Abstract:On the basis of the recent reports, immediate early gene (IEG) can be rapidly induced and expressed when a songbird is either stimulated by bird-song or in its vocal-behavior. The expression area and level in brain of IEG, such as zenk, c-fos and c-jun, are corresponding to where the neurons are related, as a songbird is stimulated, suggesting that IEG plays an important role in vocal learning and memory.

Abstract:Apoptosis-inducing factor(AIF), whose gene lies in X-chromosome, is likely an apoptogenic effector protein to mediates nuclear apoptosis directly. Once synthesized in the cytoplasm, the mouse AIF-precursor preprotein can be effectively imported into the mitochondrial intermembrane space through its N-terminal mitochondrial localization sequence (MLS). Then the MLS is cut off at position Gly 102 of the full-length preprotein, and the remains is refolded and bound with FAD group to produce the apoptogenic mature AIF, with a relative molecular mass of 57 ku. When death stimuli present, AIF is released from mitochondria to the cytoplasm and then to the nucleus, inducing peripheral chromatin condensation and large-scale fragmentation of DNA (～50 kb). These effects cannot be prevented either by the presence of wide-spectrum caspase inhibitor z-VAD.fmkor by the overexpression of Bcl-2. The evidences of gene knockout experiments indicate that AIF is essential for the cavitation of embryoid bodies during mouse morphogenesis, and the cavitation can be caused by AIF itself, independent on caspase-3 activity. Therefore, AIF-mediated cell death maybe constitutes a caspases-independent, more ancient and conserved apoptosis pathway.

Abstract:In the view of molecular biology on morphogeny of a three-dimensinal embryo, morphogenesis and development are dependent upon activation of transcription of involving genes followed by the inactivation in a specific part of embryo at a certain time. It has been demonstrated that cis-acting elements of genes can act to provide sharply defined patterns. Simultaneously, it is accepted as an important mechanism that both activation and repression of transcription factors are closely related to cell differentiation and morphogenesis.

Abstract:The discovery of DMT1 (divalent metal transporter 1, also named as Nramp2-nature resistance-associated macrophage protein 2, or DCT1-Divalent cation transporter 1) is the most important breakthrough in the field of mammalian iron metabolism in recent years. It was first identified on the basis of its homology to Nramp1 in 1995. In 1997, two groups independently identified DMT1 as the first mammalian transmembrane iron transporter. Since then, considerable research effort has been devoted to studying this newly discovered protein and the understanding of DMT1 has been greatly imporved. The current knowledge of DMT1 is summarized, including its distribution, structure and different splice forms, expression regulation, physiological function and relationship between the disruption of its expression and the pathogenesis of some diseases.

Abstract:Nuclear import and export of signaling molecules is an important step in the signal transduction pathways of cytokines and growth factors. Nuclear localization sequence(NLS) is the specific amino acid sequence on the signaling proteins to be sufficient for their nuclear translocation. In addition, nuclear pore complex(NPC),nuclear transport protein-importin and the energy-providing GTPase-Ran/TC4 are also involved in the nuclear import of the signaling molecules. NLSs are also widely located on the cytokines, growth factors or their receptors, and these ligands or receptors may act as chaperon molecules to assist nuclear translocation of other intracelluler signaling proteins through the action of NLS.

Abstract:By binding cyclin D1, functional p16^INK4a contributes to the maintenance of the pRb in its unphosphorylated or hypophosphorylated state, which in turn inhibits cell cycle progression. The expression of p16^INK4a is up-regulated by Ets and down-regulated by Bmi-1. Inactivation of p16^INK4a by deletion, mutation, methylation and aberrant splicing can lead to unlimited cell cycle progression and tumorization. Reasonably, p16^INK4a is now selected to treat some kind of tumors, but research in this field remains a long way to go.

Abstract:Tissue acidosis is a general phenomenon under physiological and pathological conditions. Through acid-sensing ion channels (ASICs), neuron can detect the drops in extracellular pH. ASICs are one of members of DEG/ENaC family. To date, six subunits of ASICs family have been identified. They are widely expressed in the peripheral and central nervous system. The homomers and heteromers channels of these subunits exhibit a variety of electrophysiological properties. ASICs play a critical role in several modalities of sensation, especially nociception.

