Abstract:Heat shock factor 1 (HSF1) is the key protein in regulating stress response. It can be activated under heat，oxidative or another stress conditions. Dominant-positive and dominant-negative HSF1 are two types of HSF1 mutants. Both of them gain the DNA binding activity in the absence of stress. In addition，dominant-positive HSF1 acquires transcriptional activity，which dominant-negative HSF1 does not acquire. In this paper，the progress of using these HSF1 mutants in the research of cancer，neurodegenerative disorders and cardiovascular diseases will be discussed.

Abstract:Artemisinin，a new and a very potent antimalarial drug，is produced by the plant Artemisia annua L. with a very low yield ranging from 0.01％ to 0.8% on a dry-weight basis. This makes artemisinin an expensive drug. Several studies reported chemical synthesis of the artemisinin，but none of them seems a viable economical alternative compared with the isolation of artemisinin from the plant. Hence， a higher artemisinin concentration in the plant is necessary for cheap antimalarial drug production. Many types of cyclic sesquiterpenes in Artemisia annua have been characterized to date，each derived from the common cyclic precursor FDP in a reaction catalyzed by a sesquiterpene synthase. Sesquiterpene synthases are widely regarded as the rate-determining regulatory enzymes in the pathways they participate，and a number of sesquiterpene synthases have been cloned from Artemisia annua up to now. This report is a brief review on the following sesquiterpene synthases: epi-cedrol synthase，amorpha-4，11-diene synthase，β-caryophyllene synthase，(E)-β-farnesene synthase，germacrene A synthase，as well as a new sesquiterpene synthase whose function remains largely unknown. The report is of help for a better understanding of metabolic engineering of Artemisia annua.

Abstract:Ubiquitin-ribosomal protein S27a(UBRPS27a) is a fusion protein of Ubiquitin and ribosomal protein. The N-terminal is ubiquitin and C-termina is ribosomal protein S27a with a high conservative zinc finger domain of the C2-C2 type. When it was expressed in eukaryotes，The intact fusion protein were rapidly processed to free ubiquitin monomer and ribosomal protein S27a(RPS27a). Ubiquitin degradated proteins particularly and selectively in cell and RPS27a is indispensable for translation. This multifunctional ribosomal protein is expressed at high levels in a wide variety of actively proliferating cells and tumor tissues and is a representative characteristic of various tumor cells. In our preliminary study of this protein in the silkworm，RPS27a also be found express highly in actively proliferating cells. The precise functional role of each ribosomal protein is largely unknown and many ribosomal proteins have extraribosomal functions apart from the particle. In this article，we review the recent research on the connection between tumor and this fusion protein，Ubiquitin-Proteasome Pathway and ribosomal protein. These research may indicate the origin and development of tumor，provide the basis for clinical diagnosis of cancer and the novel therapeutic targets for the treatment of malignant tumors.

Abstract:To produce recombinant Buthus martensii Karsch insect toxin (BmK IT)，BmK IT cDNA which fused a hexahistidine sequence at the C-terminus by PCR was inserted into pTWIN1 expression vector fused in frame with an upstream Ssp DnaB intein gene. The expression plasmid was transformed into E. coli BL21 (DE3) strain and protein expression was induced by IPTG. The CBD-Intein-BmK IT^his6 fusion protein was purified from cell lysates using Ni-NTA resin affinity chromatography. The intein was removed from fusion protein by on-column intein-mediated cleavage. BmK IT^his6 was purified through Superdex 75 gel chromatography to more than 95% homogeneity. The purified protein has both correct secondary structure and insecticidal activity.

Abstract:Recently，the interactions between hepatitis C virus (HCV) genes and the host cell factors were the focus of this field. Cell factors in the different biochemical pathway were approved to be interfered when HCV infection. To make sure which HCV gene(s) was the major factor during the interaction process，ten eukaryotic expression plasmids containing different functional genes of HCV: Core，E1，E2，p7，NS2，NS3，NS4A，NS4B，NS5A and NS5B were transfected into the CHO-K1 cells respectively. Then ten stable cell lines expressing different HCV functional proteins were constructed under the selective pressure of G418. DNA and mRNA of the HCV genes were both detected by PCR and RT-PCR respectively in the corresponding stable cell lines，freezation and anabiosis would not lose the HCV genes. Besides，the E1，E2 and NS5B proteins were detected by Western-blot which demonstrated that the HCV genes have formed stable expression in the host cells. The activity of UDP-glucose ceramide glucosyltransferase (UGCG) in the stable cell lines increased in different degree by TLC assay. For example，the activity of UGCG in CHO-K1-E2 and CHO-K1-p7 was doubled according to the control cells，and in CHO-K1-NS2 and CHO-K1-NS5A was about 1.6 times compared with the control cells. The establishment of the stable cell lines containing different single HCV gene will provide foundation for investigating the interactions between the virus and the host factors，and for the filtration of antiviral medicine.

