Abstract:Abstract: Despite the improvement of PCR technology, false-negative results from PCR detection remain as an unresolved issue, which may reduce the detection accuracy. To address this problem, an internal amplification control, which is a non-target DNA fragment, is sometime suggested at present to be included into a PCR detection system to be co-amplified with the target sequence so as to indicate the PCR inhibitors in the samples and false negative results in the detection. This is also necessary for the standardization of PCR detection system. The purpose of this article was to provide a review on the IAC-PCR in three aspects: the causes of the false-negative results appearing, the construction strategies of internal amplification controls and the applications of IAC-PCR. In addition, a brief introduction of related research work on IAC-PCR in our laboratory will be mentioned in this review, which might be helpful to develop an easy method for the construction of IAC, which could indicate the false-negative results without the decrease of detection sensitivity.

Abstract:Abstract: [Objective] Cardiovascular disease, harmful to human health, is a worldwide disease, for which fibrinolytic enzyme is usually used as an effective remedy. Fibrinolytic microbes under extreme environmental conditions may excrete some high efficient and safe fibrinolytic enzymes. So, for obtaining this fibrinolytic enzyme, we isolated fibrinolytic strains from frozen soil in the Tibetan Plateau. [Methods] We screened microbes hydrolyzing blood powder by blood powder (bovine)-agar plate, followed by detection of thrombolysis effect through thrombolytic test in vitro. After that, we detected and measured the fibrinolytic activity of those microbes screened above with fibrin plates. Finally, we classified and identified the fibrinolytic microbes by combining physiological and biochemical test and 16S rRNA gene sequence analysis. [Results] We isolated a strain DR-536 from frozen soil at 4300 m altitude in Yushu Tibetan Autonomous Prefecture, Qinghai Province. We found that the strain DR-536 not only hydrolyzed blood powder in the blood-agar plate, but also completely dissolved thrombus in vitro. We also found that strain DR-536 could hydrolyze fibrin, and the fibrinolytic activity was 51.80 IU/ml (with Urokinase as a standard). At last, results of classification and identification indicated that DR-536 is a new strain of Arthrobacter aurescens with fibrinolytic function. [Conclusion] We provided a strain for research and exploitation of novel fibrinolytic enzymes.

Abstract:Abstract: [Objective] To demonstrate the novel regulatory pathways mediated in bacterial pathogenicity and motility in Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of bacterial blight in rice. [Methods] Molecular identification and functional characterization of Tdrxoo, which interacts with GacAxoo of the two-component regulatory system (GacSxoo/GacAxoo) in Xoo, were performed through gene cloning, sequencing and disrupt analysis. [Results] tdrxoo was successfully cloned from the genomic DNA of wild-type PXO99A by using polymerase chain reactions with the degenerated primers tdrxooF/R. The tdrxoo gene was found to be highly conserved in the plant-pathogenic Xanthomonas spp. Sequence analysis showed that Tdrxoo was homological to a protein with the TonB-Dependent-Receptor (TDR) domain. Tdrxoo is probably localized in the outer membrane of bacterial cells, recognizing the signals from extracellular environment, and inducing the intracellular signal transduction. △tdrxoo, the disrupted mutant, was obtained after a single cross-over recombination event between tdrxoo and the plasmid pK-tdr with the tdrxoo segment. The mutant lost the ability of causing the disease, and was affected in growth in vitro compared to PXO99A. In addition, the motility and the extracellular enzymes production of △tdrxoo were reduced, which can be restored through complementation of the △tdrxoo mutant by introduction of tdrxoo. tdrxoo deficiency didn’t affect siderophore production. [Conclusion] According to the existence of tdrxoo in Xoo genome and phenotype of △tdrxoo, Tdrxoo, as the outer membrance protein, is proposed to be involved in regulation of pathogenicity, extracellular enzyme production, the growth and motility of Xoo.

