room temperature,and the mixture was sheared on ice.After centrifugation for30min at10,000g at4°C,the pellet was resuspended in10ml of TED-buffer containing50mM Tris–HCl(pH8.0),2mM EDTA,0.1mM dithiothreitl,and5%glycerol.The sus-pension was sonicated on ice four times for15s.After the addition of sodium deoxycholate to afinal concentration of 0.05%(w/v),the mixture was incubated for15min at4°C and then centrifuged for30min at10,000g.Thefinal pellet was solubilized in50mM Tris(pH8.0)containing8.0M urea and2mM EDTA.The insoluble pellets were removed by centrifugation at15,000g for20min at4°C.The solubilized inclusion bodies were purified by FPLC pro-cedure(AKTA Explorer100,GE).Fusion protein was loaded onto a chelating Sepharose FF column with aflow rate of1–2ml/min.The refolding of the bound protein on the column was achieved by the use of a linear urea gra-dient treatment with aflow rate of0.1ml/min.The refolded protein was then eluted using renaturation buffer with a linear imidazole gradient from20to250mM.The imid-azole and salt were removed by the Sephadex G-25 column,and the fusion protein was cleaved by recombinant enterokinase(EK)(Shanghai Haiyi Biotech,China).His-tag was removed by chelating the Sepharose FF column. After being further purified with Q Sepharose FF column, the purified rhALR was subjected to SDS-PAGE,Western blot,molecular weight(MW)determination,and N-termi-nal amino acid analysis.Characterization of Recombinant rALRSDS-Polyacrylamide Gel Electrophoresis and ImmunoblottingSDS-polyacrylamide gel electrophoresis was performed using14%(w/v)tris–glycine gels under reducingconditions.Briefly,sheared bacterial pellets with or with-out induction were subjected to gel electrophoresis. Analytic gels were stained in a solution containing30%(v/ v)methanol,10%(v/v)acetic acid,and0.5%(w/v)Coo-massie brilliant blue,and distained in a solution containing 30%(v/v)methanol and10%(v/v)acetic acid.For immu-noblotting,the purified proteins were transferred to a positively charged NC membrane(Roche)according to standard protocols.The blotted membrane was blocked overnight with5%(w/v)non-fat dry milk in TBST[10mM Tris,pH7.6;150mM NaCl;0.1%(v/v)Tween20]at4°C. The membrane was subsequently incubated at room tem-perature with a1:2,000diluted rabbit polyclonal anti-ALR antibody(multiBIND biotech GmbH,Dortmund,Ger-many)in1%(w/v)non-fat dry milk in TBST for2h and then with a1:1,000diluted peroxidase(POD)-labeled anti-rabbit IgG as second antibody(Pierce)for45min.The membrane was rigorously washed after each incubation. Color development of POD-labeled secondary antibody was performed using DAB substrate(Pierce).MW Determination and N-SequencingSequentially purified rhALR was ionizedfirstly and entered into the mass analyzer(MALDI-TOF-MS,BrukerAutoflex II).The target protein was identified,and MW was deter-mined according to the difference of mass-to-charge(m/z) ratio.The N-terminal amino acid sequencing of the protein is performed based on the Edman degradation method with two steps.Thefirst step is to cut the amino acids one by one from the N-terminal by a chemical method;the second step is to separate protein by HPLC through separating and analyzing the amino acid.Both of the above determinations were accomplished by the Research Center for Proteome Analysis(RCPA),the Chinese Academy of Sciences,Shanghai.Human Hepatocyte Proliferation MTT Assay Immortalized human hepatocytes(L-02)were obtained from the cell bank of the Chinese Academy of Sciences, Shanghai.Cells were maintained with DMEM(Gibco) supplemented with10%FCS at378C with5%CO2 atmosphere.Hepatocytes were seeded into96-well plates at 59104/ml(100l l/well).The culture medium was replaced with a fresh medium containing0.5%FCS when 60%of cells were confluent.Sixteen hours later,fresh culture medium supplemented with different concentra-tions of rhALR ranging from0.16l g/ml to20l g/ml in double dilution was used for24h.Then cell culture was continued for4h together with10l