Abstract:To study the effect of HCV core protein on the interferon-induced antiviral genes expression and its mechanisms. Methods HepG2 cells were transiently transfected with HCV core protein expression plasmid and the blank plasmid respectively. RT-PCR was used to analyze the effect of HCV core protein on PKR and 2′-5′OAS expression. The effect of HCV core protein on ISRE-medicated gene expression was detected by luciferase activity assay. Western-blot assay was performed to observe the change of mRNA and protein levels of SOCS3，STAT1 and p-STAT1 following HCV core expression. In the presence of HCV core protein，the transcription of PKR and 2′-5′OAS are down-regulated. ISRE-medicated reporter gene expression and STAT1 phosphorylation were inhibited. The transcription and expression of SOCS3 were induced compared with blank plasmid-transfected group. In HepG2 cells，HCV core protein can down-regulate the expression of some interferon-induced antiviral genes，which involves the induction of SOCS3 and the inhibition of STAT1 phosphorylation.

Abstract:In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella，the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver，respectively，the cDNAs from sporozoites of E. tenella was used tester，Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver，the cDNAs from sporulated oocysts was used tester，one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%，96% and 98% recombinant clones，respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries，respectively，thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts，eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries，nine ESTs shared significant identity with previously described，the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

Abstract:KCTD10 is a TNF-α inducible protein that can interact with the small subunit of DNA polymerase δ and PCNA. In order to study the function of KCTD10，we prepared the rabbit anti-mouse KCTD10 polyclonal antibody by using the His-tagged recombinant mouse KCTD10 protein to immune New Zealand white rabbit. Mouse KCTD10 shares significant similarity with PDIP1 (polymerase delta-interacting protein 1) and TNFAIP1(tumor necrosis factor alpha-induced protein 1) protein，and then KCTD10 polyclonal antiserum possesses cross-reactivity with PDIP1 protein and TNFAIP1 protein. The partially digested fragments of homogeneous proteins PDIP1 and TNFAIP1 were mixed and incubated with anti-KCTD10 antiserum at 4℃ for 3 h to deplete unspecific antibodies. Through this method，we removed successfully the cross-reactivity of anti-KCTD10 antibody with PDIP1 and TNFAIP1 and obtained specific anti-KCTD10 antibody. Then，the anti-KCTD10 antibody was used in immunohistochemistry experiments of mouse. The results of immunohistochemistry on whole-mount embryo and paraffin section demonstrated that KCTD10 is highly expressed in neuroepithelium of neural tube and dorsal root ganglion of 12.5 d embryos. These results suggest that KCTD10 may play roles in the development of neuroepithelium of neural tube and dorsal root ganglion.

Abstract:A gene appA encoding a novel phytase was firstly cloned from Hafnia alvei by PCR and sequenced. The gene was consisted of 1335 bp, encoding 444 amino acids. The calculated molecular weight of the mature APPA was about 45.2 kD. The gene appA was expressed in E. coli BL21(DE3). Recombinant APPA was purified and its enzymatic properties were determined. The optimum pH for the enzyme was 4.5 and the optimum temperature was 60℃.The pH stability of r-APPA is good, the relative phytase activity was above 80% after treated in buffers of pH2.0～10.0. The specific activity of r-APPA is 356.7 U/mg, and the K_m value was 0.49 mmol/L and V_max of 238 U/mg. The enzyme showed resistance to pepsin and trypsin treatment.

Abstract:The natural isoflavones biosynthetic pathway is only limited in legumes plant. To study the isoflavone in bacteria by metabolic engineering requires transformation of multi-gene of the whole pathway into the host strain to resembling the expression and metabolism of the genes. The multi-gene transformation and expression strategy become necessary because of this. This article talks about the multi-gene transformation strategy using one or many vectors, taking the five genes of isoflavonoid biosynthetic pathway to E. coli. The recombinant bacteria carry five genes with two vectors, the whole Isoflavonoid biosynthetic pathway was constructed into E. coli. Fermented with L-tyrosine as substrate and IPTG as an inducer, the recombinant bacteria can produce a new isoflavone related metabolite showed on HPLC analysis profile.

