Abstract:F-box protein is an expanding family of eukaryotic protein characterized by an F-box motif which has specificity of substrate recognition in the ubiquitin-mediated proteolysis. These proteins have been shown to be critical for many physiological process, such as cell-cycle transition, signal transduction, development, and so on.

Abstract:Mitogen-activated protein kinase (MAPK) signaling pathway plays important roles in the meiosis of oocytes. p90^rsk is the best known target of MAPK, which mediates multiple functions of MAPK during oocyte meiotic maturation, including the resumption of meiosis, the MⅠ/MⅡ transition and the sustain of MⅡ arrest. The phosphorylation of p90^rsk is the result of MAPK activation, and the dephosphorylation of p90^rsk is following the inactivation of MAPK at the end of meiosis. The progress of p90^rsk in oocytes is introduced.

Abstract:Sigmoid curve and non-additive activation by multiple regulatory sites are two typical manifestations of transcriptional synergy in eukaryotes. There are three possible mechanisms for synergistic regulation: interaction between activators, cooperative binding of activators to DNA upstream sites and cooperative interaction of site-bound activators with GTM (general transcriptional machinery) components. All these mechanisms involve direct or indirect interactions among multiple activators that bind to upstream regulatory sites. The pre-bound activators can facilitate the binding of free ones through these interactions and this underlines the basis of synergistic activation.

Abstract:Annexin Ⅰ belongs to the annexins protein superfamily that comprises a multigene family of Ca²⁺ and phospholipid binding proteins. Annexins consist of a conserved C-terminal or core domain that confers Ca²⁺-dependent phospholipid binding and an N-terminal domain that is variable in sequences and length and responsible for the specific properties of each annexin. Annexin Ⅰ is one of the structurally related，calcium-dependent，phospholipid-binding proteins that have been implicated in diverse cellular roles, including anti-inflammatory, signal transduction, cell differentiation，membrane aggregation，inhibiting the activity of cytosolic phospholipase A2，calcium channels and interaction with cytoskeletal proteins.

Abstract:In vitro molecular directed evolution, based on the combination of random mutagenesis, recombination and high throughput screening, is a new strategy of improving protein properties. With this technology, the properties of the target proteins, such as specificity, catalytic activity and affinity can be tuned without the knowledge of 3-D structure information and function mechanisms. Recently, the methodology originating from error prone PCR and DNA shuffling has been greatly developed by many new techniques. Here, based on the literature review and the experience of authors, the recent developments have been reviewed.

Abstract:The nuclear matrix is an essential component of the nucleus which is important for the nuclear structural integrity and specific genomic functions. The nuclear matrix is composed of nuclear lamina,internal nuclear skeleton and nuclear pore complex which provides structural support for several processes such as DNA replication,transcription,and RNA splicing and transport. The molecular mechanisms about the morphological and biochemical changes which take place in the cell nucleus during the apoptotic process have escaped clarification for many years.Recently, the studies on the nuclear matrix and apoptosis have made great progress. The biochemical and morphological changes and the apoptotic genes' expression detected in the nuclear matrix during the apoptotic process will be delineated. Particular emphasis will be laid on the proteolysis that some nuclear matrix proteins undergo early during the apoptotic process, which may have important biological significance in researching the molecular mechanisms of apoptosis.

Abstract:Retrotransposons are mobile genetic elements that transpose through reverse transcription of RNA intermediate. Retrotransposons are ubiquitous in plants and play a major role in plant gene and genome evolution. The types and structures of plant retrotransposons were summarized. The retrotransposons were used as genetic tools in plant biology.

Abstract:Cinnamomin is a type Ⅱ ribosome-inactivating protein (RIP) isolated from the seed of camphor tree (Cinnamomum camphora). Recently, a small RIP named as cinphorin was isolated from the seed of camphor tree. The reduced cinphorin exhibited the RNA N-glycosidase activity and inhibitory activity to protein synthesis in vitro like the reduced cinnamomin did. Cinphorin B-chain exhibited the same N-terminal 10 amino acid sequence and molecular mass as the B-chain of cinnamomin. Its A-chain exhibited the same 10 N-terminal amino acid sequence and owned a C-terminal cysteine residue linked to the B-chain, but the molecular mass of cinphorin A-chain was only half of cinnamomin A-chain. RT-PCR and Northern blotting revealed that there was no corresponding mRNA of cinphorin. Cinphorin is probably produced from cinnamomin by protein splicing.

