Abstract:Abstract: β-mannanases (β-1, 4-D-mannanase, EC 3.2.1.78), as a hemicellulose hydrolase, are widely distributed in bacteria, fungi, plants and even animals. They can randomly hydrolyze the β-1,4-mannosidic linkages in mannan and heteromannan and have great potential in the food/feed, pulp/paper, medicine, oil exploitation and detergent industries. Most β-mannanases often display a modular organization and usually contain structurally discrete catalytic and non-catalytic modules. Catalytic domains of these enzymes share a (β/α)8 –barrel fold, which play important roles in substrate binding and catalysis. Carbohydrate binding modules, as the most common non-catalytic modules, fold as β-sandwich and facilitate the targeting of these enzymes to polysaccharide. In this review, a brief introduction is given concerning structural characteristics and function of these β-mannanase modules.

Abstract:Abstract: Pigs may play an important role in the evolution and ecology of influenza A virus. The tracheal epithelium of pigs contain both SA α 2,6 Gal and SA α 2,3 Gal receptors and can be infected with swine, human and avian viruses, therefore, pigs have been considered as an intermediate host for the adaptation of avian influenza viruses to humans or as mixing vessels for the generation of genetically reassortant viruses. Evolution patterns among swine influenza viruses including evolution of host adaptation, antigenic drift and genetic reassortment, and the latter is the main one. Unlike human influenza viruses, swine viruses have different epizootiological patterns in different areas of world, which is enzootic and geographic dependence. Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2, and these include classical swine H1N1, avian-like H1N1, human-like H3N2, reassortant H3N2 and various genotype H1N2 viruses. In Europe, North America and China, influenza A viruses circulating in pigs are distinct in the genetic characteristics and genetic sources. Since 1979, three subtypes, avian-like H1N1, reassortant H1N2 and H3N2 viruses, have been co-circulating in European swine. Before 1998, classical H1N1 viruses were the exclusive cause of swine influenza in North America. However, after that, three triple-reassortant H1N2, H3N2 and H1N1 viruses with genes of human, swine and avian virus began to emerge in pigs. Genetically, the pandemic viruses emerging in human, so called influenza A (H1N1) viruses, contain genes from both Europe and North American SIV lineages. SIV is not the same as Europe and the United States in the prevalence and genetic background in China, mainly classical swine H1N1 and human-like H3N2 type virus. However, in recent years, SIV from Europe and North America have been introduced into Chinese pig herds, so more attention should be given on the evolutionary of SIV in China. Worldwide, more than 50 cases of SIV infection in human have been documented since the 1970s, which indicate that SIV is also an important zoonosis, and the potential of SIV as human pandemic virus or genes donator. In view of SIV in the importance of ecology, as well as a potential threat to human public health, it is recommended to start as soon as possible regular surveillance, paying close attention to its prevalence and molecular evolution. At the same time, we should establish the surveillance network of the whole influenza virus (including human and animal influenza virus) in China, ecologically mastering the prevalence and evolution of influenza viruses, which is of great significance for the protection of animal health and the prevention of human pandemic.

Abstract:Abstract: Anoxygenic phototrophic bacteria have evolved highly efficient systems-membrane-located pigment-protein complexes which can convert sunlight into chemical energy that they can use and also benefit other organisms. More and more attentions have been paid to pigment-protein complexes of anoxygenic phototrophic bacteria in recent years. We summarized the current opinions in the pigment-protein complexes from anoxygenic phototrophic bacteria, including their chemical compositions, crystal structures and functions, homology of protein sequence. In particular, we depicted the novel light-harvesting complexes namely LH3 and LH4. The problems and prospects about the pigment-protein complexes have also been addressed in this review.

