Transforming growth factor-ß-activating kinase 1 and its binding protein 1 participate in the innate immune responses via modulating the IMDNFκB signaling in mud crab (Scylla paramamosain).

Transforming growth factor-ß-activating kinase 1 (TAK1) is essential for diverse important biological functions, such as innate immunity, development and cell survival. In the present study, the homologs of TAK1 and TAK1-binding protein 1 (TAB1) were identified and characterized from mud crab Scylla paramamosain for the first time. The full-length cDNAs of SpTAK1 and SpTAB1 were 2, 226 bp and 2, 433 bp with 1, 782 bp and 1, 533 bp open reading frame (ORF), respectively. The deduced SpTAK1 protein contained a conserved S_TKc (Serine/threonine protein kinases, catalytic) domain, and the putative SpTAB1 protein possessed a typical PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain and a potential TAK1 docking motif. Real-time PCR analysis showed that SpTAK1 and SpTAB1 were highly expressed at early development stages, suggesting their participation in crab's development process. Moreover, the expression levels of SpTAK1 and SpTAB1 in hepatopancreas were positively stimulated after challenge with Vibro alginolyticus and Poly (I:C), implying the involvement of SpTAK1 and SpTAB1 in innate immune responses against both bacterial and viral infections. When SpTAK1 or SpTAB1 were silenced in vivo, the expression levels of two IMDNFκB signaling components (SpIKKß and SpRelish) and six antimicrobial peptide (AMP) genes (SpALF1-5 and SpCrustin) were significantly reduced, and the bacteria clearance capacity of crabs was also markedly impaired in SpTAK1 or SpTAB1 silenced crabs. Additionally, overexpression of SpTAK1 and SpTAB1 in HEK293T cells could markedly activate the mammalian NF-κB signaling. Collectively, our results suggested that TAK1 and TAB1 regulated crab's innate immunity via modulating the IMDNFκB signaling. These findings may provide new insights into the TAK1/TAB1-mediated signaling cascades in crustaceans and pave the way for a better understanding of crustacean innate immune system.

[Research advance in circular RNAs].

Circular RNAs (circRNAs) are a class of endogenous, covalently closed, single-stranded RNA without 3'-poly(A) and 5'-cap structures. CircRNAs are characterized by universality, diversity, stability and conservation, and have been found to regulate mammalian transcription and be translated into proteins. In this review, we summarized the biogenesis, classification, expression, distribution, biological functions and regulation of circRNAs. In addition, we discussed the association of circRNAs with diseases and the methods for identification and characterization of circRNAs. Finally, we speculated the application prospect and research direction of circRNAs.

Effects of both cold and heat stress on the liver of the giant spiny frog (Quasipaa spinosa): stress response and histological changes.

Ambient temperature-associated stress can affect normal physiological functions in ectotherms. To assess the effects of cold or heat stress on amphibians, giant spiny frogs (Quasipaa spinosa) were acclimated at 22°C followed by exposure to 5°C or 30°C for 0, 3, 6, 12, 24 and 48 h, respectively. Histological alterations, apoptotic index, generation of mitochondrial reactive oxygen species (ROS), antioxidant activity indices and stress-response gene expression in frog livers were subsequently determined. Results showed that many fat droplets appeared after 12 h of heat stress and the percentage of melanomacrophage centres significantly changed after 48 h at both stress conditions. Furthermore, the mitochondrial ROS levels were elevated in a time-dependent manner up to 6 h and 12 h in the cold and heat stress groups, respectively. The activities of superoxide dismutase, glutathione peroxidase and catalase were successively increased with increasing periods of cold or heat exposure, and their gene expression levels showed similar changes in both stress conditions. Most tested heat shock protein (HSP) genes were sensitive to temperature exposure, and the expression profiles of most apoptosis-related genes was significantly upregulated at 3 and 48 h under cold and heat stress, respectively. Apoptotic index at 48 h under cold stress was significantly higher than that under heat stress. Notably, lipid droplets, HSP30, HSP70 and HSP110 might be suitable bioindicators of heat stress. The results of these alterations at physiological, biochemical and molecular levels might contribute to a better understanding of the stress response of Q. spinosa, and perhaps amphibians more generally, under thermal stress.