Abstract:Mouse ribosomal protein (Rp) L6 promoter contains several transcription binding sites, in addition to common features shared by other Rp promoters, suggesting that RpL6 promoter might be induced by some extracellular elements. To access this question, Jurkat and K562 cells were cultured with α-IFN, γ-IFN or EPO and looked at RpL6 expression by Northern hybridization as well as reporter assay. Treatment with these cytokines upregulated RpL6 mRNA was detected by Northern hybridization. Reporter vectors with promoter fragments containing different transcription factor binding sites were constructed and transiently transfected to Jurkat and K562 cells cultured with α-IFN, γ-IFN or EPO. Luciferase activity was examined in the cell lysates. The promoter activity of RpL6 was elevated significantly after cytokine treatment. These results suggest that cytokines may increase the transcription level of RpL6 gene via induction of the RpL6 promoter.

Abstract:To examine the application of DNA microarray in drug discovery and development, cluster analysis for genome-wide expression data was tested after Saccharomyces cerevisiae was treated with nine antifungal agents with known and unknown pharmacological mechanisms. The results indicated that antifungal agents with similar action mode were clustered together. Amphotericin B and nystatin, ketoconazole and clotrimazole have been known to have similar antifungal mechanism respectively. Consistent with their known mechanisms, amphotericin B and nystatin were clustered together; also ketoconazole and clotrimazole were clustered together based on their expression patterns. Solasodine, which was known to inhibit the synthesis of ergosterol, had a close clustering position with ketoconazole and clotrimazole group. Analyzing the relationship among the known and unknown drugs using cluster approach, It can be infered that the pharmacological mechanisms of unknown drugs from their clustering positions relating to the known drugs.

Abstract:A novel human gene encoding a protein of 208 amino acids is identified and characterized, which has been offered by HGNC with symbol of C17orf32 and name of chromosome 17 open reading frame 32. The full-length cDNA of 1 679 bp for C17orf32 was cloned through a blast search of public databases following the identification of 1 119 bp cDNA obtained by EST assembly with full robotization of SiClone software (created by Chen RS and Ling LJ, and will be released on their website) in ShenWei Ⅳ-type supercomputer. Structurally, C17orf32 has one calcitonin / CGRP / IAPP family signature from amino acid 16 to 169, one dihydroorotase signature from amino acid 43 to 117, one tyrosine kinase phosphorylation site from amino acid 68 to 75, and one bipartite nuclear localization signal from amino acid 28 to 45. These motifs imply the potential biological importance of this gene. Genomic organization analyses show that C17orf32 gene is comprised of six exons, in the size ranging from 43 to 1 101 bp, and five introns, in the size ranging from 163 to 1 124 bp, and spanning 4.61 kb. All of the exon/intron boundaries are consistent with the GT/AG rule, and consensuses surrounding the splice boundaries are found as well. The C17orf32 gene is located on accession NT-010808.7 in the human chromosome 17, and is only linked with LOC124919, a hypothetical human gene of 889 bp mRNA encoding hypothetical protein XP-058865 of 260 amino acids supported by XM-058865. The sequence of LOC124919 has not been verified experimentally. Furthermore, the full-length ORF of 627 bp cDNA from 31 to 654 bp by RT-PCR from the single-stranded human gastric adenocarcinoma MGC803 cell line are cloned and sequenced, which is fully identical with that of the in silico cloning determined by the nucleotide sequencing. Thus, in silico cloning of C17orf31 gene with GenBank accession number of AY074907 and TPA: BK000260 is identified solely by bioinformatics analyses. The full-length cDNA sequence of 1 679 bp exhibits very good overall homology to that of LOC123722 of 899 bp mRNA, with matching percentage of 99% in 78% of total window and 57% in 57% of total window over the full-length nucleotide and protein, respectively. However, the base G in the No.401 position of LOC123722 cDNA is a redundant insert, which causes a reading frame shift in the translation of an alternative protein. The insert G of LOC123722 is not supported by the experimental clone, and is fully rejected by human EST alignment, and is shown as a redundance by genomic GT/AG organization analysis. C17orf32 gene has 9 putative promoters with possibility of 58%～97%, two TATAs, a stop codon in the upstream of ORF, two PolyA signals and a PolyA tail in the downstream of ORF, and accords with Kozak rule around the translation start of the ORF. Based on the above results, it can be concluded that a complete novel human gene is obtained. The full-length gene sequence exhibits little overall homology to any known protein at either the nucleotide or the amino acid level. The two related proteins, with 31% (in 29% of total window) and 18% (in 18% of total window) identity over the full-length protein, respectively, are hypothetical caenorhabditis elegans protein F09E5.11.p of 221 amino acids and polyphosphate kinase ［the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120］ of 736 amino acids. Taken together, by combining bioinformatics analyses with experimental verification, a novel human gene C17orf32 is successfully cloned, verified by a series of theoretical and experimental evidence. The strategy will be helpful in discovering more novel human genes, even in correcting errors appeared in NCBI GENOME ANNOTATION PROJECT REFSEQs, such as LOC124919, a model reference sequence predicted from NCBI contig NT-010808 by automated computational analysis using gene prediction method. Therefore, human genome coding region annotated by computer should be used with caution.