Abstract:Abstract: [Objective] To study the diversity and bioactivities of cultured actinomycete from feces of Ailuropoda melanoleuca, Equus burchelli and Rhizomys sinensis, and Xylocopa dissimilis body. [Methods] Fresh feces samples of animals’ and Xylocopa dissimilis were collected. We used four media to isolate the actinomycete. Strains were identified by phylogenetic analysis of 16S rRNA gene sequences. Hydrolization and enzyme activities of isolates were examined. [Results] Actinomycetes (121) from the samples were isolated on four media. Among them 47 strains belonged to 9 actinomycetes genera. Six known rare genera and one possible new genus of actinomyctes from Rhizomys sinensis were identified. Three possible new species from other three samples were identified. Some strains could hydrolyze chicken feather and cellulose. [Conclusion] It is considered that the coprophilous microorganisms not only have important function on the digestion and absorption of animal feed, but also are important resource for development of industrial products, including enzyme and bioactive metabolites.

Abstract:Abstract: [Objective] To investigate the relationship between quorum sensing (QS) and type III secretion system (T3SS) in Pseudomonas aeruginosa. [Methods] We constructed several gene knockout mutants of QS systems. The promoters of the T3SS genes, exoS , exoY, exoT and exsD-pscA-L were cloned and fused upstream of the luminescence reporter gene cluster, luxCDABE. The reporter constructs were integrated on the chromosome in the wild type strain PAO1 and the QS mutants respectively, and the expression of the T3SS genes in these strains was compared. [Results] The expression of exoS and exoT in pqsR mutant was increased significantly. The Rhl system repressed the activities of the four T3SS genes and the Las system had no effect. [Conclusion] The results indicate that the Rhl and Pseudomonas Quinolone Signal (PQS) systems play an important role in regulating T3SS gene expression in Pseudomonas aeruginosa.

Abstract:Abstract: [Objective] Construction and characterization of a spoIIID gene deletion mutant of Bacillus thuringiensis. [Methods] Scanning electron microscopy and spore formation analysis were used to detect the ability of sporulation and formation of crystal protein in both the mutant and the wild strain. SDS-PAGE analysis was used to detect the expression of crystal protein. [Results] Scanning electron microscopy and spore formation analysis showed that spores were hardly produced and the crystal existed in the spoIIID deletion strain. SDS-PAGE results showed that the expression of cry gene in the mutant was decreased in Luria-Bertani medium, but not affected obviously in Schaeffer’s sporulation medium (SSM). [Conclusion]This indicated that the spoIIID gene was one of the essential genes for the sporulation of Bacillus thuringiensis, and influenced the expression of crystal protein.

Abstract:Abstract: [Objective] To identify genes induced by plant seed exudates in Azorhizobium caulinodans ORS571. [Methods] Using promoterless kanamycin resistance gene (Kmr) on transposon as reporter gene and seed exudates as inducers, we screened genes of interest from transposon insertion mutants libraries. We streaked mutants on TY solid medium with Km, and another with Km and seed exudates correspondingly. If Kmr is inserted into a gene that can be induced by plant signals, Kmr will possibly express at the same time. Thus, mutants were selected that can grow on medium with Km and exudates, rather than on medium with Km. [Results] We identified a lysE family gene named asiE in strain Azc0 that can be induced by seed exudates and further analysis indicated that the inducing substance is canavanine(CAN). lacZ transcriptional fusion of asiE confirmed that its expression increased by ten-fold or so under the induction of CAN. Besides, lysE gene in four different species of Rhizobia can be induced by CAN. lysE mutants are all sensitive to CAN treatment whereas wild type are resistant. [Conclusion] The existence of LysE can make rhizobia better survived in the rhizosphere and may play an important role in early stage of interaction between rhizobia and host plant.