Identification and functional analysis of immune deficiency (IMD) from Scylla paramamosain: The first evidence of IMD signaling pathway involved in immune defense against bacterial infection in crab species.

Immune deficiency (IMD) pathway, one of the most essential pattern recognition receptor signaling pathways, plays vital roles in innate immune responses to eliminate pathogen infection in invertebrates. In the present study, an immune deficiency (IMD) gene and two NF-κB family members, Relish and Dorsal, were identified and characterized in mud crab Scylla paramamosain for the first time. The deduced SpIMD, SpRelish and SpDorsal protein contained conserved death domain and classical NF-κB domains, respectively. Phylogenetic analysis suggested that SpIMD was classified into the invertebrate IMD branch, and SpRelish could be classified into the type I NF-κB class while SpDorsal could be grouped into the type II NF-κB class. Tissue distribution results showed these three genes were ubiquitously expressed in all tested tissues. The expression patterns of IMD signaling pathway and NF-κB genes, including SpIMD, SpIKKß, SpIKKε, SpRelish and SpDorsal, were distinct when crabs were stimulated with Vibro alginolyticus, indicating that they might be involved in responding to bacterial infection. When SpIMD was silenced by in vivo RNA interference assay, the expression levels of IMD pathway and antimicrobial peptides (AMPs) genes, including SpIKKß, SpRelish, SpALF1-6 and SpCrustin, were significantly down-regulated (p < 0.05). Correspondingly, the bacteria clearance ability of hemolymph was extremely impaired in IMD silenced crabs. Overall, the IMD played vital roles in innate immune response by regulating the expressions of its down-stream signaling genes and AMPs in S. paramamosain. These findings might pave the way for a better understanding of innate immune system and establish a fundamental network for the IMD signaling pathway in crustaceans.

Effects of heat stress on the liver of the Chinese giant salamander Andrias davidianus: Histopathological changes and expression characterization of Nrf2-mediated antioxidant pathway genes.

Nuclear factor E2-related factor 2 (Nrf2) is a crucial transcription factor that regulates the basal and inducible expression of many antioxidant-relevant genes, and the Nrf2-mediated antioxidant pathway has been regarded as a critical switch in the initiation of cellular defence systems against oxidative damages. In this study, Nrf2 was first identified and characterized in the Chinese giant salamander (Andrias davidianus). A. davidianus was exposed to a high ambient temperature of 30 °C for various periods of time (0, 3, 6, 12, 24, 48 and 72 h). We investigated the effects of heat stress on alterations of the hepatic malondialdehyde (MDA) concentration, the activities of lactic acid dehydrogenase (LDH), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), the histology of the liver, and the mRNA expression patterns of 11 genes involved in the Nrf2-mediated antioxidant pathway in A. davidianus. The results showed that both the hepatic LDH activity and MDA content significantly increased after heat exposure, indicating that heat stress could induce cell injury and oxidative damage. Histological analysis of the liver showed that heat stress caused hepatocyte abnormalities, fat accumulation and ultrastructural alterations of the hepatocytes, endoplasmic reticulum and nuclei. The expression patterns of genes involved in the Nrf2-mediated antioxidant pathway in the liver were distinct when A. davidianus was exposed to heat stress. To the best of our knowledge, this study is the first on the characterization of Nrf2 in A. davidianus and even in amphibians. The results indicated that heat stress could induce oxidative damage, and the Nrf2 antioxidant pathway might play a critical role in the resistance against heat stress in A. davidianus. These findings will deepen and enrich the current knowledge on the evolutionary conserved antioxidant roles and mechanisms of Nrf2 in A. davidianus, or even in amphibians, in the antioxidant defence against heat stress.

Metal-free catalytic hydrocarboxylation of hexafluorobut-2-yne.

