Abstract:Epithelial surfaces of the upper alimentary tract and upper respiratory tract are swarming with protein compounds that protect itself from many kinds of damages. These compounds are consisted of innate immune molecules. From the structure and functional predication, the new member is added, plunc family, to the compounds. They are located at a narrow area less than 300 kb of human chromosome 20, including at least eight members. All the members of the family have the BPI domain and a signal peptide at the N-terminal. The sequences are highly conserved in human, mouse and rat, each expressed at the different sites of the airway epithelial, tongue, von Ebner gland and parotid gland. Some of them can be detected in the nasal lavage fluid. They may function to protect epithelial surfaces from pathogenic micro-organism and harmful gases, the wrong expression will lead to host tissues destruction and tumorigenesis.

Abstract:Tat is a HIV-1 transaction transcriptional activator protein and plays a pivotal role in viral replication and in several AIDS-associated pathologies. Its biological properties and functions make it as a good candidate for the development of an anti-AIDS vaccine and/or drug. Strategies designing vaccines and drugs for anti-AIDS include vaccines derived from Tat, extracellular Tat-binding antagonists, inhibitors of Tat-activated intracellular second messengers, anti-Tat antisense, anti-TAR antisense, decoy and antagonists, anti-Tat intracellular intrabody, inhibitors of intracellular Tat cofactors and so on. An effective anti-AIDS therapy will require a multi-targeted approach in which classic antiviral drugs and protease inhibitors are combined with novel extracellular and intracellular Tat antagonists. This approach could prevent the development of drug-resistant HIV strains and decrease the dosage and related toxicity of each single drug and lead to a cure for AIDS-associated pathologies.

Abstract:RNA silencing is a high conserved mechanism in organisms that involves sequence-specific RNA degradation. For virus induced gene silencing (VIGS), it is induced by infecting a plant with a plant virus that has had its genome modified to include a sequence identical to that in RNA transcribed from the host gene to be silenced. Up to now, various VIGS vectors based on RNA virus, DNA virus, satellite virus or DNA satellite have been established, these VIGS vectors could suppress gene expression effectively in many important plants including Arabidopsis, tomato and barley. VIGS vectors have been used to study function of genes involved in the signal transduction of N-mediated or Pto-mediated resistance, anti-virus mechanism, and plant development and metabolism. With the determination of complete genome sequences or expressed sequence tags, VIGS has recently emerged as a powerful method for identification of gene functions in plants.

Abstract:The Cre/lox P site-specific recombinase system, which has two components: Cre recombinase and two 34-bp lox P sites that Cre recognizes, has been widely used in conditional gene knockout/activation to study the structure and functions of gene. In the present study, a cell-permeable fusion protein (His6-NLS-Cre-MTS) containing a 12-amino acid membrane translocation sequence (MTS), a nuclear localization signal (NLS) and an N-terminal His6 affinity tag, was expressed in BL21 strains (e.g., DE3) transformed with pDJHisCre by induction of IPTG. The fusion protein was purified with His-Bond Ni-NTA resin. Its functionality was confirmed in a cell-free recombination assay with a plasmid (e.g., pApoE-SCS-EGFP) containing lox P-flanked gene(s), and in an intracellular recombination system using lox P-flanked STOP cassette-modified BEL-7402 cells, by assaying the expression of enhanced green fluorescent protein (EGFP). This cell-permeable Cre recombinase provides a rapid alternative means of manipulating mammalian gene structure and function in vitro and in vivo. Its advantages and potential uses are discussed.