Abstract:Abstract: [Objective] We studied the acid and bile tolerance of Lactobacillus paraplantarumⅡ32, and examined the possible mechanisms of cholesterol reduction. [Methods] StrainⅡ32 was incubated in MRS (de Man, Rogosa, Sharpe) broth supplemented with cholesterol, and the cholesterol in medium was determined using gas chromatography. We examined cholesterol reducing ability under different conditions, and the relation between growth and cholesterol reduction of strainⅡ32. [Results] StrainⅡ32 showed acid resistance, bile salt tolerance and the cholesterol- reducing ability. The results indicated that strainⅡ32 survived at pH2 after 2h culture and the living bacterial number could reach 104 cfu/mL. In addition, the growth of strainⅡ32 was delayed less than 0.5h in MRS broth with 0.3%-0.4% bile salt, when the absorbance value both increased to 0.6 unit. Cholesterol removed by dead and resting cells were 5.64 and 5.90 mg/g of dry weight compared with growing cells, which was 16.98 mg/g of dry weight. Cholesterol removal was greatly associated with growth of the bacteria. The possible mechanisms for removal of cholesterol from media were proposed: assimilation of cholesterol during growth, and binding of cholesterol to the cell surface. [Conclusion] StrainⅡ32 may be promising candidates for use as a dietary adjunct to lower serum cholesterol in vivo.

Abstract:Abstract: [Objective] Lycopene epsilon cyclase (LCYE) is the key enzyme in the lutein synthesis pathway and catalyses linear lycopene to form cyclic ε-carotene, a presucursor of lutein. We aimed to clone the full-length cDNA of LCYE gene from Chlorella protothecoides CS-41, to predict the functional sites and the three-dimensional structure of LCYE through bioinformatics analysis and to confirm its activities and functions. [Method] We used RACE (rapid-amplification of cDNA ends) essay and RT-PCR for the cloning of the full-length cDNA of LCYE from C. protothecoides CS-41. The online software such as PredictProtein, Pfam HMMs and Swiss-Model were used in bioinformatics analysis of the amino acid sequence of LCYE protein. We constructed the expression vector for LCYE gene with pET-28a(+) and transformed into Escherichia coli BL21(DE3). Furthermore, the E. coli strain containing the pAC-LYC plasmid which could accumulate lycopene was used for the functional confirmation of LCYE from C. protothecoides CS-41. [Results] A 2107 bp cDNA（GenBank Accession No.FJ752528）sequence was cloned with 1731 bp open reading frame, encoding a putative LCYE, from C. protothecoides CS-41. Homology studies showed that the deduced amino acid sequence of LCYE gene had a significant similarity with the corresponding sequences of other green algae and higher plants. It shared the highest sequence identity, up to 67%, with the LCYE gene from Chlamydomonas reinhardtii. One typical lycopene cyclase protein domain (Pfam05834) was predicted between the 48th -459th amino acid. In addition, the sequence between 261th -284th was one typical conserved lycopene cyclase protein motif. The SDS-PAGE result showed that the LCYE gene was over-expressed in Escherichia coli BL21(DE3) after the addition of IPTG. The prokaryotically expressed LCYE protein was able to transfer the color of the E. coli strain containing the pAC-LYC plasmid from pink to yellow. [Conclusion] The full-length cDNA sequence of LCYE gene was successfully cloned with the size of 2107 bp. Several typical motifs were found and the three-dimensional structure of LCYE was constructed from Bioinformatics analysis. The generated phylogenetic tree showed the closest relationship between C. protothecoides CS-41 and C. reinhardtii among the listed organisms. Finally, the expression product of the LCYE gene cloned in the study was confirmed to hold the function and activity of lycopene epsilon cyclase.