Abstract:Nanopore technology is a new method developed in recent years to directly decipher genetic information of nucleic acids. It can sequence more than 1 000 bases per second by converting strings of nucleotides directly into electronic signatures. Nanopore sequencing is more quickly, easily and cost-effectively than existing technologies. Besides prospects for nucleic acids ultrarapid sequencing, this technology can also be used for gene diagnosis of pathogen, detection of single nucletide polymorphisms, rapid, stimultaneous multianalyte detection and other areas.

Abstract:Nuclear transfer from somatic cells provides a wide range of opportunities, both in basic and applied research. However, the efficiency of the nuclear transfer procedure remains low. The fundamental reasons are that the basic mechanisms of nuclear transfer are unclear. The research work in the past on these questions have shown that two groups of factors have multiple effects on the reconstructed embryos in nuclear transfer. The first are those involved in maintaining ploidy of the reconstructed embryos and the second are in reprogramming the donor nuclei. Some of the cell cycle interactions between the donor nucleus and recipient cytoplasm are reviewed.

Abstract:The molecular recognition of macrocyclic polyamines (MP-1,MP-2 and MP-3) to RNA, and its effects on apoptosis of cos-7 cells were studied in order to explore their mechanism of anti-HIV-1 activity. Cleavage of RNA was observed by agarose electrophoresis; and apoptosis was determined by flow cytometry assay. Computer modeling was used to investigate the theoretical possibility of compounds binding to TAR RNA. Results showed that: (1) Compounds MP-1,MP-2 and MP-3 could not only cleave the polyA·polyU and TAR RNA, but also inhibit the interaction of Tat-RNA. (2) Compounds could affect the percentage of hypodiploid cell. It is proposed that compounds MP-1,MP-2 and MP-3 could recognize the polyA·polyU and TAR RNA molecules and cleave them, and affect the interaction of Tat-RNA. The compounds may affect the apoptosis of cos-7 cells.

Abstract:Both cellular cholesterol metabolism imbalance and apoptosis are related to the development of atherosclerosis. To investigate the relationship between the cellular cholesterol metabolism and apoptosis, the porcine aortic smooth muscle cells were cultured with medium 199 containing 15 mg/L oxidized low density lipoprotein (Ox-LDL) for 72 h, the ratio of cellular cholesteryl ester to total cholesterol increased from 26.2% to 64.1%, and Ox-LDL induced accumulation of cellular cholesteryl ester in a concentration dependent manner in the cells. It indicated that the vascular smooth muscle cells had transformed to foam cells. In addition, cells incubated with oxidized low density lipoprotein had characteristic of apoptosis, as determined by fluorescence microscope, laser scanning confocal microscope and flow cytometry. From this findings, it was speculated that the induction of apoptosis may be related to the raise of the ratio of cellular cholesteryl ester to total cholesterol besides oxidation of low density lipoprotein in vascular smooth muscle cells.

Abstract:In order to further probe the function of NDR2 gene, the expression of NDR2 gene was determined in different human tissues and their relevant tumors. Tissues of human brain and glioma, lung and lung cancer, colon and carcinoma of colon, stomach and carcinoma of stomach were collected for total RNA extraction and paraffin-embedment. Immunohistochemistry and RT-PCR were used to examine the expression of NDR2 both in mRNA and protein level. DNA sequencing was performed to confirm the result of RT-PCR. Immunohistochemistry results showed that NDR2 protein was extensively expressed in the tissues. RT-PCR further showed an extensive expression of NDR2 mRNA, with a higher expression level in normal brain and lung tissue than glioma and lung cancer respectively, while no differences were observed between colon and carcinoma of colon, stomach and carcinoma of stomach. These results suggest a possible involvement of NDR2 in the genesis and progression of glioma and lung cancer, which provide a clue for further studies on NDR2.