Abstract:A glucocorticoid receptor (GR) interacting protein, interferon-inducible protein P56, was isolated from the human bone marrow cDNA library by two-hybrid screening in yeast using the GR ligand-binding domain (GR-LBD) as bait. The interaction between GR and P56 and the effect of P56 on GR were investigated. PCR was performed to amplify GR-LBD fragments and it was cloned into the bait vector pGBKT7 to create the plasmid pGBKT7-GR LBD. The plasmid was used as bait to screen a cDNA library constructed in the pACT2 vector. The positive colonies were sequenced. P56 and GR-LBD cDNA fragments were cloned respectively into the vector pGEX-4T-2, pACT2,pCMV-Myc, pCMV-HA for GST pull down, yeast two-hybrid, coimmunoprecipitation analysis and CAT activity assay. 42 positive clones were obtained by yeast two-hybrid, in which one isolated cDNA from the library encoded the COOH-terminal portion of the interferon inducible protein P56 (221～1 642 bp, residues 53～478 amino acids) as shown by DNA sequencing. Yeast two-hybrid, GST pull down and CO-IP assays verified that P56 interacted with GR-LBD. Expression of P56 resulted in dose-dependent decrease in GR-CAT expression when GRα, GRE-driven reporter genes were cotransfected with P56. Thus, the study demonstrates that P56 interacts with GR-LBD in vitro and in vivo, P56 inhibites the GR-mediated transcriptional activity.

Abstract:Human intestinal trefoilfactor (hITF) cDNA has been transferred into protoplasts of Pleurotus ostreatus by electroporation. Integration of the hITF cDNA into the genome Pleurotus ostreatus was confirmed by PCR analysis. Then competitive enzyme linked immunosorbent assay (ELISA) was developed for detection and quantitative analysis of human intestinal trefoil factor. Results show that the highest hITF protein content in mycelia is 2 000～2 250 ng/g, about 1.5% of the total soluble protein. The transgenic Pleurotus ostreatus is proved to have good biological activity of preventing rats from the gastric ulcer induced by ethanol, in which both of polysaccharides and hITF have a direct effect on ulcer tissue.

Abstract:Starburst^TM PAMAM dendrimers are novel polymers with a molecular architecture characterized by regular, dentritic branching with radial symmetry. Having high density of positive charges on their surfaces in physiological condition because of the protonization of amino groups on the surfaces, and complexing with genetic materials on the basis of electrostatic interactions, those Starburst^TM PAMAM dendrimers deliver genes into alive cells.In order to characterize the potential effects of Starburst^TM PAMAM dendrimers as a carrier for DNA transfection, six different types generations of Starburst^TM PAMAM dendrimers were investigated for their capabilities in binding DNA, and the effects on both DNA transfection and maintenance of cell viability was evaluated in vitro. The experiments demonstrated that it was the full generations but not the half generations of Starburst^TM PAMAM dendrimer could transfect eukaryotic cells efficiently. The dendrimer/DNA complexes were very steady, no dissociation of the complexes was detectable in a large scope of pH(2～10). The complexation of Starburst^TM PAMAM dendrimer and DNA prevent the reaction that endonuclease dissociates the DNA. In a certain range of dendrimers to DNA charge ratios, the Starburst^TM PAMAM dendrimer with higher generations showed much better transfection efficiency than those with lower generations. The transfection efficiency was also variable in different cell lines. Starburst^TM PAMAM dendrimers complexing with DNA have no or very low cytotoxicity at the concentrations effective for DNA transfection (≤1.3×10^-1 g/L). However, the cytotoxicity of Starburst^TM PAMAM dendrimers without binding DNA could be detected at a lower concentration. The results demonstrated that Starburst^TM PAMAM dendrimers,as a novel type of low toxicity, non-viral DNA delivery vehicle,had promising potential to mediate DNA transfection in vitro. It provide primary experimental basis for the application of the nanometer material-Starburst^TM PAMAM dendrimers in vivo as DNA delivery carrier.

Abstract:In order to clone and express human cardiac troponin I-C fusion protein in application to the quality control for cTnI detection system, human cardiac troponin I and troponin C cDNAs were amplified from human cardiac using gene-specific primers designed from the published cDNA sequences by the polymerase chain reaction. The full-length of cTnI was linked with TnC by a short DNA sequence coding for 19 neutral amino acid residues. An expression construct for cTnI-C was engineered by inserting the corresponding cDNA into a pET15b plasmid. Then recombinant plasmid was transformed into E.coli BL21(DE3)pLysS cells, and protein expression was induced by isopropyl-β-D-thiogalactopyranoside（IPTG）. Soluable expression of cTnI-C in prokaryotic system was successfully obtained. Fusion protein had an N-terminal His-tag sequence which could be purified by affinity chromatography on a Ni²⁺-Sepharose column. After one step affinity chromatography the fusion protein shows homogeneity as judged by SDS-PAGE. The fusion protein was stable and easy to be purified,could be used as candidate reference material for cTnI detection systems.