Abstract:Abstract: [Objective]To obtain the complete nucleotide sequence of the plasmid pVAE259 from Vibrio alginolyticus, to analyze molecular characteristic of the plasmid, and to explore its potential function. [Methods]We got the whole sequence of pVAE259 by the steps including of enzyme digestion, cloning, analysis of the acquired sequence and putative encoded protein using the softwares followed by speculation of its potential biological function.[Results] pVAE259 was a covalently closed circular plasmid. The complete sequence consists of 6075 bp in length, with a 42.16% GC content, and it is similar to the crytic plasmid pPS41 of Vibrio sp. 41. There is an origin of transfer (oriT) region in the sequence and a replication region located in the part from 4118 to 5494 bp. Seven open reading frames that longer than one hundred amino acids were found in pVAE259, ORF1-ORF7. The protein encoded by ORF1 should be classed into the members of relaxase superfamily, which is closest related to the MobA-like protein of Escherichi coli. The protein encoded by ORF2 should be classed into the members of the replicase superfamily, which is closest related to the protein RepA of Stenotrophomonas maltophilia. The protein encoded by ORF5 is similar to the protein MobC in the plasmid pHE1 of Heliamphora longata. [Conclusion] The plasmid pVAE259 might have the ability to transfer between different bacterial strains.

Abstract:Abstract：【Objective】Construction of Botrytis cinerea T-DNA insertion mutant library through Agrobacterium-mediated transformation and for further understanding of the molecular mechanisms underlying the pathogenicity of Botrytis cinerea.【Methods】Agrobacterium containing the binary vector pCMBIA1390 was used for transformation of Botrytis cinerea. Hygromycin resistant transformants were screened out and subjected to biological and morphological observation. Detached tomato leaves were used for pathogenicity assay. The flanking sequence of T-DNA inserted in mutant genome was cloned and analyzed by using TAIL-PCR.【Results】A variety of mutants were obtained and important phenotypes including reduction in growth rate, loss or reduction in conidiation and loss of pathogenicity were screened out. The T-DNA flanking sequence of one of the mutants was successfully amplified with TAIL-PCR.【Conclusion】Agrobacterium-mediated transformation system of Botrytis cinerea was established and a T-DNA insertion mutants library was constructed. TAIL-PCR was an effective method for isolating the flanking sequence of T-DNA inserted in genome.

Abstract:Abstract: [Objective] We cloned a cell division cycle PsCdc2 from Puccinia striiformis f.sp. tritici (Pst) and analyze its expression profile. [Methods] Using PCR and RT-PCR methods, we isolated the cDNA and genomic DNA sequences of PsCdc2. We analyzed the amino acid sequence of PsCdc2 using bioinformatic softwares. In addition, Real time RT-PCR was used to analyze the gene expression pattern of PsCdc2 at different time points after wheat inoculated. [Results] A 2279 bp DNA sequence of PsCdc2 was cloned and comprised of 11 extrons and 10 introns. The cDNA sequence of PsCdc2 included a complete 885 bp open reading frame and encoded a putative protein composed of 294 amino acids, with a molecular weight of 33.14 KDa and a pI of 6.26. PsCdc2 contained two conserved kinase domains and a transmembrane domain. Phylogenetic analysis indicated that PsCdc2 showed high similarity with Cdc2 from Puccinia graminis (73.1%), Cryptococcus neoformans (72.4%) and Ustilago maydis (70.4%), respectively. Real time RT-PCR analysis showed that in compatible interaction between Pst and wheat, PsCdc2 was up-regulated at early stage of infection. The maximum induction occurred at 12 hpi, at which transcripts were 1.62 fold over that in urediniospore. From 24 to 268 hpi, the accumulation of transcripts decreased steadily. The minimum accumulation occurred at 96 h, at which transcripts were only 0.07 fold of that in uredinisopore. During incompatible interaction between Pst and wheat, PsCdc2 was down-regulated and its accumulation was lower than that in urediniospore. The maximum induction occurred at 12 h, at which transcripts were 0.34 fold of that in urediniospore. The minimum accumulation occurred at 96 h, whose transcript was only 0.02 fold of that in urediniospore. [Conclusion] PsCdc2 might be involved in primary hyphal growth and haustorium formation during early infection by regulating cell cycle of Pst. The present study would be helpful for understanding the essence of cell cycle control and provided basis for new chemical control of Pst.