Abstract:In order to investigate the possible role of mesenchyme forkhead-1 (MFH-1) in osteogenesis and osteoblast differentiation, the gene-recombination and hybridization methods are used to produce anti-mouse MFH-1 monoclonal antibody. The expression of MFH-1 induced by bone morphogenetic protein-2 (BMP-2) in myoblasts C2C12 was examined by Western blot and Northern blot analysis. The alkaline phosphatase (ALP) activity and osteocalcin were used as the markers of the osteoblasts lineage, and also were measured. The results showed that the anti-mouse MFH-1 monoclonal antibody was able to identify specifically the mouse MFH-1 protein which was expressed in human bladder carcinoma HTB 9 cell transfected with CX-MFH-1 plasmid by Western blot analysis. The myoblasts C2C12 could express the endogenous MFH-1 protein in its nucleus. MFH-1 protein and MFH-1 mRNA both increased markedly in C2C12 cells after treatment with BMP-2; after lowering the endogenous MFH-1 level by stably transfecting C2C12 cells with antisense MFH-1 sequence, the alkaline phosphatase(ALP) activity and production of osteocalcin induced by BMP-2 were significantly lowered in antisense MFH-1 cell lines than in control cell lines. It can be concluded that the results suggest that the BMP-2-induced MFH-1 protein may play an essential role in regulating the osteoblastic differentiation of myoblasts C2C12.

Abstract:In order to clone novel gene associated with tumor in human chromosome 7q31-32 harboring one or more tumor suppressor gene, expressed sequence tag(EST)-mediated positional cloning strategy was used and a novel human brain cDNA was identified. The isolated cDNA encodes a polypeptide of 653 amino acids with a theoretical molecular mass of 72.7 ku and a calculated isoelectric point of 6.58. The deduced amino acids contains seven typical leucine-rich repeats(LRRs) flanked by N- and C-terminal cysteine-rich LRR region, one immunoglobulin C2 like domain, one signal peptide at N-terminal and one transmembrane region at C-terminal. Amino acid sequence of this novel gene exhibits high similarity and similar domain organization as many other LRR proteins. Analysis of this novel gene shows no significant homology to any reported genes in database of GenBank. So it is a novel member of leucine-rich repeat superfamily and designated as LRRC4 according to the guide of HUGO Gene Nomenclature Committee(GenBank Accession No.AF196976). Northern Blot and RT-PCR analysis revealed that normal expression of LRRC4 was highly specific for brain，whereas absent or significantly down-regulated in primary brain tumors including glioma, meningioma and pituitary adenoma. In addition, mouse homology of LRRC4 has been mapped to mouse chromosome 6 by similarity analysis(GenBank Accession No.AF290542). Taking the structural properties and expression patterns into consideration, LRRC4 may play an important role in nervous system.

Abstract:The correlation of glycophorin A and band3 was confirmed by purification and identification of hydrophilic peptides of red blood cell transmembrane domain with high performance liquid chromatograghy(HPLC). Glycophorin A gene(410 bp) was amplified from K562 with RT-PCR and subcloned into the yeast BD hybrid vector pGBKT₇ and baculovirus transfer vector pFASTBac. Glycophorin A and band3 C-terminus were co-transformed into the yeast strain AH109 and the interaction was identified by nutritional selection and β-glycotosidase activity detection. The GPA expression product in Sf9 cells was analyzed on Western blot with anti-GPA and anti-human band3 antibodies, and the result showed that they have immunologic cross-link reaction. All the results above confirmed the interaction between band3 and glycophorin A.

Abstract:In order to Screening antitumor drugs tartgeted to IGF1R gene, 9 different 20-mer anti-sensitive oligo-deoxyribonucleic acid (ASODN) were designed according to the mRNA second structure of IGF1R gene and were transfected into tumor cells under various conditions in the presence of lipofectin. Cell growth activity were evaluated by MTT assay. The best sequence with antitumor activity in vitro and in vivo were analyzed. This sequence showed strong anticancer activity in vitro and in vivo and had dose -dependent relation. The sequence had no obvious toxicity on tumor -burdended nude either . IGF1R could be used as an appropriate target for tumor therapay.

Abstract:The NO-induced intracellular free calcium concentration (［Ca²⁺］_i) rise and the inhibiting effect of selenium on this process were determined by using Fura-2/AM fluorescence measurement method in a human umbilical vein endothelial cell line: ECV-304. Experimental results indicated that there was a fast rise of ［Ca²⁺］_i by the treatment of S-nitrosoglutathione(GSNO), a donor of NO. The rise of ［Ca²⁺］_i was inhibited by Hb, a scavenger of NO. These results suggested that this rise of ［Ca²⁺］_i was induced by NO. There was no effect on ［Ca²⁺］_i response to NO by removing calcium ion from bath, or by adding the non-selective calcium channel antagonist, CdCl₂（1 mmol/L）to the bath. When the cells were pretreated by sodium selenite(1 μmol/L),the rise of ［Ca²⁺］_i induced by NO was obviously inhibited. This result suggested that selenium can inhibit calcium store release.