Abstract:To investigate the components in Plastrum Testudinis which have effects on the proliferation of rat bone marrow mesenchymal stem cells( bMMSCs), the active parts of plastrum testudinis which can promote proliferation of rat mesenchymal stem cells were extracted by petroleum aether. The activities of inducing the proliferation of bMMSCs were studied by MTT assay and flow cytometry. The chemical components of extraction were analyzed by GC-MS. The results showed that the petroleum aether extraction can obviously promote the proliferation of the stem cells. The main components are long-chain fatty acids, cholesterols and cholest-4-en-3-one, and palmitic acid, stearic acid and cholest-4-en-3-one have effects on proliferation of bMMSCs. In plastrum testudinis, fatty acids can promote the proliferation of bMMSCs but not increase overly. This provide the experiment basis, and offer important reference for Traditional Chinese Medicine that researches stem cells.

Abstract:The effects of the cultivation media, plant growth regulators and inoculum size on the cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata R.Br. were investigated. The cell growth and 20-hydroxyecdysone formation reach the highest when cells are cultured in the Gamborg's B5 medium supplemented with 2.0mg/L BAP (6-benzylaminopurine) and 1.0mg/L 2,4-D. The maximum 20-hydroxyecdysone productivity, of about 1.1mg/L/day, was observed in the culture with 20%　PCV (packed cell volume) of inoculum size. These data also show that the increment of the inoculum size to 20% PCV could increase the productivity in 7-folds.

Abstract:In order to research developmental competence of transgenic somatic cell by serial nuclear transplantation, goat cloned embryos were compared with recloned embryos in ability of in vitro development. Fetal fibroblasts including human finger-domain lacking t-PA gene was microinjected into cytoplasm of the MII oocytes. Goat embryos (G₀) were cloned by this procedure. A single blastomere from 16～64-cell goat cloned embryos(G₀) was microinjected into Intracytoplasm of the MII oocytes. Goat embryos (G₁) were cloned by this procedure. Goat embryos (G2, G3) were recloned by using 16～64-cell recloned embryos. The developmental time of donor embryo affected the developmental rate of recloned embryos(G₁,G₂).The results show: the cleavage rate of cloned embryos(G₀) (76.45%±1.17%)was no difference significantly with recloned embryos (G₁ G₂ G₃) (72.18%±1.97%，76.05%±2.38%，75.99%±2.84%); the developmental rate of morulae and blastocysts of cloned embryos (47.20%±2.93%,11.00%±1.42%) were higher than these of recloned embryos(34.99%±2.66%，28.23%±2.00%，23.34%±1.99%)(3.87%±0.67%，2.08%±1.66%，0); the morulae rate(29.57%±1.53%, 24.43%±1.87%) and blastocysts rate(1.96%±1.31%, 2.01%±1.34%) of recloned embryos (G₁ G₂) from 16-cell recloned embryos were lower than those(34.32%±1.31%, 29.76%±1.66% and 3.86%±1.03%, 3.48%±0.34%)from 32～64-cell recloned embryos(Ｐ>0.05). In conclusion, nuclear transfer embryos should not were recloned mostly; and the embryos recloned by using 32～64-cell embryos achieved higher developmental ability compared with using 16-cell embryos.

Abstract:Shoot tips of a traditional table banana (Musa spp. cv. Kanthali) of Bangladesh were evaluated for in vitro propagation. Initial surface sterilization (with 0.1% HgCl₂ for 12 minutes) of shoot tips was successful but microbial contamination (mostly bacteria) at the rhizomatous base of the explants was observed within 6～15 days after inoculation which eventually killed 85% of inoculated explants. So, for contamination free culture establishment explants were soaked in two broad spectrum antibiotics namely ampicillin and gentamicin. Cent percent contamination free cultures were established by soaking the explants in 400mg/L ampicillin or 200mg/L gentamicin for 1h. Antibiotic treated explants were found to be full contamination free but failed to regenerate after 3 weeks of culture. But some of them absorbed media for up to 2^nd subculture and showed swelling of explants and some color changes from pale white to light/deep green. Finally, a few days after 3^rd subculture, no growth of explants was observed and all treated explants eventually started to die. Among the untreated alive explants the best medium for single shoot development was MS+4.0mg/L BA+0.5mg/L KT+15% CW and average time required for shoot development was 18～21 days. But the regeneration percentage was very low (30%). The best medium for shoot multiplication was MS+4.0mg/L BA+2.0mg/L IAA+15% CW and only average 3～4 shoots were formed per shoot. Finally, in vitro proliferated shoots produced roots with maximum frequency (90%) in half strength of MS medium fortified with 0.5mg/L IBA.