Abstract:Abstract: 【Objective】Since protein Y3 encoded by gene y3 reduced TMV activity, we intended to clone the full length of gene y3 in order to study gene y3 and its inhibitory function against TMV in in vivo conditions.【Methods】We amplified the unknown 5′- terminal cDNA sequence of gene y3 with 5′-RACE and using 5′- Full RACE Core Set (TaKaRa), and then the full length of gene y3 was obtained by RT-PCR amplification of total RNA of Coprinus comatus. The expression plasmid of y3 was constructed on the basis of gene y3 and transformed into Nicotiana tabacum via agrobacterium- mediation. 【Results】We obtained the full length of y3 successfully. It consisted of 534 bps including one ORF that encodes 130 amino acid residues. The cDNA sequence and its deduced amino acid sequence, which were obtained in this study, showed high similarity (up to 94%) to known fragment of gene y3 sequence. According to the full sequence, we cloned gene y3 and constructed the expression plasmids. The plasmids were constructed via inserting gene y3 sequence, CaMV 35S promoter, and NOS terminator, which were obtained from pBI121, at the MCS of pCAMBIA1301. The plasmids were used for gene transformation, resulting in transgenic Nicotiana tabacum plantlets. Gene y3 transcription was characterized by Northern blot analysis. Inoculation tests of the transgenic plantlets with TMV demonstrated inhibitory activity against TMV. 【Conclusion】Gene y3 transcription was expressed in transgenic tobacco plants. Gene y3 demonstrated some inhibitory function against TMV in vivo. The full length of gene y3 obtained in this study and its expression in Nicotiana tabacum plantlets could provide background information and benefits for future study on gene y3.

Abstract:Abstract: [Objective] ε-Poly-L-Lysine (ε-PL) is a natural amino acid homopolymer. The study aimed at isolating new ε-PL-producing strains. [Methods] ε-PL-producing strains were screened from the soil by using a new isolation approach which had three steps, (1) enrichment culturing ε-PL toleranting strains; (2) screening by improved Nishikawa’s method; (3) selection of strains with higher ε-PL tolerant ability. [Results]A new ε-PL-producing strain TUST-2 was isolated from the soil collected from Hainan province, China. Chemotaxonomic and morphological characteristics of the isolate were typical of strain of the genus Streptomyces. The strain TUST-2 was found to belong to Streptomyces diastatochromogenes by comparative 16S rRNA gene sequence analysis. The purified fermentation product of the strain TUST-2 was confirmed as ε-PL by characteristic analysis, hydrolysate analysis, infrared spectrum, 1H NMR Spectrum, 13C NMR Spectrum, and MALDI-TOF-MS. [Conclusion] On the basis of 16S rRNA gene sequence analysis and its morphological and physiological characteristics, ε-PL-producing strain TUST-2 is a new isolate of Streptomyces diastatochromogenes, named as Streptomyces diastatochromogenes TUST-2.

Abstract:Abstract: [Objective] To screen for monoamine oxidase (MAO)-inhibiting lactobacillus from healthy human dejecta in an in vitro model, and to provide reference for anti-ageing study in vivo of lactobacillus in the future. [Methods] The monoamine oxidase inhibitory model in vitro was applied to screen fermented supernatant and cell-free extracts originated from lactic acid bacteria, and two indices based on screened samples were determined, including biological dosage-effect and the effect of pre-incubated time between MAO and inhibitor on suppression rate. Meanwhile, with membrane separation technology, MAO inhibition from different molecular weight range of samples was measured. Screened strain JH-23 was investigated as target probiotic lactobacillus, identified through the sequence analysis of 16S rDNA gene and API system. [Results] The MAO inhibitory rate of cell-free extracts produced by strain JH-23 reached 33.7 %. After samples being vacuum freeze-dried, MAO inhibitory rate was up to 53.2 % when the reactive concentration was 16 mg/mL. The inhibition was significantly enhanced with the duration of pre-incubation increased, and then the inhibitory effect became steady after 30 minutes. Crude samples were dialyzed for 48 hours, MAO inhibitory effect of dialysates was significantly increased compared with pre-dialysis. The result of bacteria identification demonstrated that strain JH-23 was ascribed to Lactobacillus casei. [Conclusion] A new screening model in vitro, regarding monoamine oxidase as a target enzyme, was explored in this study. This model was characterized by convenience, rapidity and high sensitivity, and it could be useful for the following anti-aging research in vivo. The cell-free extracts of Lactobacillus casei JH-23 was inhibitory to MAO, and the intracellular small-molecules played a major role in the inhibition.