Abstract:Abstract：[Objective] We studied the effects of different organic solvents and inhibitors on the decolorization of recombinant triphenylmethane dyes decolorization enzyme (TpmD), expressed extracellularly from Pichia pastoris. [Methods] The recombinant TpmD was purified by ultrafiltration and Ni2+ affinity chromatography. The decolorization activity for malachite green was determined by UV-visible spectrophotometer. We studied the effects of some organic solvents and inhibitors on TpmD activity through the rate of decolorization decreased using malachite green as the substance. We also compared the difference between dithicthreitol (DTT) and NADH as the cofactor in assisting the decolorization by monitoring the dissolved oxygen consumption and the end products. [Results] The effect of methanol on the enzymatic activity was weak since TpmD still retained its high activity in the reaction environment containing 10%- 20% methanols. The ethanol and acetone made the enzymatic activity fade away quickly. Dimethylsulfoxide in low concentration was propitious to keep the TpmD activity, although 30% dimethylsulfoxide inhibited the enzymatic activity lost in a half. Ethylenediaminetetraacetic acid (EDTA) of low concentration, L-cysteine and NaN3 all exhibited only weakly inhibitory effects; higher concentrated (25mmol/L) EDTA could strongly inhibit enzymatic activity and sodium dodecylsulfonate (SDS) inhibited the activity completely. The effect of DTT on TpmD activity was beyond the expectation. It could substitute of coenzyme NADH to assist and accelerate the enzyme in decolorizing reaction. We found that the dissolved oxygen consumption behaviors and reaction end products measuring by UV-Visible full wave-scan were completely different between the reactions assisted with DTT or NADH. This is the first report about DTT which can act as a cofactor for a decolorization enzyme. [Conclusion] The effects of different organic solvents and inhibitors on the enzymatic activity of TpmD are very different. By the ?dissolved oxygen assay and the end products analysis, we concluded that the decolorizing reaction assisted with DTT was a novel reaction process. The mechanism of reaction with DTT as cofactor is completely different from that with NADH as coenzyme.

Abstract:Abstract: ［objective］To explore population dynamics of endophytic bacteria and obtain antagonistic endophytic bacteria toward Verticillium dahliae Kleb (Vd), Fusarium oxysporium f.sp. Vasinfectum (Fov) from cotton.［methods］Root, stem and leaf samples were surface-disinfested, and subsequently used to isolate endophytic bacteria by diluting plate counting method. We assayed antagonism of the isolated endophytic bacteria toward three pathogens: Vd (V107, which is a highly virulent defoliating isolate and V396, which is a mildly virulent non-defoliating isolate), Fov (F108) by dual culture method, and analyzed the 16S rDNA sequence of doubly antagonistic endophytic bacteria (DAEB) isolates toward both Vd and Fov.［results］The population size of endophytic bacteria in root was significantly larger than that in leaf and stem. The populations at seedling stage were generally lower than those at the flowering/maturing stage in root, the populations in stem and leaf were fluctuant at different development stages, but variation law was not observed obviously. Furthermore, although no significant differences of the population densities in root were found among 6 cotton cultivars, the population densities in stem and leaf showed cultivar differences. The proportion of endophytic bacteria antagonizing Vd and Fov in root was higher than that in stem/leaf, moreover, the amount of endophytic bacteria antagonizing toward V107 was less than that toward V396/F108. Based on 16S rDNA sequence analysis, all 44 DAEB isolates consisted of two phyla, i.e., Bacteroidetes (1 out of 44) and Proteobacteria (43 out of 44), and fell into 8 genera. The genus enterobacter (18 out of 44) and Pantoea (15 out of 44) were predominant. Notably, ten DAEB isolates demonstrated <97% sequence similarity with the most similar sequences of strain deposited in the Ribosomal Database, these DAEB isolates might be potential novel species.［conclusion］This article suggested that plant genotype, development stage, and tissue influenced the population of endophytic bacteria. We discovered that DAEB with predominant and various genus existed in cotton. Endophytic bacteria in cotton could serve as a pool for discovering biocontrol agent toward cotton pathogens.

Abstract:Abstract：[Objective] To study the effect of inorganic nitrogen fertilizer on nirS-type denitrifiers’ community in the wheat soil.[Methods] We constructed nirS gene libraries of two different treatments of soil: soil fertilized with inorganic nitrogen fertilizer (B-UN) and without nitrogen fertilizer (B-NN). And we used restriction fragment length polymorphism (RFLP) method to analyze the nirS-type denitrifiers’ diversity. [Results] There were 27 operational taxa units (OTUs) in each treatment after MspI and AfaI digestion and only nine OTUs existed in both treatments. The ecological indexes such as Shannon-Wiene index, Simpon index, richness index and evenness index of two different soils were nearly equivalent. However, significant difference was found between the OTU clusters from clone libraries of different treatments. Eleven representative clones were sequenced. The similarities of ten sequences were from 73% to 95% to the sequences of the database. There was one sequence which had no similar nucleotide identities in database. [Conclusion] The application of inorganic nitrogen fertilizer altered significantly the nirS –type denitrifiers’ community in the wheat soil.