Abstract:The chitosan thermosensitive hydrogel is liquid at room temperature but gels rapidly when heated to body temperature. This hydrogel are wildly used for cell encapsulation, drug delivery or tissue-engineered scaffolds. The system can sustain the release of macromolecules over a period of several hours to a few days. However, with low-molecular-weight compounds, the release is generally completed within 24 h. To prepare a functional chitosan thermosensitive hydrogel for slow release both broad-spectrum antibiotic chlorhexidine and growth factor recombined human bone morphogenetic protein\|2 (rhBMP-2),The β-cyclodextrin was used to prepare an inclusion complex with chlorhexidine, and then the latter was incorporated into the chitosan thermosensitive hydrogel system. Simultaneously, rhBMP-2 was added into thecoelastic properties of system with or without objective factors. And the in vitro release kinetics of chlorhexidine and rhBMP-2 was investigated by HPLC (high performance liquid chromatography) and ELISA (enzyme-linked immunosorbent assay) respectively.The results showed that the addition of chlorhexidine/β-cyclodextrin inclusion complex to the thermosensitive solution did not change the gelling behavior of the thermosensitive system. Further, the in vitro release profiles demonstrated that the release rate of chlorhexidine and rhBMP-2 from hydrogel became slower, controlled delivery over at least 1 month.By first preparing chlorhexidine/β-cyclodextrin inclusion complex, and then mixing the IC and rhBMP-2 into the chitosan thermosensitive hydrogel, a functional chitosan thermosensitive hydrogel system with ability of slow release both rhBMP-2 and chlorhexidine is successfully made.

Abstract:A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21×10⁵u/L and 7.38×10⁴u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39×10⁵u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50℃ and at a pH range from 3 to 10. However, the specific activity of rLacDx (1,761u/mg) for ABTS was higher than that of rLacDe (1,122u/mg), and the apparent K_m value of rLacDx (427 μM) was less than that of rLacDe (604 μM).

Abstract:With sodium alginate as a carrier and glutaraldehyde as the crosslinking agent, an improved immobilization method of β-glucosidase for production of soybean genistein was developed. As compared with entrapment or entrapment-crosslinkage, crosslinkage-entrapment that β-glucosidase was treated with glutaraldehyde and then entrapped in sodium alginate remained high loading efficiency and activity recovery, Effects of bead sizes, concentrations of alginate and glutaraldehyde as well, on the loading efficiency and activity recovery were assessed. When compared with the free enzyme, the optimum temperature, pH value and K_m of the immobilized β-glucosidase were respectively shifted from 50℃ to 40℃, 4.5 to 4.0 and 2.57 μg/mL to 2.02 μg/mL. The stabilities of the immobilized β-glucosidase were considerably better than that of the native enzyme. The immobilized β-glucosidase was employed to genistein production, 84.94% of the activity and 56.04% of conversion were kept after consecutive use of 6 times.

Abstract:The synthetic characteristic of extracellular polysaccharide (EPS) of Ganoderma lucidum performed in batch fermentation was studied. The result showed that the production EPS was partially growth-associated.The cell dry weight (CDW) and EPS reached 15.56g·L^-1<,3.02g·L^-1<respectively. The yield of EPS to cell dry weight (Y_p/x)was 0.19.Based on the test result of batch fermentation, a kinetic model was proposed by using the Logistic equation for cell growth, the Luedeking Piret equation for EPS production and the Luedeking-piret-like equation for consumption of glucose as substrate. The calculated results of models were compared satisfactorily with experimental data under various glucose concentrations, the average relative error was no more than 5%. The kinetic model had practically guiding producing PES in fermentation of Ganoderma lucidum.