Abstract:Abstract: [Objective] To investigate the effects of the cell wall of Streptococcus mutans on the expression of Toll-like receptor 4 (TLR4), IL-6 and IL-8 in EAhy926 cells (The human endothelial hybridoma of human umbilical vein endothelial cells (HUVEC) and the human epithelial cell line A549, characterized by endothelial phenotype and biology). [Methods] EAhy926 cells were treated with different concentrations of the cell wall of Streptococcus mutans. The proliferation of EAhy926 cells was determined by MTT assay. The mRNA expression of TLR4, IL-6 and IL-8 in EAhy926 cells was detected by reverse transcription polymerase chain reaction (RT-PCR). TLR4 was analyzed by flow cytometry. The production of IL-6 and IL-8 in the cultured supernatants was measured by biochemical method and ELISA respectively. TLR4 blocking assay was used to investigate the relationship between IL-6, IL-8 and TLR4 mRNA expression. [Results] The proliferation of EAhy926 cells was significantly enhanced by the cell wall of Streptococcus mutans at 6 h (P<0.05), and inhibited after 12 h (P<0.05), both time- and dose-dependent. The expression of mRNA and protein for TLR4 in EAhy926 cells was increased after stimulated by the cell wall of Streptococcus mutans, peaking at 16 h (P<0.05), and then decreased. The expression of IL-6 and IL-8 mRNA was significantly induced when exposed to the cell wall, reaching the maximal level at 16 h and 24 h, respectively (P<0.05). Meanwhile, the cell wall of Streptococcus mutans induced the production of IL-6 and IL-8 (P<0.05). The expression of IL-6 and IL-8 mRNA in EAhy926 cells was blocked by anti-human TLR4 antibodies significantly. [Conclusion] The cell wall of Streptococcus mutans upregulated the expression of TLR4 and induced the production of inflammatory cytokines IL-6 and IL-8, indicating that the expression of TLR4 of EAhy926 cells may elicit a TLR4-mediated innate immune response and contribute to production of inflammatory cytokines IL-6 and IL-8

Abstract:Abstract: [Objective] The strain No.V3.4504 of Lecanicilliurn lecanii (Zimmermann), an entomopathogenic fungus, was studied on the effect of successive multi-generation culture in seven different media on its colony growth characteristics, extracellular enzyme activities and the virulence against scale insects. [Methods] The strain No.V3.4504 of L. lecanii was original isolated from a natural infected scale insect. The two species of scale insects used were Rhodococcus sariuoni Borchsenius and Ceroplastes japonicus Green. Seven media were used and fungus colony characteristics, growth rate and sporulation, extracellular protease and chitinase activity, and infective effect against the two species of scale insects were conducted. [Results] The fungus cultured on PDA medium for successive nine generations showed the most fast in colony growth, the minimal in sporulation, straight decline of extracellular protease and chitinase activity with generation increasing, and the minimal mortality of the scale insects. There was no significant effect to promote virulence of the fungus by increasing peptone into medium. On the media D, E and F, that with the body materials of the two scale insects, although the fungus appeared lower in the colony growth rate, its sporulation was higher upward 8.83×106-9.13×106 spores/cm2, extracellular protease and chitinase activities averagely reached 2.16-2.13 U/g and 1.01-1.03 U/g respectively, and the mortalities of the two scale insects were 55%-58% and 39%-42% respectively. Cultured three generations in vitro of the two scale insects, the fungus exhibited the highest activities in its protease and chitinase that were 3.08 -2.92 U/g and 1.45 -1.42 U/g respectively and the best infection effect against the two scale insects with mortalities of 71.30% and 58.89% respectively. A linear correlation was found between extracellular protease and chitinase activities of the fungus and the mortalities of the scale insects. [Conclusion] Cultured on PDA medium successive multiple generations made retrogradation of the strain No.V3.4504 of of L. lecanii. It was significant effect on keeping the vigor and higher virulence of the fungus adding the body materials of the scale insects into the medium. The vitro by using live scale insects as medium materials was the best way for the rejuvenation of the entomopathogenic fungus and promoting its virulence.