Abstract:The orientation modulation properties of 597 cells in cat dLGN were studied using drifting sinusoidal grating as stimulus, while the feedback project from cortex was blocked through cortical ablation (including area 17, 18, 19 and LS). The average orientation bias of neurons was 0.154 in dLGN of decortiate cats, similar to that of normal cats (0.155). The optimal orientation tended to horizontal in decortiate cats as that in normal cats. However, the cells lossed the tangent distribution of the optimal orientation in decortiate cats. Similar results were observed in cats whose visual cortex were silenced by GABA or KCl. The results suggested that the tangent distribution of the optimal orientation in normal dLGN was generated from the feedback projection from visual cortex.

Abstract:The two-photon excitation microscopy has become an important tool of noninvasive imaging due to the better penetration and relative harmlessness of the longer wavelength. However, the high photon flux in two-photon excitation can potentially lead to higher-order photobleaching within the focal volume. The relationship between the photobleaching rate and the excitation power for rhodamine 123 and rhodamine B in vivo and in vitro were measured. The coincidence of the results in vivo and in vitro demonstrated the correctness of the method. As expected, the photobleaching rate increased near-linearly with the excitation power for one-photon excitation. However, the two-photon photobleaching rate increased with high-order power (≥3.5) of excitation power, indicating the presence of high-order photon interaction in two-photon excitation microscopy. The same results are obtained by photobleaching experiments of the green fluorescence protein. As a consequence, the use of multi-photon excitation microscopy in the study may be limited by increased photobleaching.

Abstract:Fourier spectra of 120 short coding sequences (＜1 200 bp) show that not all coding sequences are characterized by 3-base periodicity. Statistical analysis suggests that whether a coding sequence has 3-base periodicity may be related to the composition and distribution of bases, the usage and the order of the amino acids of the encoded protein as well as the synonymous codon usage. Generally, the content of A+U is higher than that of G+C in non-period-3 sequences, inversely in period-3 sequences. In the three codon positions, the base distribution in the non-periodic-3 sequences is more uniform than in the periodic-3 sequences. The usage biases of the amino acids and the codons in non-period-3 sequences are weaker than that in period-3 sequences. All of these phenomena should be considered sufficiently in predicting the genes and exons of DNA sequences by Fourier analysis method.

Abstract:In order to investigate its alteration in the molecular events and early carcinogenesis mechanism of esophageal epithelium cell in the high incidence area of esophageal cancer, content of DNA，telomerase and multi-gene p53，p16，cyclin D1 expression in esophageal precancerous cell were quantitative detected by flow cytometry with indirect immunofluorescence technique and DNA propidium iodide fluorescence staining methods. The detected results showed the DNA content increased singnificantly and the heteroploid rate was 87.9% in occurred carcinogenesis. The p53 protein accumulated and p16 was deleted in the early carcinogenesis of esophagus. The positive rate of p53 was 100%(5/5) in the cancea group. The telomerase and oncogene cyclin D1 were overexpression in the cancer group and their positive rates were 100%(respectively 6/6,7/7), the results indicate that DNA content and heteroploid rate increased, tumor suppressor gene p16 deleted and p53 protein accumulated while telomerase and cyclin D1 protein overexpressed in the early carcinogenesis of esophageal epithelium. There were multiple molecular events occurred when the esophageal carcinoma happened.

Abstract:In order to investigate the mechanism of the essential role of leukemia inhibitory factor (LIF) in blastocyst implantation, blastocyst was incubated with LIF and its specific antibody. The effect of LIF on matrix metalloproteinases (MMPs) of pre-implantation blastocyst was analysized by RT-PCR and immuno-bloting techniques. The results showed that gene expression and secretion of MMP9 were significantly induced by LIF treatment, after blocking by LIF antibody, those effects were decreased, and the decreasing tendency was weakened with the increase of the incubation time. However the effect of LIF on tissue inhibitor of metalloproteinase1(TIMP1) was not obviously. The results indicate that LIF may play its role by inducing the gene expression and increasing the secretion of MMP9 to facilitate blastocyst implantation.