l/well3-(4,5-dim-ethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT) (Amesco)solution(5mg/ml).The medium was replaced with DMSO(Sigma)100l l/well,and the plate was vor-texed for15s.Absorbance of each well was then detected at wavelength550nm.The experiment was triplicated and repeated three times.Rescue of CCl4Induced Acute Hepatic Injury in Mice Totally60male Chinese Kunming mice(age:6weeks; body weight:25–30g each,Shanghai Experimental Ani-mal Center)were randomly subdivided into four groups (n=15/group),i.e.,group A1:CCl4-induced injury with rhALR treatment(200l g/kg);group A2:CCl4-induced injury with rhALR treatment(40l g/kg);group A3:CCl4-induced injury without rhALR,but with same volume of normal saline instead;group A4:model mock group(with the same volume of olive oil instead of10%CCl4).For the CCl4-induced injury model,CCl4was injected intra-peritoneally at10ml/kg CCl4in10%CCl4olive oil solution.Four hours later,different dosages of rhALR or control normal saline were administrated intraperitoneally every12h.Eight hours after the fourth administration, blood samples were collected,and sera were obtained for alanine aminotransferase(ALT)and aspartate amino-transferase(AST)determination with a routine automatic biochemistry analyzer(model7600,Hitachi,Japan).His-tological study of slices of10%formaldehyde-treated liver tissue was performed with standard hematoxulin and eosin(HE)staining protocols.The study was repeated three times.Protective Effect of rhALR on CCl4-Induced Lethal Injury in MiceAnother60male Chinese Kunming mice(age:6weeks; body weight:25–30g each,Shanghai Experimental Ani-mal Center)were randomly subdivided into four groups (n=15/group):group B1:CCl4-induced lethal injury with rhALR treatment(200l g/kg);group B2:CCl4-induced lethal injury with rhALR treatment(40l g/kg);group B3: CCl4-induced lethal injury without rhALR treatment,but with the same volume of normal saline treatment instead; group B4:model mock group(with the same volume of olive oil instead of50%CCl4).CCl4was injected intra-peritoneally at10ml/kg CCl4in50%CCl4olive oil solution.The rhALR or control normal saline at the same volume was administrated once intraperitoneally1h later. The number of survival mice was recorded for each group over the following7days.Statistical AnalysisResults were expressed as mean±SD for MTT assay and liver function test.Statistical analysis was performedusing SPSS software(SPSS10.0).Student’s t-test was used to determine whether there were any statistical differences between the rhALR-treated group and con-trol.A P value of less than0.05was considered significant.ResultsConstruction of a pET28(a+)Plasmid Encoding Recombinant Human ALRWe cloned a378-bp human ALR coding region corre-sponding to126amino acids into the six His-tagged pET28(a+)vector.By our cloning strategy,an EK diges-tion site immediately before N-terminal of ALR was generated so as to remove the His-tag away from the His-ALR fusion protein.The pET28(a+)vector is a pBR322-based plasmid-containing promoter and transcription ter-minator signal from bacteriophage T7.The nucleotide sequence of the pET28(a+)derivative was confirmed by DNA sequencing.The encoded126-amino acid peptide has a calculated MW of15,027.9.Purification and Identification of Recombinant hALRFor expression of the rhALR,plasmid pET28(a+)-hALR was transformed into the bacterial host strain BL21 (DE3).T7RNA polymerase was induced by IPTG,and recombinant protein synthesis was monitored by SDA-PAGE under reducing conditions.The T7RNA poly-merase directed the synthesis of a fusion protein containing some purification tag with an apparent molecular weight of20,000.The synthesized fusion pro-tein was obtained largely in the insoluble fraction of the bacterial extract,which constitutes30%of total bacterial protein(Fig.1).Purification of rhALR was achieved by sequential detergent washes,Sepharose FF,and Sephadex G-25chromatography,EK digestion,and chelating chro-matography.The purified rhALR was subject to SDS-PAGE,indicating over90%purity,and had a molecular weight of around15,000after