Abstract:Ebola virus (EBOV) causes hemorrhagic fever, resulting in mortality rates as high as 90% among infected humans and non-human primates (NHPs). The 2014 Ebola epidemic in West Africa is the severest in history, leading to WHO taking all control measures to stop any possibility of cross-border outbreaks.?Because no licensed vaccines or effective therapeutics against EBOV are available, the current outbreak management has been limited to palliative care and barrier methods to prevent transmission. Several promising experimental EBOV vaccines have demonstrated protection in NHPs against lethal EBOV challenge, and some progresses have been made through clinical trials of EBOV vaccine candidates. It is believed there will be some licensed vaccine available in the near future to control EBOV outbreaks. In this review we provide some insights for further development of EBOV vaccines.

Abstract:Chlorella is nutritious and has been used as a functional food much earlier than the other microalgae. C. pyrenoidosa, the potential microalgae which is currently cultured and developed for the new strategic industry of biofuels production and biological CO2 fixation, is a new resource food announced by the Ministry of Health of the People’s Republic of China late 2012. Accumulation of high value-added substances in C. pyrenoidosa during the cultivation for lipid makes it possible to reduce the costs for C. pyrenoidosa-based biofuels production. Among these potential substances, hot water extract of Chlorella (CE), commercially known as “Chlorella growth factor”, is the unique one that makes Chlorella more precious than the other algae, and the market price of CE is high. It is believed that CE is effective in growth promotion and immunoregulation. However, there is no systematic analysis on the research status of CE and its bioactivity. The present report summarized recent research progress of CE and its bioactivity. Generally, besides the main effect on immunoregulation and tumor inhibition, CE was efficient in improving metabolic syndrome, scavenging for free radicals, protecting against ultraviolet damage, chelating heavy metals, and protecting liver and bowel. Several major challenges in CE research as well as its prospects were also analysed in the present report.

Abstract:Filamentous fungi have been used in industrial fermentation extensively. Based on non-phosphorylating electron transport process, alternative respiration pathway (ARP) acts as an energy overflow, which can balance carbon metabolism and electron transport, allow the continuance of tricarboxylic acid cycle without the formation of ATP, and permit the turnover of carbon skeletons. Alternative respiration pathway also plays an important role in the stress response of fungi and the physiological function of conditioned pathogen. Alternative oxidase (AOX) is the terminal oxidase responsible for the activity of alternative respiration pathway, which exists widely in higher plants, parts of fungi and algae. Owing to the property that alternative oxidase (AOX) is sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of cytochrome respiration, alternative respiration pathway by AOX is also named as cyanide-resistant respiration (CRR). In recent years, the study of the alternative respiration pathway and alternative oxidase has been a hot topic in the area involving cellular respiration metabolism. In this review we summarized the latest research advances about the functions of alternative respiration pathway and alternative oxidase in industrial fungi.

Abstract:Insecticidal protein genes from Bacillus thuringiensis are currently the most widely used insect-resistant genes. They have been transferred to many crops for breeding and production. Among them, cotton, maize, potato and other insect-resistant crops are commercialized, creating considerable economic benefit. In this review, we summarized advances in identifying functional genes and transgenic crops for insect resistance, compared different strategies for enhancing vigor of insecticidal protein and utilizing gene stacking as well as listing valuable groups of stacked genes. In addition, the methods for multiple gene transformation was discussed.

Abstract:New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.

Abstract:In order to develop monoclonal antibodies (McAbs) against the gp90 protein of reticuloendotheliosis virus (REV), the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas (3G5-B8, 3G5-A10 and 1G12) that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.