An efficient method for stereoselective synthesis of trifluorinated enol esters catalyzed by base was introduced. The DFT calculations and experimental results both supported the nucleophilic addition process. The protocol featured mild reaction conditions and showed a wide functional group tolerance. The one-pot simultaneous etherification and esterification of the salicylic acids further demonstrated the prospective synthetic application.

Thin-Film Composite Membrane Prepared by Interfacial Polymerization on the Integrated ZIF-L Nanosheets Interface for Pervaporation Dehydration.

Thin-film composite (TFC) membranes are attracting wide attention because their ultrathin selective layer usually corresponds to the higher membrane flux for pervaporation. However, the direct preparation of the TFC membranes on ceramic substrates confronted with the great difficulties because the larger pores on ceramic substrate surfaces are detrimental to the formation of an intact polyamide (PA) selective layer produced by interfacial polymerization (IP) reaction. Here, the integrated ZIF-L nanosheets were proposed to be used as an assistance interlayer for the first time to eliminate the existence of the pores of the ceramic support, and provides a better basis for the formation of an intact PA selective layer by IP reaction between TMC and ethylenediamine (EDA). The experimental data obtained in pervaporation (PV) show that the increased flux from 1.1 to 2.9 kg/m2h corresponds to the decreased separation factor from 396 to 110 when the feed concentration of ethanol decreases from 95 wt % to 80 wt % at 50 °C. In addition, the membrane flux increases from 0.8 to 2.5 kg/m2h with a change of the separation factor from 683 to 111 when the operational temperature varies from 30 to 60 °C. These results demonstrate the great potential of the fabricated TFC membranes in practical application for PV dehydration of organic solutions.

Functional differences of three CXCL10 homologues in the giant spiny frog Quasipaa spinosa.

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in acting as a bridge between the innate and adaptive immune responses. In this study, we identified three CXC motif chemokine 10 (CXCL10) homologues (QsCXCL10-1, QsCXCL10-2 and QsCXCL10-3) from giant spiny frog Quasipaa spinosa. All three deduced QsCXCL10 proteins contained four conserved cysteine residues as found in other known CXC chemokines. Phylogenetic analysis showed that QsCXCL10-1, 2, 3 and other CXCL10s in amphibian were grouped together to form a separate clade. These three QsCXCL10s were highly expressed in spleen and blood. Upon infection with Staphylococcus aureus or Aeromonas hydrophila, the expressions of QsCXCL10s were markedly increased in spleen and blood during biotic stresses. Meanwhile, the QsCXCL10s transcription in liver could also be up-regulated under abiotic stresses such as cold and heat stresses. The recombinant proteins of frog CXCL10 homologues were produced and purified in E. coli and possessed similar but differential bioactivities. Both rCXCL10-1 and rCXCL10-2 had strong effects on the up-regulation of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-8) in vivo, whereas rCXCL10-3 induced a weak expression of these cytokines. Moreover, the rCXCL10-1 and rCXCL10-2 could strongly promote splenocyte proliferation and induce lymphocytes migration, while rCXCL10-3 had limited effects on these biological processes. All three frog chemokines triggered their functional activities by engaging CXC motif chemokine receptor 3 (CXCR3). Taken together, these results revealed that the three QsCXCL10s had similar but differential functional activities in mediating immune responses and host defenses, which might contribute to a better understanding of the functional evolution of CXCL10 in vertebrates.

The first evidence of four transcripts from two Interleukin 18 genes in animal and their involvement in immune responses in the largest amphibian Andrias davidianus.