removing the unrequired tag in the target protein under reducing conditions (Fig.2).The accurate MW was15,029for monomer and 30,136for dimer by MALDI-TOF-MS determination.The MW for monomer is quite similar to the calculated one (Fig.3).Five N-terminal amino acids were M-R-T-Q-Q, exactly the same as anticipated for hALR.To demonstrate the immunological identity of the protein,an immuno-blotting analysis was performed(Fig.4).As expected,the expressed protein migrating at about15,000showed excellent antigenicity,indicating the excellent immuno-logical identify of the recombinant protein.Biological Assay of rhALR In Vitro and In VivoEffect of rhALR in Vitro on Human Hepatocyte Proliferation(MTT Assay)Recombinant hALR was found to have a strong stimulus on normal human hepatocyte proliferation as measured by MTT assay(see Fig.5).Also,a dose-dependent effect of rhALR on hepatocyte proliferation was observed.The significant stimulative effects appeared at10l g/ml and20 l g/ml.In Vivo Protective Effect of rhALR on Acute Hepatic Failure and Lethal Injury in CCl4-Induced MiceALT and AST were significantly elevated in all10%CCl4-treated mice compared to control,indicating acute liver injury occurred because of the toxic effect of CCl4on liver. Administration of rhALR at40l g/kg could decrease the ALR from441.7U/l to215U/l(P\0.01)and AST from 824U/l to478U/l(P\0.05).Application of rhALR at higher dosage(200l g/kg)could decrease the ALT level significantly,too(P\0.05).So rhALR at low(40l g/kg) or high dosage(200l g/kg)showed the positive effects on hepatocyte function improvement(Fig.6).Upon histolog-ical study,HE staining of the liver tissue section indicated heavy necrotic and degenerative change of hepatocytes in 10%CCl4-induced intoxication.In therhALR-treated Fig.1SDS-polyacrylamide gel electrophoresis of total protein extracts from BL21(DE3)transformed with pET28(a+)-hALR.The bacteria were harvested3h after induction of the T7RNA polymerase.Fusion protein prior to(1)and after induction(2)with IPTG was fractionated on a14%SDS-polyacrylamide gel under reducing conditions and detected by staining with Coomassie brilliant blue.Sizes of protein marker(KD)are given in the left margingroup,fewer necrotic hepatocyes were found,and some regenerative hepatocytes appeared (Fig.7).Among 50%CCl 4-intoxicated mice,40%of them survived after 7days without rhALR treatment,indicating the lethal toxic effect of CCl 4occurred for more than half of the mice,while a protective effect was observed since 80%of mice survived after administration of rhALR at 40l g/kg or at 200l g/kg (Fig.8).DiscussionMammalian augmenter of liver regeneration protein (ALR)was initially identified as a secondary growth factor involved in liver regeneration [1,10].The human gene was found to map to human chromosome 16[13].Its sulfhydryl oxidase activity and involvement in iron homeostasis have been recently demonstrated [3,6,7].ALR is expressed in a broad range of peripheral organs [3].The hepatocyte was the predominant liver cell in which ALR was synthesized and stored [2].ALR appears to be constitutively expressed in hepatocytes in an inactive form and released from the cells in an active form in response to partialhepatectomyFig.2SDS-polyacrylamide gel electrophoresis of rhALR during the process of purification.Sizes of protein marker (KD)are given in the left margin (from upper to lower:97,66,44,29,20,and 14).Lane 1–3:Samples of bacteria lysate (1),inclusion body (2),and washed inclusion body solution (3);lane 4:flow-through liquid from chelating Sepharose FF ne 5:Elution liquid from chelating Sepharose FF ne 6:Final purified sample after EKdigestionFig.3Molecular weight (MW)determination of rhALR by MALDI-TOF-MS.Sequentially purified rhALR was ionized firstly and entered in the Mass Analyzer (BrukerAutoflex II).Target protein was identified,and MW was determined according to the difference of mass-to-charge (m/z)ratio.The MW was 15,029for monomer and 30,136for dimer,with the homodimer predominatingnativelyFig.4Immunoblot