Abstract:It has been reported that there is a significant increase in PAI-1 expression level in obese subjects. To explore the linkage between PAI-1 gene expression and obesity, the restriction enzymes and DNA recombination technologies were used to construct the chimeric plasmids with luciferase and different lengths of PAI-1 promoter. After transfection of the chimeric plasmids into 3T3-L1 preadipocyte and detection of luciferase activity, the results indicated that a positive dexamethasone cis-acting element （bases －690 to －850） may be present in mouse PAI-1 promoter. In addition, computer analysis using Match-Search Software found that a new motif of DexRE (dexamethasone response element) 5′ GGTAACCTCTGTTCTCAT 3′ and a putative C/EBPs binding site (cis-motif) exist respectively in the fragment (nucleotides －751 to －770) of, and a sequence (bases －720 to －740) of, mouse PAI-1 promoter,and GMSA and competition assays identified that the trans-acting factors induced by dexamethasone can specifically bind to those cis-motifs. Meanwhile, the site-directed mutagenesis by PCR was performed to detect the influence of mutant DexRE and C/EBPs cis-motif on PAI-1 gene expression. Similarly, the chimeric plasmids containing luciferase as a reporter gene and a fragment of mouse PAI-1 promoter comprising the mutant cis-motifs were constructed, and then transfected into 3T3-L1 preadipocytes. The measurement revealed that the luciferase activities were markedly lowered by mutant DexRE and mutant C/EBPs cis-motif compared with their wild counterparts, implicating that the DexRE and C/EBPs cis-motif identified may control the expression of PAI-1 gene in 3T3-L1 adipocyte. The study is very helpful to elucidate a molecular mechanism through which the dexamethasone may regulate the expression of PAI-1 gene in 3T3-L1 adipocyte.

Abstract:To confirm whether Epstein-Barr virus latent membrane protein 1 (LMP1) induced telomerase activity of nasopharyngeal epithelial cells through nuclear factor-kappa B(NFκB), NFκB activity analysis was performed with pNFκB-luc reporter plasmid and telomerase activity was tested by PCR-ELISA in primary nasopharyngeal epithelial cells and Tet-on-LMP1 HNE2 cells and other cellular model. The results showed that LMP1 could induce telomerase activity and the C terminus of LMP1 could promote both of NFκB transactivity and telomerase activity. In addition, phosphorothioate olrgonucleotides of antisense NFκB p65 and dominant negative mutant of IκBα could inhibit telomerase activity that was induced by LMP1. It was implicated that LMP1 could regulate telomerase activity via NFκB in nasopharyngeal epithelial cells.

Abstract:In order to illustrate the mechanism of AP1 signalling pathway mediated by latent membrane protein 1 (LMP1) encoded by EB virus, the expression of phospho-JNK in Tet-on-LMP1-HNE₂ cell line (L7) was determined at different time by Western blotting, JNK activation increased with the induction of LMP1 in a time dependant manner was abserved. The expression of phospho-JNK and AP1 activity were analyzed by Western blotting and reporter gene in nasopharyngeal carcinoma cell lines which stably expressing LMP1 and the three kinds of its mutants containing different mutation in carboxyterminal activating region. The result showed that no difference existed between HNE₂-LMP1 and HNE₂-LMP1ΔCTAR1, but significant difference between HNE₂-LMP1ΔCTAR2 and HNE₂-LMP1, HNE₂-LMP1ΔCTAR1,2. The phospho-JNK expression and AP1 activity of HNE₂-LMP1 cell lines were individually transfected by dominant negative TRAF(TRAF-DN) and dominant negative TRADD(TRADD-DN). The results showed that the transfection of TRAF-DN or TRADD-DN made JNK expression and AP1 activity decreased significantly. The results suggested that the functional domain CTAR2 of LMP1 encoded by EB virus can mediate JNK signalling pathway through cooperation with TRAF/TRADD complex.