Abstract:Mip gene of Legionella pneumophila and ctxB gene of Vibrio cholerae were PCR amplified respectively. The amplified fusion DNA was ligated to pcDNA3.1(+) vector. The recombinant plasmid, which was named pcDNA3.1-mip/ctxB, was identified by restriction analysis, PCR and further confirmed by sequence analysis. NIH3T3 cell was transfected by recombinant plasmid pcDNA3.1-mip or pcDNA3.1-mip/ctxB with Lipofection strategy. Transient and stable products of mip/ctxB fusion gene was detected by immunofluorescence and Western blot. The results showed that NIH3T3 cell was transfected successfully and stable products can be detected in the transfected cell. To evaluate immunonicity of pcDNA3.1-mip and pcDNA3.1-mip/ctxB, BALB/c female mice were immunized intramuscularly with them and antigen specific antibodies, lymphocyte proliferative response, IFN-γ production and cytotoxic T-lymphocyte response of immunized mice were detected. The results showed that immunogenicity of pcDNA3.1-mip or pcDNA3.1-mip/ctxB immunized mice were higher than control and immunogenicity of pcDNA3.1-mip/ctxB immunized mice were higher than pcDNA3.1-mip immuneized mice. Statistic analysis by one way ANOVA showed that there was significantly difference between groups (P＜0.01).The results provide experimental proof to study of mip/ctxB fusion gene DNA vaccine.

Abstract:The purpose was to investigate the influence of brain asymmetry on hypothalamic-pituitary-adrenal(HPA) axis and the functions of macrophage under predator stress. The paw preference test was used to select right-pawed, left-pawed, and ambidextrous-pawed mice. After acute(45 min, once), chronic (45 min, once a day, for two successive weeks) predator stress by cats, mice were sacrificed and the blood and peritoneal macrophages were collected. The levels of plasma corticosterone and nitric oxide(NO), IL-1β in the supernatants of cultured peritoneal macrophage were respectively detected by enzyme immunoassay (EIA), nitrate reducase method and enzyme-linked immunosorbent assay (ELISA). The results showed: (1) The level of plasma corticosterone. In the acute predator stress group, the levels of plasma corticosterone were higher in right and ambidextrous-pawed mice than that of left-pawed mice as well as corresponding normal control group (P＜0.05), and that of the right-pawed mice was the highest, but that was decreased in left-pawed mice; after chronic predator stress, in right and ambidextrous mice，the levels of corticosterone were higher than those of the corresponding normal control (P＜0.05)，and the left-pawed and ambidextrous-pawed mices were also higher than those of the corresponding acute predator stress group (P＜0.05). (2) Level of NO. In the acute predator stress group, the level of NO in the supernatants of cultured peritoneal macrophage in right pawed mice was significantly higher than that of left-pawed mice (P＜0.05); after chronic predator stress, those in left, right, and ambidextrous mice were higher than those of the corresponding normal control group (P＜0.05)，and in right-pawed and ambidextrous-pawed mice were also higher than those of the corresponding acute predator stress group (P＜0.05). (3) Level of IL-1β. In the acute predator stress group, the levels of NO in the supernatants of cultured peritoneal macrophage in left-pawed mice were significantly lower than that of the corresponding normal control (P＜0.05); after chronic predator stress, that in right-pawed mice was significantly higher than that of the corresponding normal control group (P＜0.05).In conclusion, brain asymmetry may affect the activity of HPA axis and the functions of macrophages in Balb/c mice exposed to their predator, cat, stress.