Abstract:Based on homologous recombination, recombinant plasmid pRKG was constructed by replacing the internal fragment of 18S rDNA of pRJ-5 with a copy of γ-glutamylcysteine synthetase gene (GSH1) from the industrial brewing yeast strain G03 and a copy of G418 resistance gene (Kan) used as the dominant selection marker respectively. The fragment 18s rDNA::(Kan-GSH1) obtained through the PCR reaction was integrated to the chromosomal DNA of G03 strain, and recombinants were screened by G418 resistance. It was shown that the GSH content of beer fermented with the recombinant strain SG1 was 16.6% higher than that of G03, and no significant difference in routine fermentation parameters was found. To test the genetic stability, strains SG1 was inoculated into flasks and transfered continuously 5 times. The intracellular glutathione content of strain kept constant basically. It is an instructive attempt of genetically modifing industrial brewing yeast, as GSH1 was obtained from the host itself.

Abstract:Marine Streptomyces GB-2, isolated from marine samples collected in the intel tidal zone of Lianyungang, was found to produce antibacterial substance which exhibited significant inhibitory effects on 11 Gram-positive bacteria and 4 Gram-negative bacteria. The antibacterial substance was proved to be neutral and water-soluble according to paper chromatogram analysis, and its production was significantly associated with aritificial seawater. The stability analysis of the fermentation broth of Streptomyces GB-2 showed that it was very stable at pH1 and pH12 under 121℃ and changed very little under ultraviolet treatment. The substance produced by strain GB-2 exhibited potential use in the areas of bio-control, food and medical application.

Abstract:May the structure information of protein be obtained from the corresponding nucleotide sequence? For this question, a computer program was used to conduct a statistical analysis about the clustering phenomena of amino acids. In this method,20 kinds of amino acid were classified into 2 types according to the number of hydrogen bonds formed by middle base of their correlation codons.It can be seen that amino acid has a rather great possibility of neighboring on another of its class and this assembly has a tendency to forming specific secondary structure. A sequence was designed and its secondary structure was predicted by this prediction software.

Abstract:Receptors play a crucial role in determining the pathogenesis and tissue tropism of virus. Foot-and-mouth disease virus（FMDV）has been showed to use four integrins, αvβ1, αvβ3, αvβ6 and αvβ8 as receptors to initiate infection. In this study, the porcine integrin αv gene was cloned by RT-PCR from the lung tissue of healed pig infected experimently with FMDV, and compared its nucleotide and deduced amino acid sequence with the αv gene of other animals. The 3141bp cDNA of bovine integrin αv encodes a polypeptide of 1046 amino acids consisting of a 30-residue putative signal peptide, a 955-residue ectodomain, a 29-residue transmembrane domain, and a 32-residue cytoplasmic domain. The ectodomain contains 11 potential N-linked glycosylation sites（NXT/NXS）, 2 calcium binding domains(DX［D/N］XDGXXD) and 18 cysteine residues. The nucleotide sequence similarities of integrin αv between pig and cattle, human, rheses monkey, house mouse, chicken, dog are 93.3%, 91.5%, 91.4%, 85.6%, 73.2% and 89.9% respectively; and the amino acid sequence similarities are 96.3%, 94.6%,94.1%, 90.8%, 81.6% and 93.8%, respectively. The αv gene of cattle and pig exhibited the highest sequence homology. It is possible that host tropism of FMDV may related to divergence in receptors among different species.

Abstract:Total RNA was isolated from kidney of BaMei pig，a local strain of Chinese pig, and then the cDNA sequence of SOCS-2 gene was cloned by RT-PCR (GenBank accepted number is EF121242). Then the cloned SOCS-2 gene was inserted into PMD19-T vector by T/A cloning, transformed into DH-5α, tested by PCR and sequenced. The data show that the homology of the cloned porcine SOCS-2, including 822 bp,is more than 93% and that of the deduced amino acid sequence is 89% when compared with human, rat and mice. And the molecular weight of SOCS-2 protein is about 22.25kD and PI is 8.03. The cloning of SOCS-2 gene is useful for the further research on the molecular mechanism by which regulating growth and development of organism.