Abstract:Abstract: [Objective] To construct an attenuate Actinobacillus pleuropneumoniae serovar 10 strain apxIC-/p36+ for new vaccine development. [Methods] The mutant was constructed by transconjugation and counter-selection and then verified by PCR, western blot and sequence analysis. A transconjugation plasmid pEICALDH was constructed and transformed into donor strain Escherichia coli X7213. After mixing the donor cells with A. pleuropneumoniae acceptor cells, we cultivated the mixture for 6 hours and plated on solid medium containing chloromycetin. Then the CmR positive clones were picked and inoculated into liquid medium without any antibiotic. Cultures were pelleted, plated on sucrose plates and incubated overnight. Finally, Sucrose-resistant colonies(SucBR) were selected and considered as mutant. [Results] Compared with parental strain, the mutant have the same growth rate in vitro and reduced virulence in mice; additionally, the animal experiment indicated that the mutant strain can successfully induce as good immune response as the parental strain, despite of deletion of apxIC gene. [Conclusion] In conclusion, we successfully constructed the attenuate strain apxIC-/p36+ of Actinobacillus pleuropneumoniae serovar 10, and this mutation system will facilitate development of live attenuated vaccines.

Abstract:Abstract: [Objective] To explore the biological function of the interferon stimulation reaction element (ISRE) like motif CTGAAAACGAAAGA within porcine circovirus type 2 (PCV2) Rep promoter. [Methods] Two recombinant PCV2 strains, namely PCV2 1740G-C and PCV2 1741A-T, were constructed by transfecting PK15 cells with site-mutated infectious clone of PCV2 strain Denta. Replication character, genetic stability and reactive character to porcine interferon alpha (poIFN-α) were compared among parental PCV2 and the two mutant viruses. [Results] The ISRE like motif in Rep promoter was not necessary for the replication of PCV2 because two site-mutated viral genome clones both produced infectious virus. In contrast to parental PCV2, the viral antigen positive PK15 cells of the two site-mutated PCV2 were decreased. PCV2 1740G-C was genetically stable in the PK15 cell while PCV2 1741A-T was found to have another two nucleotide mutated from 1744AC1745 to 1744TT1745 between 3th and 7th passage in the PK15 cell. After treated with 100U/mL porcine interferon alpha, the viral antigen positive PK15 cells and virus genomes of parental PCV2 and two site-mutated viruses were both increased. But the enhancement rate of the two site-mutated PCV2 was significantly lower than parental PCV2. [Conclusion] Site-mutation of ISRE like motif in Rep promoter decreased the replication and poIFN-α induced enhancement of PCV2 in PK15 cells. According to these above results, it maybe speculated that ISRE like motif in PCV2 Rep gene promoter contain a functional element and it may contribute to the interferon inducible enhancement of virus replication in PK15 cells.

Abstract:Abstract: [Objective] The study aimed at establishing the bacterial luciferase: FMN-NADH oxidoreductase bioluminescent system in vitro and evaluating its potential for quantitative detection of NADH. [Methods] By optimizing the amount of substrates, we set up the coupled luciferase:FMN-NADH oxidoreductase bioluminescent system in vitro, based on the crude extract from Photobacterium leiognathi YL. [Results] The in vitro coupled bacterial luciferase:FMN-NADH oxidoreductase bioluminescent system was: 1mL crude extract, 27 mmol/L Dodecane 100 μL, 10 mmol/L FMN-Na 0.5 μL and 0.14 mmol/L NADH 300 μL. Furthermore, we developed a method for quantitative detection of NADH according to the bioluminescence of NADH catalyzed of bacterial luciferase: FMN-NADH oxidoreductase system in vitro. A good linear relationship of NADH concentration was in a range of 1.0×10-10 to 1.0×10-8 mol/L. [Conclusion] The bacterial luciferase:FMN-NADH oxidoreductase system used to measure NADH concentration was a good attempt to detect living bacterial cells in the fields of environment, food sanitation and other related.