Interleukin 18 (IL-18), a member of IL-1 cytokine superfamily, is an important proinflammatory cytokine with multiple functions in both innate immunity and acquired immunity. However, the characteristics and functional roles of IL-18 remain largely unknown in amphibians, which were classed as major group of vertebrates. In the present study, two IL-18 genes (AdIL-18A and AdIL-18B) and four transcripts (AdIL-18A1, AdIL-18A2, AdIL-18B1 and AdIL-18B2) were firstly identified and characterized from Chinese giant salamander (Andrias davidianus). To the best of our knowledge, this is the first report on the presence of more than one gene copy or two transcripts of IL-18 in one species. The complete open reading frames of AdIL-18A1, AdIL-18A2, AdIL-18B1 and AdIL-18B2 were 588 bp, 603 bp, 591 bp and 606 bp, respectively. The putative AdIL-18 proteins possessed the typical IL-1 domains and phylogenetic analysis indicated that AdIL-18s grouped together with other vertebrate IL-18 proteins. The expression profiles of AdIL-18s were investigated under the challenges of Aeromonas hydrophila, Staphylococcus ureae and Poly (I:C) respectively, and the results suggested that AdIL-18s were involved in the immune responses against both bacterial and viral infections. Moreover, the expression levels of two NF-κBs (P100 and P105) and four proinflammatory cytokines (IL-1ß, IL-6, TNF-α and IFN-γ) were inhibited in AdIL-18A1/A2-silenced cells when treated with bacteria and viral RNA analog. Additionally, the transcription levels of these immune-related cytokine genes were markedly induced when the lymphocytes were treated with recombinant AdIL-18A1 or AdIL-18A2 proteins, implying the involvement of AdIL-18s in triggering NF-κB signaling and proinflammatory responses. These results might provide new insights into the origin or evolution of IL-18 in amphibians and even in vertebrates.

The nuclear factor interleukin 3-regulated (NFIL3) transcription factor involved in innate immunity by activating NF-κB pathway in mud crab Scylla paramamosain.

NFIL3 is a transcriptional activator of the IL-3 promoter in T cells. In vertebrates, it has been characterized as an essential regulator of several cellular processes such as immunity response, apoptosis and NK cells maturation. However, the identification and functional characterization of NFIL3 still remains unclear in arthropods. In this study, the NFIL3 homologue was firstly cloned and characterized in mud crab Scylla paramamosain. The full-length of SpNFIL3 was 2, 041 bp in length with an open reading frame of 1, 509 bp, containing a conserved basic region of leucin zipper domain. The qRT-PCR analysis indicated that SpNFIL3 was significantly highly expressed in hepatopancreas and in hemocytes. Moreover, the SpNFIL3 transcription could be up-regulated after the challenge of Vibrio alginolyticus or virus-analog Poly (I:C). The dual-luciferase reporter assays revealed that SpNFIL3 could activate NF-κB pathway. The immunofluorescence assay indicated SpNFIL3 was located in nucleus. After NFIL3 was interfered in vivo and in vitro, the expressions of two NF-κB members (SpRelish and SpDorsal), six antimicrobial peptide genes (SpCrustin and SpALF2-6) and pro-inflammatory cytokine SpIL-16 were suppressed, and the bacteria clearance capacity of crabs was also markedly impaired in NFIL3 silenced crabs. These results indicated that SpNFIL3 played crucial role in the innate immunity of S. paramamosain and it also brought new insight into the origin and evolution of NFIL3 in arthropods and even in invertebrates.

The Activin-like ligand Dawdle regulates innate immune responses through modulating NF-κB signaling in mud crab Scylla paramamosain.

Activins, members of transforming growth factor ß (TGF-ß) superfamily, are pleiotropic cytokines with critical roles in mediating cell proliferation, differentiation, homeostasis, apoptosis and immune response. However, the structural characteristics and specific functions of Activins remain largely unknown in invertebrates. In the present study, an Activin-like ligand Dawdle (Daw) was firstly identified and characterized from mud crab Scylla paramamosain. The obtained cDNA sequence of SpDaw was 2, 196 bp long with a 1, 149 bp open reading fame, which encoded a putative protein of 382 amino acids. The putative SpDaw protein contained a signal peptide, a TGF-ß propeptide region and a TGF-ß domain. Real-time PCR analysis demonstrated that SpDaw was predominantly expressed at early embryonic development stage and premolt stages, implying its participation in development and growth. Furthermore, SpDaw responded to both Vibro alginolyticus and Poly (I:C) challenges, suggesting the involvement of SpDaw in innate immune responses. Knockdown of SpDaw in vivo dramatically increased the expressions of NF-κB signaling genes and anti-lipopolysaccharide factor (ALF) genes, and the bacteria clearance efficiency was also markedly enhanced in SpDaw-silenced crabs. Moreover, the in vitro experiment further demonstrated that recombinant SpDaw protein could block the increased transcription of IKKs, NF-κBs and ALFs induced by pathogen challenges. Taken together, these results indicated that SpDaw not only participated in development and growth processes but also played an immune-regulatory role in crabs' innate immunity, which may pave the way for a better understanding of TGF-ß superfamily members in crustacean species.