Abstract:The random amplified polymorphic DNA (RAPD) technique was used to amplify DNA fragment, aiming at finding markers linked to the sex trait in Cycas tanqingii D. Y. Wang. A total number of 160 random primers were screened in the RAPD-PCR and more than 2500 RAPD fragments were generated from the male or the female plants. One fragment of about 500bp was amplified steadily and repeatedly by the S₀₄₆₅ (CCCCGGTAAC) primer only from female plants but not male plants. The RAPD marker was then converted into female-linked dominant SCAR (Sequence Characterized Amplified Regions) marker named STQC-S465-483. The development of this sex-linked SCAR marker provides a possibility of identifying the sex of Cycas tanqingii before sexual maturation, which is very important to in situ or ex situ conservation.

Abstract:HC-pro gene of Watermelon Mosaic virus was obtained by RT-PCR was 1371bp in length. It was cloned into pPIC9K, then the eucaryotic recombinant expression plasmid pPIC9K-WHC was constructed. After being linearized with restriction endonuclease SalⅠ, the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The high copy transformants with Mut⁺/His⁺ phenotype were selected by RT-PCR and screening on G418, MD and MM medium. Induced by methanol for 5 days, the culture supernatant was analyzed by SDS-PAGE，the results showed that a specific protein with a molecular weight of about 66kD was expressed. Western blot analysis proved that the expression protein could specifically bind to HC-Pro polyclonal antibody. Far western blot analysis proved that the expression protein could bind to coat protein, given support to “bridge" hypothesis that HC-Pro help aphid transmission of non-persistent viruses.

Abstract:According to published nucleotide sequences, ORF4 gene of barley yellow dwarf virus GAV (BYDV-GAV) was synthesized by reverse transcription-polymerase chain reaction (RT-PCR).The BYDV-GAV ORF4 gene was expressed in baculovirus -insect cell expression system efficiently, and western bolt analysis confirmed its expression product. Confocal laser scanning microscopy showed that GFP: ORF4 fusion protein was associated with the nuclear envelope of insect cells. By expressing the N- and C-terminal regions of ORF4-encoding product (P4) in insect cells combined with structure prediction, it was found that the N-terminal region of P4 containing four α-helices is required for targeting P4 to the nuclear envelope. These results provide a base for biological function of ORF4 gene during systemic infection of BYDV-GAV in host plants further.

Abstract:Human leukocyte elastase is an important selection target of inflammation and cancer. In this paper, a high throughput screening model was established for screening human leukocyte elastase inhibitors from thousands of strains of actinomycetes. As a result, a strain, N01WA-735 with potent suppression activity was isolated. Firstly, the strain N01WA-735 was identified as Streptomyces according to morphology and biochemical analysis. The Streptomyces N01WA-735 was processed by solvent extraction, silica column chromatography, Sephadex LH-20 column chromatography and crystallization to get a pure active compound named N01WA-735E. Its chemical structure was elucidated as the same as that of the compound named BE-52440A by physicochemical properties and spectral data of UV, MS, ¹H-NMR and ¹³C-NMR respectively. The compound showed a strong inhibitory activity against human leukocyte elastase with IC₅₀ of 3.02μmol/L. The compound is reported as a human leukocyte elastase inhibitor for the first time.

Abstract:To reduce the huge labor-cost in the screening in traditional monoclonal antibody generation, We established a new system for monoclonal antibody generation integrating with protein array. BALB/c mice were immunized by eight recombinant proteins respectively, and the positive hybridoma cells were obtained by cell fusion and ELISA screening. All the eight kinds of positive hybridoma cells were mixed, cloned, screened by protein array, and definite dilution cloned. Results: 175 single cell clones were obtained by complex cloning, and 119 of those were positive clones. Then 8 positive cell lines were generated by the following 2 rounds definite dilution cloning. By comparing with the traditional method, we got 8 monoclonal antibodies using the combined protein array screening and multiplex cloning method in 1 cycle, and fewer amounts of antigens were used. As a result, the combined protein array and multiplex cloning method could be used as an economical, rapid and simple tool applying in high throughput monoclonal antibody generation.