Abstract:To apply the small interfering RNAs targeting survivin to inhibit expression of endogenous survivin gene in human hepatocarcinoma cell SMMC-7721, the recombinant plasmid pshRNA-survivins were transfectted into SMMC-7721. The expression level of survivin was determined by Western blot and immunofluorescence staining and the transcription of survivin gene was detected by semi-quantitative RT-PCR. The introduction of plasmids pshRNA-survivin was showed to efficiently and specifically inhibit the expression of survivin according to results of Western blot and immunofluorescence staining, with inhibitory rates at 62%～78％peaking 72 h. Semi-quantitative RT-PCR showed that mRNA transcription of survivin gene was reduced by nearly 57%～64%. On the contrast, the control plasmid did exhibit no inhibitory effect on the protein expression and mRNA transcription of survivin gene. The results demonstrate that the small interfering RNA targeting survivin gene shows dramastic inhibition on protein expression and RNA transcription.

Abstract:Kidney gene expression profile of db/db diabetic nephropathy mice was analyzed by using Affymetrix oligonucleotide genechip. Mice diabetic nephropathy related genes (and ESTs) were found out. A 1.7 kb cDNA was amplified and cloned by using RACE method, based on the sequence of EST. Homologous analysis of the sequence was carried out. And the result shows that it is part of mouse expression sequence AL023001, which function is unknown. To further investigate the relationship between AL023001 and diabetic nephropathy, the function of AL023001, expression levels of AL023001 were analyzed quantitatively using RT-PCR method. Result of RT-PCR shows that AL023001 is expressed differentially in liver, muscle, brain and fat, while the expression pattern of AL023001 in kidney is just like that revealed by gene chip. These results suggest that AL023001 is related to mice diabetic nephropathy, though its function and possible role played in the development of mice diabetic nephropathy still to be investigated.

Abstract:In order to study the biological function and conservation of homology to the human prostate and colon cancer-associated gene (hPC-1), the complete cDNA sequence from mouse kidney was cloned. The gene was named mPC-1 (GenBank ACC No.AY048852), and its cDNA was 2 193 base pairs. The whole cDNA locus was mapped to mouse chromosome 3A1～A2 by BLAST analysis to the mouse genome. The longest ORF of mPC-1 encoded the putative protein of 224 amino acids, which is 82% identical to hPC-1 coding region, with a coiled-coil domain and PEST domain in each. Bioinformatics analyses show that mPC-1 is comprised of six exons and highly homologous to mD 52 and its first exon is a highly special gene sequence thereof. Special promoter of mPC-1 has been verified experimentally, therefore, it was assumed that mPC-1 is a gene overlapping with mouse mD 52. RT-PCR performed in different mouse tissues or organs and various embryogenesis showed that the expression of the gene was strongly in prostate, kidney and eye, slightly low in stomach and smooth muscle, but not or very weakly in other tissues, however, the mD 52 gene almost expressed ubiquitously in mouse tissues or organs. Consequently, the gene sequence between mPC-1 and mD 52 is markedly overlapping in large-scale but modulated independently. Taken together, the results suggest that mPC-1 is a novel gene in coincidence to hPC-1 either structure or function.

Abstract:By analyzing the cDNA sequences of carboxyltransferase alpha subunits(α-CTs) from Arabidopsis thaliana and Glycine max,and combination RT-PCR, 3′-rapid amplification of cDNA ends (3′-RACE) and Genome walking techniques, the full length cDNA of plastid α-CT from embryos of Brassica napus between 20 and 29 days after flowering was cloned. Homologous analysis showed that the amino acid sequence deduced from it shares 85% and 59% homology with those of α-CT in Arabidopsis thaliana and Glycine max, respectively. The sequence encoding mature peptide that was absent from chloroplast transit peptide has been inserted into expression vector pHBM625 and expressed successfully in Escherichia coli by Western blotting analysis.

Abstract:Bio-cell sensor and bio-cell chip techniques have been important tools in post-gene era.They can detect functional information of cells as well as nature of matters detected.Living cells would be detected as a unit or sensetive element by exchanging between biological signals and physical and electrochemical signals.The detection using these tools is raptive,in real-time,quantitive and qualitive. Bio-cell sensor and bio-cell chip techniques have been applied in fields of the cellular biology,the monitoring of envirement and the developments of drugs.The developments of methods,applications in three years and the trends are reviewed.