Identification and functional analysis of transforming growth factor-ß type I receptor (TßR1) from Scylla paramamosain: The first evidence of TßR1 involved in development and innate immunity in crustaceans.

The transforming growth factor-ß (TGF-ß) receptor-mediated TGF-ß signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-ß receptor (TßR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTßR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTßR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTßR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTßR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTßR1 was silenced. Additionally, the expression levels of SpTßR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TßR1 antagonist or agonist for 24 h. These results indicated that TßR1 not only contributed to the crabs' development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Effect of cadmium exposure on hepatopancreas and gills of the estuary mud crab (Scylla paramamosain): Histopathological changes and expression characterization of stress response genes.

Cadmium (Cd) is a heavy metal that accumulates easily in organisms and causes several detrimental effects, including tissue damage. Cd contamination from anthropogenic terrestrial sources flows into rivers, and through estuaries to the ocean. To evaluate the toxic effects of Cd on estuary crustaceans, we exposed the mud crab Scylla paramamosain to various Cd concentrations (0, 10.0, 20.0, and 40.0mg/L) for 24h. We also exposed mud crabs to a fixed Cd concentration (20.0mg/L) for various periods of time (0, 6, 12, 24, 48, and 72h). We observed that after exposure to Cd, the surfaces of the gill lamellae were wrinkled, and the morphologies of the nuclei and mitochondria in the hepatopancreas were altered. We analyzed the expression profiles of 36 stress-related genes after Cd exposure, including those encoding metallothioneins, heat shock proteins, apoptosis-related proteins, and antioxidant proteins, with quantitative reverse transcription PCR. We found that exposure to Cd altered gene expression, and that some genes might be suitable bioindicators of Cd stress. Gene expression profiles were organ-, duration-, and concentration-dependent, suggesting that stress-response genes might be involved in an innate defense system for handling heavy metal exposure. To the best of our knowledge, this study is the first one of histopathology and stress-response gene expression pattern of Scylla paramamosain after Cd exposure. Our work could increase our understanding of the effect of environmental toxins on estuary crustaceans.

Identification and characterization of six peroxiredoxin transcripts from mud crab Scylla paramamosain: The first evidence of peroxiredoxin gene family in crustacean and their expression profiles under biotic and abiotic stresses.