Abstract:In order to study the effect of activation of transcription factor AP1 and NF-κB by EB virus encoded latent membrane protein 1 on invasion and metastasis in nasopharyngeal carcinoma(NPC). NPC cell line SUNE-1 and its subclones were used. Transcriptional and DNA binding activity were analyzed by reporter gene assay and electrophoretic mobility shift assay (EMSA) respectively. Western blot was used to determine the protein expression; the potential of metastasis of NPC cell lines was determined by nude mice tumorigenesis. The results showed the positive relationship of AP1 and NF-κB transcriptional activity, DNA binding activity, LMP1 expression, c-Jun-N-terminal kinase(JNK) activity to malignancy in NPC cell lines. Activation of AP1 and NF-κB by LMP1 may involved in invasion and metastasis of NPC.

Abstract:A large naïve phage displayed human single-chain variable fragments antibody (scFv) library and an in vitro immune library were constructed in parallel conditions, based on the PBLs from healthy and sero-negative blood donors, part of which were in vitro immunized by peptide PreS1 conjugated to BSA. After 3 rounds of panning against PreS1, measurement of antibody-antigen reaction revealed: a scFv specific to PreS1 from the immune library was obtained, which affinity (k=10^-7～10^-8 M) was higher than that from the naïve one (k=10^-6～10^-7 M). Sequencing of the two scFv showed they were human antibodies, which may be of interest in therapy of Hepatitis B. This investigation also illustrated that the method of in vitro immunization results in antibody library more satisfied even than the large naïve one.

Abstract:Four kinds of hepatitis B virus（HBV）DNA vaccines were constructed, two of which encode HBV envelope and core fusion gene，and the others encode HBV envelope or core gene. These DNA vaccines were intramuscularly vaccinated into BALB/c mice respectively. The serum antibodies, T lymphocyte proliferative response and cytotoxic T lymphocyte response of mice were detected. The results showed that the fusion gene DNA vaccines induced weaker antibody, but stronger and longer cellular immune responses than single gene DNA vaccines did, which indicated that the envelope and core fusion gene DNA vaccine may be more useful than single gene DNA vaccine for therapy of hepatitis B.

Abstract:By using four methods (histogram, chaos game representation, discrepancy of distance and discrepancy of entropy) at genomic level, the composition of short oligonucleotides and their compositional complexities in three different regions(introns, intergenic DNAs and exons) of genomic DNA from Arabidopsis thaliana, Caenorhabditis elegans and Drosophila melanogaster were studied. It can be concluded that: (1) although the genome sizes and gene numbers are quite different, the compositional complexities of exons are similar, while that of noncoding regions are quite different between eukaryotic genomes. From quantitative perspective, this finding means that the degree of organismal complexity is mainly reflected by noncoding regions, but not by exons; (2) in the same regions of genomic DNAs, composition and compositional complexity are highly similar between chromosomes or contigs within eukaryotic genomes; (3) composition differ remarkably little between introns and intergenic DNAs. This suggests that the effects of transcription, splicing, second structure contribute minimally to the constraints operating on these sequences.

Abstract:A series of pyridinone derivatives were synthesized and examined for inhibition of 5-lipoxgenase. The 6-substituted pyridinone derivatives(2a～2e) exhibited a prominent inhibitory activity on 5-lipoxygenase. Among them, 6-phenylthio-1-phenyl-2-methyl-3-hydroxy-4(1H)-pyridinone (2a) showed the most potent inhibitory activity (IC₅₀=2.52 μmol/L). As removal of substituent at 6- position of pyridinones, the inhibitory abilities of these compounds were almost vanished. The inhibition mechanism of pyridinone derivatives was discussed.

Abstract:The SpltNPV DNA can induce the apoptosis of SL-1 was discovered. Microscopic examination of Spodoptera litura (SL-1) cell transfected with DNA of Spodoptera litura nucleopolyhedrovirus revealed progressive cell blebbing starting at 6 h postinfection and culminating in total cell destruction at 18 h postinfection. The fragmentation of the infected cell nuclei and apoptotic body were observed by stained with the specific fluorescent dye DAPI. Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder. All these supported that the transfected SL-1 cells undergo apoptosis. Neither apoptosis nor polyhedra was observed in the SL-1 infected with the virions of SpltNPV.