Abstract:Abstract: [Objective] To construct an E.coli-L.lactis shuttle expression vector with nisI as a food grade selection marker. [Methods] According to the sequence of Nisin resistant gene nisI reported by GenBank, the nisI fragment was amplified by polymerase chain reaction with pLEB590 as template. After being sequenced, the amplicon was confirmed by Blast from NCBI. Then, the nisI was subcloned into the E.coli-L.lactis shuttle vector pMG36e, resulting in the plasmid pMG36e-NisI. The recombinant strain MG1363/pMG36e-NisI was obtained when the plasmid pMG36e-NisI was transformed into L.lactis MG1363 competent cell by electroporation. [Results] When the medium contained 20 IU Nisin/mL, the recombinant strain carrying pMG36e-NisI showed the same growth curve and genetic stability as L.lactis MG1363. [Conclusion] The nisI gene could be used as a selection marker for construction of a food grade expression vector.

Abstract:Abstract: [Objective] In order to study the taxonomic characteristic and physiological, biochemical properties of anaerobic bacteria from hot springs in Tengchong Rehai, Yunnan Province, China. [Methods] Using Hungate anaerobic technique We isolated seven strains from hot springs in Tengchong Rehai, Yunnan province, and analyzed their 16S rRNA gene sequences. [Results] The seven isolates were rod-shaped, Gram-negative, obligate anaerobe, and spores formation was not observed. All strains could grow well at 70℃. Growth of strain RH0802 occurred between 60 and 80℃, optimally around 70℃. The pH range for its growth was between 5.5 and 8.5, with an optimum around 7.0. Strain RH0802 grew on a wide range of carbon sources, including glucose, starch, mannitol, mannose, ribose, maltose, cellobiose, xylose, fructose, galactose, xylan and glycerol, but it could not utilize sucrose or pyruvate. 16S rRNA gene phylogenetic analysis showed that the maximum similarity between the five strains and the strains of genus Caldanaerobacter was up to 98%, except RH0804 and RH0806, which reached to 96% and 93%,respectively. The two isolates were presumed to be potential novel species. The GenBank accession numbers of RH0802 to RH0808 were FJ748766, FJ748762, FJ748761, FJ748763, FJ748765, FJ748764 and FJ748767. [Conclusion] The results showed that the seven thermophilic anaerobes belonged to the genus Caldanaerobacter.

Abstract:Abstract: [Objective] To identify two marine fungi and evaluate the inhibitory effects of their crude extracts on Tobacco mosaic virus and two tumor cell lines. [Methods] Crude extracts was obtained by extracting with MeOH and evaporated in vacuo. The extracts was water-soluble fraction which was dissolved in water, and the other fraction was water insoluble. The fungi were identified by morphology and Internal Transcribed Spcer (ITS) rDNA molecular methods. The inhibitory effect on Tobacco mosaic virus was evaluated by indirect enzyme linked immuno- sorbent assay, and the anti-tumor activity was tested by methyl thiazolyl tetrazolium method. [Results] The fungi were identified as Penicillium oxalicum and Neosartorya fischeri There crude extracts inhibited Tobacco Mosaic Virus and two tumor cell lines. The active fraction named 0312F1 inhibited Tobacco Mosaic Virus and tumor cell lines and was water-soluble. The fraction named 1008F1 inhibited Tobacco Mosaic Virus and was insoluble in water, whereas the fraction inhibited tumor cell lines was water-soluble. [Conclusion] The active fraction named 0312F1 inhibited Tobacco Mosaic Virus was different from that named 1008F1 inhibited Tobacco Mosaic Virus. The active fraction named 0312F1 inhibited tumor cell lines was the same as that named 1008F1. Furthermore, the inhibitory activity of water-soluble fraction named 0312F1 against BEL-7404 cell line was much higher than that against SGC-7901 cell lines, whereas the inhibitory activity of active fraction named 1008F1 against SGC-7901 cell line was much higher.