The peroxiredoxins (Prxs) define a novel and evolutionarily conserved superfamily of peroxidases able to protect cells from oxidative damage by catalyzing the reduction of a wide range of cellular peroxides. Prxs have been identified in prokaryotes as well as in eukaryotes, however, the composition and number of Prxs family members vary in different species. In this study, six Prxs were firstly identified from the mud crab Scylla paramamosain by RT-PCR and RACE methods. Six SpPrxs can be subdivided into three classes: (a) three typical 2-Cys enzymes denominated as Prx1/2, 3, 4, (b) two atypical 2-Cys enzymes known as Prx5-1 and Prx5-2, and (c) a 1-Cys isoform named Prx6. The evolutionarily conserved signatures of peroxiredoxin catalytic center were identified in all six SpPrxs. Phylogenetic analysis revealed that SpPrx3, SpPrx4, SpPrx5s and SpPrx6 were clearly classified into Prx3-6 subclasses, respectively. Although SpPrx1/2 could not be grouped into any known Prx subclasses, SpPrx1/2 clustered together with other arthropods Prx1 or unclassified Prx and could be classified into the typical 2-Cys class. The comparative and evolutionary analysis of the Prx gene family in invertebrates and vertebrates were also conducted for the first time. Tissue-specific expression analysis revealed that these six SpPrxs were expressed in different transcription patterns while the highest expression levels were almost all in the hepatopancreas. Quantitative RT-PCR analysis exhibited that the gene expression profiles of six SpPrxs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and hypoosmotic salinity, suggesting that the SpPrxs might play different roles in response to various stresses. The recombinant proteins including the SpPrx1/2, SpPrx4, SpPrx5-1 and SpPrx6 were purified and the peroxidase activity assays indicated that all these proteins can reduce H2O2 in a typical DTT-dependent manner. To our knowledge, this is the first study about the comprehensive characterization of Prx gene family in Scylla paramamosain and even in crustaceans. These results would broaden the current knowledge of the whole Prx family as well as be helpful to understand and clarify the evolutionary pattern of Prx family in invertebrate and vertebrate taxa.

Dynamic expression of Runx2, Osterix and AJ18 in the femoral head of steroid-induced osteonecrosis in rats.

OBJECTIVE: To study dynamic changes in gene expression and protein synthesis of runt-related transcription factor-2 (Runx2), Osterix and AJ18 in the femoral head of steroid-induced osteonecrosis in rats. METHODS: Forty mature Wistar rats, 250-270 g (mean, 260 g) in weight, were randomly divided into model (30 rats) and control groups (10 rats). An early rat model of femoral head necrosis (FHN) was created by weekly injections of dexamethasone (20 mg/kg) into alternate sides of the gluteus maximus and twice-weekly training on a laboratory animal treadmill for 8 weeks. Hematoxylin and eosin (HE) staining was used to determine whether osteonecrosis had been successfully induced, and the model was then divided into equal 8, 10 and 12 week groups. At the end of the appropriate time period, total RNA and total protein were extracted from the femoral heads, and then real-time quantitative polymerase chain reaction and Western blot were performed to detect dynamic changes in the expression of Runx2, Osterix and AJ18 and protein synthesis in femoral heads with steroid-induced osteonecrosis in rats. RESULTS: At the post-modeling eighth, tenth and twelfth week, expression of Runx2 mRNA, Osterix mRNA and related protein synthesis were significantly down-regulated compared to that of the control group, which showed a downward trend with time; while expression of AJ18 mRNA and protein synthesis in the model group was much higher than in the control group, which showed an upward trend with time. CONCLUSION: Glucocorticoids may induce femoral head osteonecrosis by down-regulating Runx2/Osterix mRNA and up-regulating AJ18 mRNA.

[Fermentation and purification of recombinant alpha-galactosidase from Pichia pastoris].

In order to obtain an adequate supply of alpha-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean a-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30 degrees C, 250- 300r/min until a maximum optical density at 600nm (OD600) between 2.0 to 6.0 was attained. Entire 400 mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30 degrees C, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000 x g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20 mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1 mol/L NaCl. The fractions containing recombinant a-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified alpha-galactosidase at 26 degrees C for 2 4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the alpha-galactosidase activity against artificial substrate PNPG (PNP-alpha-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target alpha-galactosidase was eluted with 40% 1mol/L NaCl and showed a 41kD band on SDS-PAGE. After concentration, the final recovery was about 41%. The Michaelis constant of the recombinant alpha-galactosidase was determined as 0.275 mmol/L, which slightly lower than the nature enzyme and suggested a higher affinity with specific substrate. When human blood type B erythrocytes pretreated with 100u/mL recombinant alpha-galactosidase reacted with bood type B antiserum, no hemagglutination occurred. This suggested that the B antigens had been removed by the enzyme successfully. These results demonstrated that the recombinant alpha-galactosidase could be produced in largescale and made it possible to explore the application of alpha-galactosidase in more fields.