Abstract:In order to study the co-expression effect of antisense thrombin receptor (ATR) and p21 genes on preventing restenosis, an adeno-associated virus(AAV) vector carrying the two genes with different promoters was constructed. Simultaneously, rAAV vectors containing ATR or/and p21 gene were constructed respectively. BHK-21 cell lines carried different plasmids were established by lipofectamine transfection and G418 selection. During the process of cell clones selected, double gene showed stronger inhibition to cell proliferation than single gene. The speed of clones formation decreased meanwhile the morphological alteration of cells occurred. Southern blot confirmed that expression vectors containing ATR or/and p21 gene had been steadily integrated into BHK-21 cells. The antisense position of TR gene has been maintained. Semi-quantitative RT-PCR showed that the expression of TR decreased and the expression of p21 increased. Co-expression of double gene was obtained. rAAV virions were packaged through recombinant herpes simplex virus (rHSV-rc/ΔU12) infecting BHK-21 cell lines carried various vectors. The titre of rAAV (particles/ml) determined by dot blot analysis. The particles of ATR and p21 in rAAV/AP double gene vector were 1.02×10¹³ particles/ml and 1.08×10¹³ particles/ml respectively. The particles of single gene rAAV /ATR or rAAV /p21 were 6.54×10¹² particles/ml or 1.06×10¹³ particles/ml.

Abstract:Previously,an EST(expression sequence tag) fragment, W123 (Genbank accession No.AF150631) differentially expressed between gastric cancer and normal tissues, was cloned using differential-display PCR technique. A few homologous EST sequences were captured when processing similarity search in human EST database. To search for additional W123 homologous genes in gastric tissue, a primer for 3′-RACE(rapid amplification of cDNA end) from highly conserved region among the above ESTs was designed and seven EST fragments with poly(A) tail were cloned. Compared with ESTs in GenBank, the seven EST fragments represented novel genes with a common sequence. Northern blot was applied to detect the expression of these genes between gastric cancer and normal tissues. The results suggested that a combination of bioinformatics and RACE technique is a rapid and effective method for seeking for disease related genes in specific tissue.

Abstract:One kind of protein from earthworm（Lumbricus bimastus）with the molecular mass about 30 ku was extracted by SDS-PAGE. Then the N-terminal of the protein was sequenced. The PCR primer was designed according to the N-terminal sequence, and its cDNA fragment was obtained by RT-PCR. The cDNA was cloned into pGEMT-vector and sequenced. The sequence showed that the fragment was 888 bp,with the ORF of 726 bp and the 3′ untranslation terminal area. The ORF encoded a protein of 242 amino acids, so the protein was named as PV₂₄₂.The prediction of protein structure shows that the PV₂₄₂ has two domains，between which is the active sites-His44 and Ser191.At the same time, the pI 4.33 of PV₂₄₂ was detected, and it is a protease in trypsin family of serine protease superfamily. PV₂₄₂ was expressed in pTrxFUS expression system as the fusion protein TrxA-PV₂₄₂ in soluble form, the TrxA was a molecular chaperon which made the expressed protein soluble. The fusion protein was purified by ion exchange chromatography. The purified fusion protein TrxA-PV₂₄₂ has fibrinolytic activity.

Abstract:In order to study whether tacrine and bis(7)-tacrine can prevent cell apoptosis induced by staurosporine in NG108-15 and HeLa cell lines. Phase-contrast and fluorescence microscopes were used to examine cell morphological changes. MTT assay was used to examine if staurosporine impairs cell metabolism. DNA was isolated and electrophoretically separated on 1% agarose gel to observe if there were DNA fragments. Western blot was made to analyse protein levels of anti-apoptotic Bcl-2 and proapoptotic Bax. The results showed that NG108-15 cells treated with 0.1 mol/L staurosporine for 12～24 h exhibited marked cell death and DNA fragmentation. Pre-treatment with 0.1 μmmol/L tacrine provided approximately 40% protective effect and resulted in obvious inhibition or delay DNA fragmentation. Moreover, NG108-15 cells treated with tacrine became elongated and polarized, and showed longer processes than control cells. Pretreatment with 0.1 mmol/L tacrine significantly increased the expression of Bcl-2 protein level and delayed the staurosporine-induced increase of Bax protein expression. However, bis(7)-tacrine did not show any protective effect on the cell impairment induced by staurosporine in NG108-15 cells. In HeLa cells 0.1 μmol/L staurosporine also induced significant cell injury, but pretreatment with tacrine and bis(7)-tacrine did not provide any obvious protective effect against this cell damage. These data suggest that tacrine markedly protect against apoptosis of NG108-15 cells but bis(7)-tacrine did not. Both tacrine and bis(7)-tacrine were not show to inhibit apoptosis of HeLa cells. It appears that the protective effects of tacrine against apoptosis not be mediated through AChE inhibition and has cell selective specifity.