Abstract:Abstract: [Objective] Lactobacillus acidophilus NCFM is a probiotic strain that promotes human health. We evaluated the influence of Lactobacillus acidophilus NCFM on the genetic expression patterns in the Caco-2 cells. [Methods] Caco-2 cells were treated with Lactobacillus acidophilus NCFM for 2h. The total RNA of cells was extracted. Hybridization with Human Genome U133 Plus 2.0 Array was performed. The image scanning and data analysis were performed subsequently. Genes with significant expression changes were selected and analyzed. To support the microarray data, three striking difference genes were studied using Real-time RT PCR. [Results] Microarrays analysis showed that the expression levels of 508 genes were altered as compared with the control 473 of them were up-regulated, and 35 were down-regulated. It was supposed that many genes in Caco-2 cell were induced, so that Lactobacillus acidophilus NCFM could play a part in immune system process, antioxidant activity, biological adhesion, cholesterol absorption and so on. Three striking difference genes CCL2, PTX3 and TNFRSF9 which involved immune system process were validated by Real-time RT PCR. And the results of Real-time RT PCR showed the same expression trend as in microarray. [Conclusion] Based on the results of gene expression alterations, the beneficial function of Lactobacillus acidophilus NCFM could be more greatly and deeply understood than ever before, and the mechanism of lactic acid bacteria would be revealed.

Abstract:Abstract: [Objective] To construct a universal baculovirus vector for efficient gene expression in both invertebrate and vertebrate cell lines. [Methods] Using the Bac-To-Bac system, we genetically engineered the immediately-early 1 gene promoter (ie1 promoter) from White spot syndrome virus into a baculovirus vector that was pseudotyped with Vesicular stomatitis virus glycoprotein (VSV G). We placed the enhanced green fluorescent protein (EGFP) gene under the control of ie1 promoter in the baculovirus vector to get the reporter recombinant baculovirus, vAc-G-EGFP. We tested the reporter EGFP gene expression in tested cell lines through virus infection or transduction experiments using direct fluorescence microscopy and Western blot analysis. [Results] Under the control of ie1 promoter, vAc-G-EGFP could efficiently express the EGFP reporter gene in both invertebrate and vertebrate cells. The steady-state expression level of EGFP in vertebrate cell lines were different from that in invertebrate Sf9 cells as reflected by Western blot assays. [Conclusion] The ie1 promoter-based and VSV G-pseudotyped baculovirus vector presents a unique and effective tool to express target genes simultaneously in various cell systems; the novel baculovirus-mediated gene expression system developed in this study has the potential to be widely used in both basic and applied research.

Abstract:Abstract: [Objective] To construct the recombinant baculovirus expressing Infectious bursal disease(IBDV) VP2 gene in the chicken primary myoblast cells.[Methods] A proteinase K digestion and phenol-chloroform extraction method was used to extract dsRNA genome from IBDV. VP2 gene was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) with the genome RNA as template. The pFastBac-pCMV-VP2 baculovirus transfer vector was constructed by inserting VP2 gene under the immediate-early promoter of cytomegalovirus. The VP2 recombinant bacmid was obtained by Bac-to-Bac system and transfected sf9 insect cell to acquire VP2 recombinant baculovirus. After amplification of recombinant baculovirus on cell passages, the recombinant virus was seeded on chicken primary myoblast cells with 50 multiplicity of infection (MOI), and the cells were harvested at 72 hours after infection. [Results]Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) and Western blot results showed that the VP2 gene was successfully expressed in chicken primary myoblast cells. The product was a 48kDa protein and could be recognized by anti-IBDV serum.[Conclusion] The recombinant baculovirus could efficiently delivery IBDV VP2 gene into chicken primary cells and that CMV, a mammalian-cell-active promoter, was functional in chicken primary cells and could direct the expression of VP2 antigen protein.The research can be a potential basis for the development of baculovirus vector vaccines for IBDV and other avian infectious disease.