Abstract:Classical swine fever (CSF), caused by classical swine fever virus (CSFV), has been epidemic or endemic in many countries, and causes great economical losses to pig industry worldwide. Attenuated vaccines (such as C-strain) have played an important role in the control of CSF. Recently some new phenomena appear, such as atypical and persistent infections of CSF, immunization failure and so on. Meanwhile, eradication programs have been implemented in many countries, restricting the widespread applications of attenuated vaccines. Thus, currently the priority is to strengthen the research in pathogenesis and transmission mechanisms, as well as to develop marker vaccines. Recently, the applications of reverse genetics technology open up a new way for research of structure and function of CSFV proteins and development of novel vaccines against CSF. This review focuses on the progress of applications of reverse genetics in the functional analysis and marker vaccine development of CSFV, and also discusses the problems confronted now and prospective aspects in the study of CSFV.

Abstract:Chitosan has natural abundance, unique bioactivity and attractive physicochemical properties. Recent years, the synthesis of chitosan-based metal nanomaterials has attracted increasing attention. The synthesis of metal nanoparticles utilizing biomelocular or organism offers a mild medium, and thus a greater degree of control over the nanoparticles produced, along with higher reproducibility. In particular, preparation of metal nanoparticles based on biomelocular or organism has its unique facility in integrating “minimum feature sizes” into labile biological components to an excellent synergy and bifunctional effect and consequently a more broad application. Herein, we review the new development of chitosan, chitosan-based synthesis of metal nanomaterials, and their application.

Abstract:Marker-free plants have been public concern. Co-transformation and site-specific recombination system are more important methods in self-gene excision. We reviewed the Cre/lox site-specific system and its applications in plants, also, we discussed perspectives of the system in according with our experience.

Abstract:In order to construct the recombinant retrovirus vector of bovine sox2 gene and obtain infectious retroviral particles, we successfully amplified the ORF (open reading frame) of bovine sox2 gene from the primodial genital ridges of bovine embryo by RT-PCR. The cDNA of ORF was subcloned to pMD18-T vectors and verified that its sequence was highly homologous to the GenBank counterpart (GenBank Accession No. NM-001105463) by sequencing. The correct fragment was digested by EcoR I/Bgl II from recombinant pMD18-T vector and inserted into the same restriction sites of retroviral vector pMSCVneo. We got recombinant retrovirus vector pMSCV-sox2 which was transfected into PT67 by lipofectamine 2000 with pMIG (including green fluorescence protein) as a control. Flow cytometry analysis showed that its transfected efficiency was 68.3%. Subsequently, we established the stable cell strain by G418 selection which could produce virus. Its viral titer was up to 8.16×107 CFU/mL. This greatly facilitates the further study of bovine induced pluripotent stem cells induced from bovine somatic cells by defined factors.

Abstract:To prepare recombinant fox growth hormone (fGH), we amplified its cDNA from silver fox pituitary tissue by RT-PCR and cloned into yeast shuttle vector pPIC9K down stream of α-factor signal peptide sequence by SnaB I and Not I restriction sites. The recombinant secretion vector pPIC9K/fGH, linearized by Sal I, was transformed into histidine-deficient Pichia pastoris strain GS115 by electroporation. We selected His+-transformed methylotropic (His+, Mut+) yeast using histidine-absent medium containing dextrose (MD) or methanol (MM) as the only carbon source, and then screened the recombinant GS115 with multi-copy fGH genes by G418. The secretive expression of fGH was performed under the induction of methanol in shaking flask culture. The results showed that the fGH cDNA sequence amplified in this paper was basically in consistence with the published in GenBank. We achieved the secretive expression of recombinant fGH identified by SDS-PAGE and Western blotting. The fGH expression level was 119 mg/L, accounted for 34% of total proteins in fermentation medium.