Abstract:Abstract: [Objective] Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is an important enzyme which is applied in nucleoside medication and intermediate biosynthesis. In this paper, we aimed to obtain the PNP gene from cold-adapted marine bacterium Pseudoalteromonas sp. XM2107 and study the characteristics of enzyme for applying in nucleoside medication and intermediate biosynthesis. [Methods] Purine nucleoside phosphorylas gene which amplified from the cold-adapted marine bacterium Pseudoalteromonas sp. XM2107 genome by homology-based PCR cloning was cloned, sequenced and expressed at E. coli BL21 (DE3) by using expression vector pET-His. The recombinant purine nucleoside phosphorylas enzyme (XmPNP) was purified by metal chelate chromatography and its several characteristics were determined completely. [Results] Analysis of entire sequences of XmPNP revealed that the whole sequence is 702 bp and coded a peptide of 233 amino acids with a calculated molecular mass of 25 kDa. Compared with mesophilic counterparts, XmPNP showed a lower temperature optimum (50℃). The optimal pH for inosine phosphorolysis catalyzed by XmPNP was around 7.6 at sodium phosphate buffer. XmPNP showed the highest activity toward inosine (Km value, 0.382 mmol/L, at 37℃) and the activity decreased in the order of guanosine and adenosine. Furthermore, XmPNP still expressed high catalytic activity and excellent thermalstability at ordinary temperature. [Conclusion] Both high catalytic activity and good thermalstability at ordinary temperature indicated that it will provide attractive candidate for prodrug activation and nucleoside medication biotransformation.

Abstract:Abstract: [Objective] To clone Penicillum expansum CICC 40356 lipase (PEL) gene cDNA and to over-express active lipase in Pichia pastoris GS115. [Methods] Primers were designed according to the nucleotide sequence of reported lipase gene from Penicillum. Ten rare codons of PEL and nine of the α-signal peptide were optimized by PCR. The native and codon-optimized PEL genes were respectively cloned into pPIC9K, pPIC9KM, and pPIC3.5K vectors. The properties of recombinant lipase were also determined. [Results] Nucleotide sequence analysis revealed that the PEL cDNA contained an 858 bp open reading frame. The deduced amino acid sequence corresponds to 286 amino acid residues, including a potential signal peptide sequence of 20 amino acid residues. The hydrolysis activity of PEL was enhanced with codon-optimization. Its optimal temperature and pH were 35 ℃ and 9.5. It favored medium chain esters (C8-C12) and showed the maximal activity toward C8 acyl-chains. It could be stimulated by Ca2+ and Mg2+, but strongly inhibited by EDTA and slightly repressed by Fe2+, Zn2+ and Cu2+. [Conclusion] The activity of PEL was improved 2.3-2.5 folds compared to that of the wild type, suggesting that the codon optimization is an efficient measure to produce the active PEL in P. pastoris system.

Abstract:Abstract：[ Objective ] We expressed the lipase gene lpsA2 from Streptomyces avermitilis in Escherichia coli and characterized the enzymatic properties of LpsA2. [ Methods ] We extracted the genomic DNA of S. avermitilis and amplified lpsA2 gene by PCR with specific primers. We then expressed lpsA2 gene in E. coli and determined the enzymatic properties of LpsA2. We also analyzed the evolutionary relationship between LpsA2 and lipase family by using phylogenetic tree based on the alignment of amino acid sequences. [ Results ] According to the alignment of amino acid sequences, we found that LpsA2 had the active site (Ser130-Asp221-His253) which was consistent with the typical characteristics of lipases that their active centers always consisted of Ser, His and Asp, and Ser always located in the conserved five peptide structure (Gly128-His129-Ser130-Gln131-Gly132). We constructed the evolutionary tree and found that LpsA2 belonged to Subfamily I.7 of lipase Family I. Furthermore, the optimal pH and temperature of LpsA2 were pH 8.0 and 50oC, respectively. The best substrate of LpsA2 was p-nitrophenol myristate. The activation energy (Ea) of LpsA2 between 10oC and 50oC was 6.3 kcal/mol. The enzyme activity of LpsA2 was increased to 250% or above by 1 mmol/L Co2+, Hg2+ and Zn2+, respectively. The activity was also elevated to 110.7% and 138.0% by 15% dimethylformamide and dimethyl sulfoxide, and 352.7% and 189.7% by 0.1% and 1% Span-20, respectively. [ Conclusion ] The enzymatic properties of LpsA2 from S. avermitilis were characterized that may provide the fundament to the screening of bio-engineered bacteria and the industrial applications in food processing and drug synthesis.