of purified rhALR.Purified protein was prepared from IPTG-induced (lane 1–4)bacteria harboring expression plasmid pET28(a +)-hALR.Non-induced (lane 5)bacterial extract was used as negative control.Rabbit polyclonal anti-hALR antibody (multiBIND biotech GmbH,Dortmund,Germany)was used in 1%(w/v)non-fat dry milk in TBST for 2h and then with a 1:1,000diluted peroxidase(POD)-labeled anti-rabbit IgG as second antibody (Pierce)for 45min.Color development of POD-labeled secondary antibody was per-formed using DAB substrate (Pierce).Sizes of prestained protein markers (New England Biolabs)are on the left margin (upper:20KD,lower:14KD)and under other circumstances of liver maturation (as in weanling rats)or regeneration [2].Previous study supports that recombinant ALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2cells in vitro [15,16].Recombinant ALR plasmid DNA injection was helpful in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing the level of AST and ALT in CCl 4-intoxi-cated rats [17].Hepatic ALR mRNA expression was higher in liver disease patients than in non-liver disease controls,and a correlation was found between serum ALR values and hepatic levels of ALR mRNA [4].ALR was producedby and released from the liver at the time of hepatic injury.Thus,circulation of ALR is also regarded as the markers for liver cirrhosis and HCC [4].Besides the effect as hepatocyte mitogen or potential marker for liver cirrhosis and HCC,ALR induces hepatocyte regeneration via inhi-bition of liver NK cells,since cytotoxicity of liver NK cells against regenerating hepatocytes has been reported as a possible mechanism of regeneration failure in fulminant hepatitis [8,9].The inhibition of the liver-resident NK cells by ALR is achieved via reduction of IFN c by liver resident NK.The administration of IFN c in 70%hepatectomized rats is followed by a significant reduction both of the mitochondrial transcription factor A expression and of liver regeneration,indicating ALR as a growth factor and as an immunoregulator by controlling,through IFN c levels,the mitochondrial transcription factor A expression and the lytic activity of liver-resident NK cells [8,9,18,19].Though ALR was first identified and cloned by Hagiya et al.in 1994[10],and since then more and more studies have focused on the distribution [3,4],functional identi-fication [5,8,9],crystal structure analysis [6],as well as recombinant expression as mentioned above [12,15–17],the low expression efficiency,incomplete characterization of recombinant human ALR (rhALR),and the extraordi-nary effect of rhALR on liver regeneration motivated us to establish a high effective expression system and then per-form a serial of identification and functional studies so as to provide a solid basis for future clinical application of rhALR.The cNDA sequence of human ALR used in this study had been especially optimized to prokaryotic expression,the purification system,and its biological activity withtheFig.5The effect of rhALR on hepatocyte proliferation by MTT.Immortalized human hepatocytes (L-02)were obtained from the cell bank of the Chinese Academy of Sciences,Shanghai.Cells were maintained under standard condition and seeded into 96-well plates at 59104/ml (100l l/well).The culture medium was replaced with a fresh medium containing 0.5%FCS when 60%cells were confluent,and fresh culture medium supplemented with different concentrationsof rhALR ranging from 0.16l g/ml to 20l g/ml in double dilution were used 16h later.Then cell culture was continued for 4h together with 10l l/well MTT (Amesco)solution 24h later.The medium was replaced with DMSO (Sigma),and the plate was vortexed for 15s.Absorbance of each well was then detected at wavelength 550nm.The experiment was triplicated and repeated threetimesFig.6ALT and AST levels of CCl 4-intoxicated mice with or without rhALR administration.Mice were injected intraperitoneally at 10ml/kg CCl 4with 10%CCl 4olive oil solution.Four hours later,different dosages of rhALR or control normal saline were