Abstract:Biodiesel fuel produced with the enzyme-catalyzed esterification and transesterification of high acid value waste oil through ultrasonic assistant was explored. Propyl oleate, biodiesel, converted from high acid value waste oil and 1-proponal catalyzed with immobilized lipases from Candida antarctica and Aspergillus oryzae in conditions of ultrasonic assistant. Commercial immobilized lipase Novozym 435 from C. antarctica was used as biocatalyst catalyzing high acid value waste oil and 1-proponal esterification and transesterification to propyl oleate under the ultrasonic assistant conditions and different conditions such as lipases amounts, initiatory molar ratio of propanol to oil, frequency of ultrasonic and power of ultrasonic were investigated and optimized. It is revealed that the enzymatic activity of Novozym435 is enhanced and, in particular, enzyme-catalyzed transesterification activity is enhanced obviously under the ultrasonic assistant conditions. Low frequency and mild energy ultrasonic is a key factor for enhancing enzymatic activity, emulsifying oil-propanol system and accelerating the speed of produce diffusing in the system. Under the optimal ultrasonic assistant reaction conditions, such as Novozym435 amounts 8% by oil quantity, initiatory molar ratio of propanol to oil 3:1, frequency of ultrasonic 28 KHz, power of ultrasonic 100 W and temperature of water batch 40～45℃, the conversion ratio to propyl oleate reached to 94.86% in 50mins in comparison with the highest conversion ratio to propyl oleate 84.43% under the conventional mechanical agitation conditions. Furthermore, it is demonstrated that various short chain linear and branched alcohols (C₁～C₅) show high conversion ratio to fatty acid alkyl esters (biodiesel) under the optimal ultrasonic assistant reaction conditions. On the other hand, ultrasonic energy is propitious to reduce the adsorption of product propyl oleate, by-product glycerol and other emplastics in system on the surface of immobilized lipase Novzym435 and recyclable Novozym435 possess clean appearances, well decentralizations, no agglomeration and easy washing and well operational stability.

Abstract:In order to establish a fast and accurate method for novel DMIs fungicide screening, lanosterol 14α-demethylase of Magnaporthe grisea expressed in E.coli was used as target enzyme and the DMI fungicides diniconazole, tebuconazole, triadimenol and triadimefon were used as representative fungicides，the effects of enzyme activity, enzyme purity and concentration on the binding spectra were investigated. The results showed that active enzyme, elimination of interference of other P450s and proper enzyme concentration were necessary for obtaining accurate binding spectra. The K_d values of diniconazole, tebuconazole, triadimenol and triadimefon were 0.143μmol/L, 0.24μmol/L, 0.257μmol/L and 0.307μmol/L respectively, which significantly correlated to their 120h-EC₅₀ values on the growth of Magnaporthe grisea. The results indicated that the binding spectra of fungicide and lanosterol 14α-demethylase can serve as a reliable and fast method for novel fungicide screening.

Abstract:Nanoemulsion-encapsulated lzp3 DNA vaccine（pCDNA3-Aat-COMP-lzp3-C3d3, lzp3） was prepared, and its quality was evaluated. The interfacial emulsification method was employed to make the nanoemulsion-encapsulated plasmid, and its matography was used to separate the nanoemulsion from the plasmid based on the charge characteristics of plasmid. The determination method for encapsulation efficiency of the plasmid nanoemulsion was established by anion exchange chromatography. The results indicated that the average size of the plasmid nanoemulsion is (23±10)nm, and the encapsulation efficiency is 80.5%. The separation and determination conditions of nanoemulsion-encapsulated plasmid was as follows: eluent buffer was 0.05mol/L Tris-HCl solution at a flow rate of 0.7mL/min, while detection wavelength for plasmid was 260nm, the temperature of column was maintained at 30℃, and injection volume was 2mL. On this condition, nanoemulsion and plasmid can be separated on the column of anion exchange chromatography significantly. This method is simple, sensitive and reproducible with respect to good linearity in the range of 0.05～0.80mg/mL (r=0.9983)，which can be applied to determine the entrapment efficiency of nanoemulsion-encapsulated plasmid

Abstract:The Spindle-view，a specialized instrument for observing spindle image，was applied to observe the meiotic spindles of vitro matured porcine oocytes at 36、42、44、48h，and enucleation from porcine, comparing to the previously methods (McGrath-Solter's method and two-step-squeezing method) in the enucleated. The results showed that:①there was no noticeable differences at vicinity of spindle images and 1st polar body among in vitro matured porcine oocytes at 40～48h under the instrument；②Spindle-view is suitable for the observation of meiotic spindles of matured oocytes and enucleation from porcine; the modified Spindle-view method for enucleation is significantly better than McGrath-Solter's method and two-step-squeezing method in the enucleated rates (95.5%,42.1%,74.2%,P<0.01)of absolutely removing nuclei matter;③the spindle images could be used to monitor the oocyte qualities.