Abstract:Abstract: [Objective] In order to study the microbial diversity and the characteristics of cultivable microorganisms in bioleaching reactors. [Methods] Both conventional culture technique and analysis of a 16S rRNA sequence library were used. The pure cultures were isolated by enriching the samples under different cultural conditions. The morphology characters, physiology and biochemistry characters, genetic characters and oxidation efficiency to different minerals of cultured isolates were analyzed. [Results] The microbial community in bioleaching reactors identified through the gene clone libraries were closely related to bacteria including Leptospirillum sp., Sulfobacillus sp., Acidithiobacillus sp., Spingomonas sp. and Archaea including Sulfolobus sp., Ferroplasma sp. Five strains were isolated after enriching and culturing, which were corresponding to Acidithiooxidians caldus, Acidithiooxidians thiooxidans, Acidithiooxidans ferrooxidans, Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans. These isolates could oxidize sulfur or Fe2＋and different sulfide ores. [Conclusion] The bioleaching reactors were dominated by a small number of microbial species that were commonly found at thermoacidophilic environments.

Abstract:Abstract: [Objective] We investigated the influence of Carnobacterium sp. Hg4-03 on intestinal microflora of the 4th instar Hepialus gonggaensis larvae, an economically important insect. [Methods] The effect was observed by molecular techniques based on the sequence of 16S rDNA V3 region gene and PCR/DGGE (Denaturing gradient gel electrophoresis). The health larvae were divided randomly into treatment group1, treatment group2 and control group, and fed on their natural food added with different amount of Carnobacterium sp. Hg4-03 ( group 1 and group 2) or ddH2O ( control). After 14 days and 28 days, 6 larvae from each group were randomly selected to be dissected and contents of guts were collected for bacterial diversity analysis. [Results] The result showed that bacterial diversity index of treatment groups with Hg4-03 was higher than control group, and the species of bacteria increased. Comparing the bands of DGGE profiles between three groups, the intensity of band stand for Carnobacterium sp. Hg4-03 have enhanced. Another band with swelled intensity found in profiles come from treatment group2 after 28 d fed, the sequence analysis showed it belongs to Bacillus subtilis. [Conclusion] Feeding with Carnobacterium sp. Hg4-03 could balance the bacteria in larvae gut and increase bacteria diversity, that means the bacteria made a positive effect on the bacterial diversity of H. gonggaensis larval gut. Hg4-03 may be used as probiotic bacteria for rearing H. gonggaensis larvae in controlling conditions or the larvae’s hemi-wild rearing.

Abstract:Abstract: [Objective] Single chain antibody-mediated delivery is a novel approach for targeting shRNA to appropriate cells. In this report, we studied whether this shRNA delivery strategy would be effective against rabies virus. [Methods] Rabies virus scFv(G) gene and ETA-GAL4 gene were amplified by PCR from vector scFv(G)-T and PE40-GAL4-T respectively. Then, the chimeric gene scFv(G)-ETA-GAL4 was constructed by lapextension PCR and cloned into the prokaryotic expression vector pET28a(+). Recombinant expression plasmid of pET28a(+)-scFv(G)-ETA-GAL4 was constructed and then transformed into the competent E.coli BL21(DE3) cells for expression under the induction of IPTG. scFv(G)-ETA-GAL4 protein was purified by Ni-NTA His Bind Resin affinity chromatograph and identified by SDS-PAGE gel and Western blot assay. Binding of the fusion protein scFv(G)-ETA-GAL4 to rabies virus was determined by ELISA. Complexes which formed spontaneously by the fusion protein scFv(G)-ETA-GAL4 with plasmid pRNATU6.3-shRNA were added to BHK-21 cells culture medium that infected with RV. Green fluorescent protein (GFP) was observed after 35h and judged the transferring efficiency of the complexes. The inhibition of RV replication by shRNA was detected by direct immune fluorescence test. [Results] A 1557 bp DNA encoding scFv(G)-ETA-GAL4 protein gene was cloned and successfully expressed in inclusion body with approximate molecular weight of 57.0 KDa, which could be recognized by anti-His mAb. The scFv(G)-ETA-GAL4 proteins were purified by Ni-NTA column , and after renatured with the yield of 2.8 mg/mL. The ELISA results showed that when concentration of the scFv(G)-ETA-GAL4 protein ranging from 2.8 nmol/L to 1000 nmol/L, binding affinity is directly related with RV. The GFP expressed in BHK-21 cell after transfection with the complexes and effectively inhibited RV replication in BHK-21 cell. [Conclusion] scFv(G)-ETA-GAL4 fusion protein could mediated plasmid pRNATU6.3-shRNA transferred into BHK-21 cell infected with RV, and then inhibited RV replication.