Abstract:After treatment of PC12 cells with various doses of dopamine (DA), the apoptotic cells were observed with TdT-mediated dUTP nick end labelling method (TUNEL) and DNA gel electrophoresis. Meanwhile, Bcl-2, Bax and cytosolic cytochrome c as well as cleaved caspase-3 P₂₀ were detected with Western blotting assay. The results showed that during the process of DA-induced apoptosis, the cleaved caspase-3 P₂₀ and the release of cytochrome c into cytosol were elevated markedly compared with the controls. The protein levels of Bcl-2 were decreased, however, Bax protein was increased significantly. After pretreatment of PC12 cells with cyclosporine A at 1 μmol/L for 24 h, the release of cytochrome c and caspase-3 activation were blocked almost. But it didn't show effect on protein levels of Bcl-2 and Bax. The results suggested that Bcl-2 family and cytochrome c and caspase-3 might be involved in the progression to apoptosis induced by DA, furthermore, the release of cytochrome c from mitochondria might be an essential event.

Abstract:The increasing data show that the soluble form of Flt-1 have the ability to block the biological activity of VEGF due to its high affinity to VEGF and formation of homologous and heterogenous dimmer with transmembrane Flt-1 and KDR. The binding activity of Flt-1 focuses on its Ⅱand Ⅲ extracellular domain. cDNA fragments encoding for signal peptide and flt-1 Ⅰ～Ⅳ extracellular domain were amplified through RT-PCR with a pair of specific primers from human umbilical vein endothelial cells. Then, the sflt-1 gene was subcloned into pcDNA3-EF-1 vector, and the expression unit of EF-1-flt-1 was subcloned into shuttle vector of pAdTrack-CMV, linearized pAdTrack-EF-1-flt-1 was co-transformed into BJ5183 cells with adenoviral genomic plasmid of pAdEasy-1. The identified recombinant DNA was transfected into 293 cells to package adenovirus. From the supernatant and cell lysis, the presence of recombinant adenovirus was proved by PCR, and RT-PCR detection demonstrated the transcription of flt-1 in MGC803 cells infected with Ad-Track-EF-1-flt-1, and immunoprecipitation reveals that soluble Flt-1 can be secreted into the culture supernatant of infected MGC803 cells. These results indicated that tumor cells infected with the prepared recombinant adenovirus vector Ad-Track-EF-1-flt-1 can secrete soluble Flt-1, which will be further used for in vivo antiangiogenesis experiments.

Abstract:BRD7 gene is a good candidate tumor suppression gene associated with NPC. In order to construct prokaryotic expression vector of BRD7 and express BRD7 in E.coli, The coding region with SalⅠ and NotⅠ restriction sites of BRD7 was obtained from pGEM-T Easy/BRD7 plasmid by PCR. PCR product and plasmid PGEX-4T-2 were digested by corresponding restrict endonucleases respectively. The fragments were ligated by T4 DNA ligase to gain recombinant expression vector. Endonuclease digesting and DNA sequencing confirmed that the coding region of BRD7 gene was correctly inserted into the vector. The recombinant plasmid PGEX-4T-2/BRD7 was transferred into competent Jm105 strain. The GST/BRD7 fusion protein was expressed in the bacteria under induction of IPTG. After induction, a new protein band of 90 ku appeared on SDS-PAGE. The result was confirmed by Western blot. The recombinant protein of 90 ku amounted to 28.48% of the total bacterial protein after inducing with IPTG for 4 h at 37℃. It existed not only in supernatant but also in precipitation of broken bacteria. The successes in construction of expression vector of BRD7 and expression of BRD7 in E.coli make it possible to study further on its biological function and antibody preparation.