Abstract:Bombyx mori bidensovirus (BmBDV) has been identified as causing chronic densonucleosis in Bombyx mori specifically. The replication mechanism of BmBDV remains unknown. Its genome comprises two single stands DNA (VD1 and VD2). In order to rescue infectious virions in vitro, we obtained the total viral DNA extracted from the BmBDV-infected larvae midguts, subsequently cloned the full-length sequence of BmBDV genome fragments by PCR and constructed recombinant plasmids pMD18T-VD1 and pUC-VD2. The linear genome fragments were obtained by digesting recombinant plasmids with corresponding restriction enzymes, and then collectively transfected BmN cells by the method of liposome-embedding. We determined the replication of the virus gene by PCR with the template of demethylated total DNA extracted from the post-transfect BmN cells. Meanwhile, we collected the total proteins from the post-transfect BmN cells and the larvae midgut of feeding the post-transfect BmN cells to perform Western blotting analysis, and detected the expression of viral genes. Here we firstly confirm that infectious virions can be rescued in BmN cells by linear co-transfect method.

Abstract:To generate an epitope-mutated foot-and-mouth disease virus (FMDV) as a marker vaccine, the infectious clone pAsia 1-FMDV containing the complete genomic cDNA of Asia 1 type FMDV was used as backbone, the residues at positions 27 and 31 in the 3D gene were mutated (H27Y and N31R). The resulting plasmid pAsia 1-FMDV-3DM encoding a mutated epitope was transfected into BHK-21 cells and the recombinant virus rAsia 1-3DM was rescued. The recombinant virus showed similar biological characteristics comparable with the parental virus. In serological neutralization test the antisera against recombine virus have a good reactivity with parental virus. The antisera against the mutant virus were shown to be reactive with the mutated epitope but not the wild-type one. The results indicated that the two virus strains could be distinguished by western blotting using synthetic peptides. This epitope-mutated FMDV strain will be evaluated as a potential marker vaccine against FMDV infections.

Abstract:Thymidine is a commercially useful precursor for production of antiviral compounds such as stavudine and azidothymidine. Biosynthesis of thymidine by Escherichia coli BL21 (DE3) was studied using metabolic engineering methods. The deoA, tdk and udp of the salvage pathway were disrupted from E. coli BL21 to construct BS03 that produced 21 6 mg thymidine per liter. Additional deletion of pgi and pyrL increased the supply of thymidine precursors and the resulting strain BS05 produced 90.5 mg thymidine/L. At last, ushA, thyA, dut, ndk, nrdA and nrdB of thymidine biosynthetic pathway were overexpressed, and the resulting strain BS08 produced 272 mg thymidine/L. In fed-batch fermentation, BS08 accumulated 1 248.8 mg thymidine/L. Metabolically engineered strain E. coli has potential applications for thymidine production.

Abstract:Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoRⅠ/XbaⅠ, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZαA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.

Abstract:IFIT family genes are a kind of interferon stimulated genes (ISGs), and play important roles in antiviral sector and immunity regulation. To study the regulatory effect of IFIT family genes during influenza A virus (IAV) infection, we used RNA-sequencing analysis (RNA-Seq) technique and found that when 293T cells were infected by A/WSN/33 (WSN), the concentration of IFIT family genes were increased. Further study reveals that overexpression of IFIT2 or IFIT3 could inhibit IAV replication and transcription, and cause the dose-dependent inhibition of polymerase activity of vRNP. In addition, IFIT2 and IFIT3 encoding protein could colocalize with NS1 in 293T cells infected by WSN, indicating that they might interact with each other. The results suggest that IFIT family genes can inhibit the replication and transcription of IAV, which contributes to our understanding of the regulatory effect of host factors during influenza virus infection.

Abstract:Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10?15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaTcell was also detected, and the repression of H2A.X foci formation was not due to hCRY1’s cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.

Abstract:Cell growth profiles were evaluated in shake-flask culture to improve sclareol production by the engineered yeast strain Saccharomyces cerevisiae S7. Product formation was tightly coupled with cell growth. High cell density cultures were performed with different carbon sources using a dissolved oxygen level feedback-control strategy in a 3 L bioreactor. The titers of sclareol were 253 mg/L, 386 mg/L and 408 mg/L, respectively, when glucose, ethanol and glucose/ethanol mixture were used as the carbons sources. The maximal titer was 27-fold higher than that obtained under shake-flask culture conditions. The results suggested that the presence of ethanol was beneficial to sclareol production. These results provided useful information for optimization of yeast cell factory and efficient production of terpenoids.