Abstract:In order to deliver αB-crystallin (αB-C) into cardiomyocytes,the full-length cDNA fragment encoding the human αB-crystallin was cloned into the bacterial expression vector pGEX-MTS containing membrane-translocating sequence(MTS) which could mediate intracellular delivery of peptides and expressed as a fusion protein coupled to glutathione S-transferase(GST).After glutathione affinity chromatography and cleaved from GST by factor Xa,the recombinant MTS-αB-C was separated from GST and factor Xa by anion exchange chromatography.Recombinant MTS-αB-C was characterized by SDS-PAGE and Western immunoblot analysis.The purified MTS-αB-C migrated on SDS-PAGE as a single band to an apparent molecular mass (23 ku) that corresponded to total native αB-C and MTS,and was recognized on Western immunoblot by anti-human αB-crystallin antibody. Both MTS-αB-C and GST- MTS-αB-C displayed chaperone like function by disaggregating the denatured and aggregated actin induced by H₂O₂ treatment in an ATP-containing buffer at 37℃.It was observed under fluorescence microscope that FITC-labeled MTS-αB-C had gone into neonatal rat cardiomyocytes by MTS mediation after the cells were incubated with the MTS-αB-C for 8 hours.

Abstract:The autofluorescence of 5-Hydroxytryptamine (5-HT) loaded rat mucosal mast cells (RBL-2H3 cells) was imaged with multi-photon excitation laser scanning microscope (MPELSM). Multi-photon excitated 5-HT relative visible fluorescence is observed in live cells for the first time, and the generating mechanism of 5-HT relative visible fluorescence is preliminarily studied. The spatial distribution of 5-HT in live cells was imaged at high spatial resolution in this present, which provides a new way to study the correlation between 5-HT spatial distribution and content, and the cellular functional state in live tissue or cells.

Abstract:Based on the experimental data of metallothionein-I (MT-I) tertiary structures, interatomic distance constraints of two characteristic structures (CXC, CXXC sequence pattern and metal-Cys chelate tertiary structure) were constructed. Then the distance geometry method was used to work out a number of possible structures, from which those with remarkably lower target function value were selected by a statistical analysis as the predicted tertiary structure models. The predicted structure of blue crab by this method was similar to the experimentally determined structure showed that this method could be used to MT prediction. The tertiary structure of a metallothionein CAP3 from Colletotrichum gloeosporioides was modeled. The predicted structure is similar to those of its homologous MT proteins in metal-Cys combination, and reasonable in its structure energy as well as in its fold motif.

Abstract:Asymmetric microbial reduction of acetyltrimethylsilane to (－)-1-trimethylsilyl-ethanol with immobilized Saccharomyces cerevisiae cells in water/organic solvent biphase system was studied. The effects of shake speed, the hydrophobicity of organic solvent, volume ratio of water phase to organic phase, pH of aqueous phase and reaction temperature on the initial reaction rate, maximum yield and enantiomeric purity of the product were systematically explored. All the factors mentioned above have important effects on the reaction. n-Hexane is found to be the best organic solvent for the reaction. The optimum shake speed, volume ratio of water phase to organic phase, pH and reaction temperature are 150 r/min, 1/2, 8 and 25～30℃ respectively for the reaction, under which the maximum yield and enantiomeric purity of the product are as high as 96.8% and 95.7%(ee) respectively.

Abstract:In order to isolate and screem tissue-specific genes of human nasopharynx and new tumor suppressor genes of nasopharyngeal carcinoma(NPC),a directional cDNA library from human embryo nasopharyngeal epithelia was constructed by SMART(switching mechanism at 5′ end of RNA transcript) technique. The total RNA and mRNA were separated from primary cultural human embryo nasopharyngeal epithelia and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer(contained sfiⅠB site) while the SMART oligonucleotide (contained sfiⅠA site) was utilized as a template so that the first-strand cDNA could be extended over the 5′end of mRNA. The double-strand cDNA was amplified by LD-PCR(long-distance PCR) with the above two primers and then digested by sfiⅠ(ⅠA & ⅠB) restriction enzyme.After cDNA size fractionation through CHROMASPIN column,the double-strand cDNA was ligated into the sfiⅠ-digested λTripIEx2 vector and then the recombinant DNA was packaged in vitro. The unamplified human embryo nasopharynx cDNA library consists of 1.0×10⁶ independent clones in which the percentage of recombinant clones is about 96%.The titer of the amplified cDNA library is 7.8×10⁹ pfu/ml and the average exogenous inserts of the recombinants is 1.2 kb.The full-length cDNA of NAG4, a candidate tumor suppressor gene related with nasopharyngeal carcinoma, was amplified in the cDNA library by PCR. These results show that the human embryo nasopharynx cDNA library has an excellent quality and lays solid foundation for screening and cloning new tumor suppressor genes of nasopharyngeal carcinoma and tissue-specific genes of human nasopharynx.