Abstract:Abstract: [Objective] Gangliosides on the cells surface can act as the Newcastle disease virus receptors, but the differences of receptor specificities between Newcastle disease virus strains isolated from different avian species have not been determined. Accordingly, we attempted to determine the difference of binding specificity to gangliosides between two Newcastle disease virus NA-1 (goose-origin) and F48E9 (chicken-origin). [Methods] Gangliosides were extracted from chicken embryo fibroblast cells (CEF) and goose embryo fibroblast cells (GEF). The extracted gangliosides were characterized and quantified by high performance thin-layer chromatography. We performed virus overlay assays and hemadsorption inhibition assays to assess the binding specificity of NA-1 and F48E9 to different gangliosides. [Results] The results showed that ganglioside compositions of the CEF and GEF were different. In the thin-layer chromatographic binding assay, we analyzed the binding of the virus to different gangliosides, detecting that NA-1 mainly bound to GD1a, F48E9 mainly bound to GM1, GD1a, GD1b. [Conclusion] In conclusion, our results suggest that two viruses used different receptors for entry into different target cells.

Abstract:Abstract: [Objective] Bursin (BS) could greatly promote the production of monoclonal antibody in hybridoma cell. We studied the interaction between BS and hybridoma cell. [Methods] Fluorescence microscopy, Laser Scanning Confocal Microscope (LSCM) and fluorescence activated cell sorter (FACS) were used. [Results] Fluorescence microscopy revealed specific binding of FITC-BS to hybridoma cell. FACS analysis demonstrated that the binding was specific, saturable and reversibility. BS was used as target protein to screen its binding peptides from 12-mer random phage display peptide library. After four rounds of biopanning, 20 phage clones were randomly selected and identified. ELISA and competitive inhibition test results indicated that 2 phage clones were identified as positive clones. The amino acid sequences analysis shown that the sequences were ACTKHLCLLQPL or MSCNDTLCLLPN, which sharing a conservative sequence LCLL. In vitro experiments suggested that the two binding peptides can inhibit BS specific binding to hybridoma cell. [Conclusion] These results confirmed that existence of BS receptor in the membrane of hybridoma cell, which involved in hybridoma cell secreting monoclonal antibody signal pathway.

Abstract:Abstract: [Objective] Most of microbial extracellular polysaccharide (EPS) has the favorable functionality and peculiarity, but little research has been done about EPS that were synthesized by yeasts. Screening of EPS-producing yeasts and studies on the optimal culture composition are the main purpose of this study. [Methods] Yeasts were obtained by screening from natural samples using spread-plate method, Yield of EPS synthesized by yeasts was estimated by phenol-sulfate acid method, Based on 5.8S rDNA sequence determination, three strains were identified and selected for the optimization of fermentative medium. [Results] 132 yeast strains were isolated from grapes, preserves and topsoil. Three yeast strains were proved to be as high exopolysaccharide-producing yeasts. The results of 5.8S rDNA sequence determination showed that similarities of two yeasts isolated from “Zuoyouhong” grape (Z14 and Z20) were over 99 % compared with Issatchenkia orientalis, meanwhile another yeast L25 isolated from topsoil of Larix gmelini was identified as Cryptococcus humicolus, with the similarity of 98.8 %. The optimal medium for strain Z20 for producing EPS was as followed: 8 % glucose, 0.2 % (NH4)SO4 , 0.1 % KH2PO4 , 0.1 % yeast extract powder and 0.01 %CaCl2 . Under the condition of initial pH 6.0, 28 ℃ and 160 r/min for 4 d, yield of EPS could reach to 2.046 g/L, which was 79.9 % higher than the control (1.137 g/L). [Conclusion] Reports that yeasts belong to some genera possessed the abilities to synthesize EPS could be seen in some related references, according to the study, we could get the conclusion that yeasts belong to Issatchenkia can synthesize EPS and EPS yield of stain Z20 could be increased by changing culture composition.