administrated intraperitoneally every 12h.Eight hours after the fourth administra-tion,blood samples were collected,and sera were obtained for alanine aminotransferase (ALT)and aspartate aminotransferase (AST)deter-mination with routine automatic biochemistry analyzer (model 7600,Hitachi,Japan)addition of EK digestion sites for cutting six His-tags away from the recombinant fusion protein (patent pending).The N-terminal amino acid sequence by HPLC,molecular weight by MALDI-TOF-MS,and antigenicity identity by immunoblotting analysis confirmed the identity of the recombinant human ALR as expected.Also,as anticipated,native rhALR was a homodimer since two MWs (15,029and 30,136)were obtained with 30,136(homodimer)pre-dominating (Fig.3),similar to the result by Giorda and Hagiya [13].Sequential detergent washes and gel-filtration as well as chelating chromatography helped to obtain rhALR with high purity (more than 90%),as was shown by SDS-PAGE under reducing conditions (Fig.2).The recombinant ALR could stimulate the proliferation of the human hepatocyte dose dependently,similar to the research on rat hepatocytes [15].The significant stimulation effects appeared at 10l g/ml and 20l g/ml,with proliferation activity more than doubled,indicating the strong mitogen-like effect of rhALR on hepatocytes in vitro.We used 10%CCl 4and 50%CCl 4injections to induce acute liverinjuryFig.7HE staining of the liver tissue from CCl 4-intoxicated mice with or without rhALR administration.Mice were injected intraper-itoneally at 10ml/kg CCl 4with 10%CCl 4olive oil solution.Four hours later,different dosages of rhALR or control normal saline were administrated intraperitoneally every 12h.Eight hours after the fourth administration,liver sections were prepared from the mice and then HE staining was performed.In CCl 4-intoxicated mice administrated normal saline,there was evident and extended centrilobular necrosis with the hepatocellular degeneration of the surrounding zones (a )(9100).The sinusoidal collagen reticulum framework around thecentrilobule collapsed,and the hepatocytes had disappeared with infiltration of abundant inflammatory cells and hyperplasia of Kupffer cells (b )(9400).In CCl 4-intoxicated mice administrated rhALR (40l g/kg),centrilobular necrosis was limited to individual lobules (c )(9100);hepatocyte degeneration of surrounding zones was not prominent,and regeneration of some hepatocytes was revealed (d )(9400).The effect of administration of rhALR at 200l g/kg was similar to that at 40l g/kg.The results indicate that rhALR improves liver structurehistologicallyFig.8Survival curve of 50%CCl 4-intoxicated mice with or without rhALR administration.Mice were injected intraperitoneally at 10ml/kg CCl 4with 50%CCl 4olive oil solution.The rhALR or control normal saline at the same volume was administrated once intraper-itoneally 1h later.The number of survival mice was recorded for each group over the following 7days.The results reveal that rhALR improves the survival rate of CCl 4-intoxicated miceand lethal intoxication,respectively,in mice.Two doses of rhALR were administrated in vivo.The administrations of rhALR intraperitoneally further confirmed that rhALR could decrease ALT(ALR at40l g/kg or200l g/kg)and AST(ALR at40l g/kg)significantly in10%CCl4-induced intoxicated mice;liver function improvement was also confirmed histologically.In50%CCl4-induced lethal intoxication,rhALR could improve the survival of intoxi-cated mice from40to80%,indicating the recombinant human ALR obtained in this study is obviously effective in rescuing CCl4-intoxicated mice via improving liver func-tion and stimulating hepatocyte proliferation.The initial1–3days were especially important for the survival in lethal intoxication,so the administration of the rhALR should be in the very early stage of the injury according to the sur-vival curve(Fig.8).In conclusion,we constructed a highly effective pro-karyotic recombinant expression system pET28(a+)-hALR.The 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