Abstract:Pichia pastoris is a system to express a lot of foreign proteins, however the expression levels of transformants vary widely from one to one clone. In situ two-film method is a rapid method based on immunology to screen Pichia pastoris transformants expressing high-level protein, in which yeast colonies are lifted from solid culture onto a cellulose acetate film. Then a nitrocellulose film is used to capture the secreted proteins that pass through the cellulose acetate film. The proteins binding on the nitrocellulose film are stained by immunology methods. Pichia pastoris transformants expressing high-level hFL are abtained by using this method, and the expression level in liquid culture is about 20 mg/L. The result of ELISA shows that the staining intensity is correlated to the expression level in liquid culture. Western blot of culture supernatant shows obvious band at 25 ku, while the control doesn't show any band. The expression quantity increases as the inducing time increases.

Abstract:The probing proteins were immobilized onto the surface of glass slides modified by aldehyde. The target proteins were labeled by either Cy5 or Cy3 for specific cases. A high-precision robot was designed to print protein samples onto glass slides to form the microarrays. The activity and the specific affinity interaction of the proteins were not compromised after fluorescent dye labeling process and covalent immobilization. To detect the antigen, arrays of antibodies attached to the glass substrate was used. Likewise, for the assessment of the property of antibody, antigen-based array was utilized. The concentration of the proteins immobilized onto slides is an important factor for determining the sensitivity in detecting the interaction of antigen-antibody. The interactions of goat-anti-mouse and goat-anti-rabbit antibodies with their corresponding antigens were proven specific as revealed by the results generated from the present protein arrays.

Abstract:A method of using colony PCR products to sequence directly by controlling primer design. Less time and cost than the normal way (without DNA extraction and sequencing prior purification). It can also detect the false insert fragments by electrophoreses of the PCR products. One BAC DNA of the Oryza sativa indica 4^th chromosome has been sequenced by this method, the GenBank Accession number: AL512542.

Abstract:Tryptophan analogs were biosynthetically incorporated into the tryptophan sites of DsbA protein to investigate the spectroscopic characteristics, local environments of tryptophans. This technique by using tryptophan-analog labeling may be of potential application to studying structure-function relationships and protein-protein interactions. 5-hydroxytryptophan labeled DsbA protein presents a fluorescence excitation wavelength at 315 nm and an emission near 340 nm. The chemical shifts of Trp76 and Trp126 in 5-fluorotryptophan labeled DsbA were identified in ¹⁹F-NMR spectra, and change of the chemical shift of Trp76 can reflect the conformational change of DsbA protein during redox reaction. The future work is to use specific fluorescence and ¹⁹F-NMR of labeled DsbA protein to study its redox properties and interaction with substrates.

Abstract:Ubiquitin associated protein 1(UBAP1) is a novel member of UBA domain protein family, located at human chromosome 9p21-22 where loss of heterozygosity frequently occurs in nasopharyngeal carcinoma. Through integrated analysis of public database such as EST, Unigene in silico and direct sequencing of cDNA clone, full-length cDNA sequence of murine homologoue of human UBAP1 gene was identified, which is 2 676 bp long and encodes a putative protein of 441 amino acids with a UBA domain. There is an average of 89% identical residues between mouse and human UBAP1 protein. Digitalized expression analysis based on EST data showed that mouse UBAP1 gene expressed ubiquitiously and strongly in most of mouse normal tissues.

Abstract:Cyanogen bromide has been widely used as an activator to some resins while preparing affinity chromatography column and immobilized enzymes. Nevertheless, it is a hazard poison with a volatilization feature, which should be carefully used. Here, 1,1′-carboxyl-diimidazole is used as an activator to Sepharose CL-6B, in order to immobilize earthworm fibrinlytic enzyme Ⅲ-1(EFE-Ⅲ-1), m-aminophenylboronic acid and to detect glycated D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with a low poison, a high coupling rate and a convenient process.