Abstract:On the basis of conventional enzyme-linked immunosorbent assay (ELISA) and dienzyme substrate recycle, An highly specific and ultrasensitive biochemical assay, named ELISA-dienzyme substrate recycle was developed. The detective range of conventional ELISA for measuring recombinant tau and abnormally phosphorylated tau purified from Alzheimer disease brain was 1ng to 32 ng. It was 0.75pg to 200pg and 0.5pg to 50pg by ELISA-dienzyme substrate recycle. Compared with the conventional method, the sensitivity of the technique established in the present study increased 400 and 1300 times, with a enlarged detective range of 8.5 and 10 times, respectively. It could detect abnormally phosphorylated tau in cerebrospinal fluid of Alzheimer disease and used effectively for the diagnosis of the disease.

Abstract:An optimized method has been established to simultaneously detect multiple antibodies of HIV, TP and HCV in the serum by the way of microarray. Chimeric antigens of HIV, TP and HCV are covalently bound to the surface of glass slide; serum samples are diluted and added to the well of microarray, unbound antibodies are washed away after incubation, and Cy3 labeled second antibody is added to the well followed by incubation and washing. The microarray is scanned by laser confocal scanner, and the resulting images are treated by the software of Imagene (produced by Bio-discover Co.). The data obtained is then treated by the software produced by own; the evaluated result of each sample is finally gained automatically by the Cutoff value of each antigen. 400 normal serum sample are detected and Cutoff value of each antigen are determined. National panels of HIV, TP and HCV had also been detected by this system and the results were compared with those detected by ELISA method. The coordinate ratios of positive and negative samples of HIV panel are both 100%(20/20); these kinds of the coordinate ratios of positive and negative samples of HCV are both 95%(38/40) and the coordinate ratios of positive and negative samples of TP are 100%(10/10) and 100%(20/20) respectively. This result is highly in accord with that of ELISA method.

Abstract:The functional fragment V_H/κ of an anti-tumor antibody AA98 was refolded in vitro by dilution refolding, gel filtration chromatography and urea gradient gel filtration, respectively. Concerning various factors in the renaturing buffer, such as GSH/GSSG ratio, arginine concentration, pH, flow rate, protein concentration in denaturing buffer and linear gradient, a urea gradient gel filtration method for V_H/κ refolding was established. As a result, compared with the traditional dilution refolding and gel filtration, the activity recovery of V_H/κ obtained by urea gradient gel filtration was significantly improved.

Abstract:In order to detect the proteases in the 1 mol/L NaCl extract of plant PSⅡ particles and its 1 mol/L NaCl extract, gelatin-SDS-polyacrylamide gel electrophoresis was used. Six proteases with molecular mass of 34,37,50,54,58 and 68 ku were detected. Influence of reducer and ion strength on the activity of proteases was analyzed preliminary. The new approach is convenient and highly sensitive for separation and detection of proteases at the same time.

Abstract:Microsatellite DNA analysis was performed using ten pairs of goat microsatallite polymorphic DNA primers on two somatic cloned Jining grey goats, donor grey goat, recipient goats and three related control Jining grey goats. The result suggests that the amplification products are remarkably polymorphic in five pairs of goat microsatallite polymorphic DNA primers: SR-CRSP1, SR-CRSP5, SR-CRSP6, SR-CRSP7 and SR-CRSP24.The amplification products were silver stained after running on the 6% SDS-PAGE. The microsatellite DNA fingerprints of the two somatic cloned goats are the same as the donor, but are different from the recipient goat and all the control grey goats. It proves that the genomes of the two somatic cloned goats come from the donor cells.

Abstract:In order to establish a sensitive method for detecting cellular growth of fibroblasts. Three kinds of fibroblasts were used, including normal rat kidney (NRK), human embryo lung fibroblast cell (MRC-5) and normal human fibroblast(NF). In those cells, mitochondrion dehydrogenase together with phenazine methosulfate (PMS) reduces 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-［(phenylamino) carbonyl］-2H-tetrazolium hydroxide (XTT) and a water soluble and light brown formazan product is formed. Cell growth and viability were obtained by measuring the metabolic product and compared with the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. It was shown that three kinds of cells have different metabolic response to XTT. XTT method directly detected water soluble formazan product. Its sensitivity was higher than MTT. These results indicate that it is a simple, rapid, sensitive and stable method. XTT colorimetric method establishes a new detective method for keloid research of traumatic surgery.