Abstract:We prepared the nano-sized magnetic fluid in aqueous phase solution to modify the killed yeast cells (Saccharomyces cerevisiae). Subsequently, the yeast cells with good magnetic response was obtained.The infrared spectroscopy analysis showed that the modified yeast cells increased obviously the absorption at the Fe-O characteristic peak of 581 cm-1. Moreover, the transmission electron microscopy picture indicated the magnetic nanoparticles presented individually or aggregately on the yeast cell surface. Under the experimental conditions, the magnetically labeled yeast with 160 μL showed the adsorption percentage up to 100% in 1 mL direct scarlet dye solution with the concentration of 0.4 mg/mL, and arrived at the adsorption equilibrium within 8 min. Besides, the adsorbed dyes can be eluted in 70% alcohol with a desorption percentage of 99.18%. Because of the strong adsorption capacity, the high adsorption speed and easy of magnetic separation, the magnetically modified yeast cells are the promising biosorbent candidates for removal of water-soluble dyes.

Abstract:Dehydroascorbate reductase (DHAR) plays an important role in the recycling of ascorbic acid. In this work, we isolated the full length cDNA clones of two different DHAR genes (tentatively named as TaDHAR1 and TaDHAR2, respectively) from common wheat. Semi-quantitative PCR experiments showed that TaDHAR1 and TaDHAR2 were transcribed in many vegetative and reproductive organs examined in this work. Transient expression analysis using wheat protoplasts indicated that the protein products of TaDHAR1 and TaDHAR2 may be located in the cytoplasm. The cDNAs of TaDHAR1 and TaDHAR2 were expressed in the bacterial cells, and resultant histidine tagged recombinant proteins could be efficiently purified using nickel chelate affinity chromatography. In vitro enzyme activity assays revealed that the recombinant TaDHAR1 and TaDHAR2 proteins could all convert dehydroascorbate (DHA) to AsA. The two proteins exhibited higher activity levels at 37oC than at 25oC. Under the two temperature conditions, the optimal pH for TaDHAR1 and TaDHAR2 was both around 7.5. The major difference between TaDHAR1 and TaDHAR2 is the activity under pH 6.0 and 7.0 at 25oC. The results and resources obtained in this study may be useful for further research into the physiological role of TaDHAR genes in AsA metabolism in crop plants under normal or stressed conditions.

Abstract:Crude elicitor of one endophytic fungi (belong to Cunninghamella sp., named AL4) induced multiple responses in Atractylodes lancea suspension cells, including rapid generation of nitric oxide (NO) and hydrogen peroxide(H2O2), sequentially followed by enhancement of essential oil production. Adding NO-specific scavenger 2-4-carboxyphenyl-4,4,5, 5-tetramethylimidazol ine-1-oxyl-3-oxide (cPTIO) and H2O2 scavenger catalase (CAT) could block elicitor-induced NO and H2O2 generation respectively, but could all partly block elicitor-induced essential oil biosynthesis. Adding NO-donor sodium nitroprusside (SNP) and H2O2 could all promote essential oil accumulation in A. lancea cells, but the effect of both was different. These results strongly suggested that NO and H2O2 may all act as signaling molecule to mediate AL4 elicitor promoting essential oil accumulation in suspension cells of A. lancea. Furthermore, adding cPTIO and CAT contemporarily could not completely inhibit essential oil accumulation induced by AL4 elicitor. This result suggested that AL4 elicitor could also promote essential oil accumulation in suspension cells of A. lancea by other means.

Abstract:We formulated a selective enrichment broth (SVV) for simultaneous growth of Salmonella, Vibrio parahaemolyticus, and Vibrio cholerae by single factor experiment and response surface method. We evaluated the enrichment effect of SVV by conventional culture method and real-time PCR assay. We obtained the SVV broth by supplementting the Buffered Peptone Water (BPW) with bile salt no. 3, potassium tellurite, and sodium citrate as inhibitors, and glucose, mannitol, snhydrous sodium sulfite and sodium pyruvate as accelerants. We also modified the concentration of sodium chloride in BPW. When mixed at equal or varied proportions, the target pathogens had a great accumulation (105-108 CFU/mL) after incubated in SVV for 18 h at 37oC with shaking. It can also effectively inhibit the competitive microflora. We detected 10 artificial simulated samples and 608 real samples using SVV with real-time PCR. After enriched in SVV for 18 h, the quantity of the bacteria in samples were above the detection limit. The SVV with PCR assay showed higher tested positive (4.06%) compared to that of the converntional detection method (3.78%) and there was no false report. In summary, SVV is a promising new multiplex selective enrichment broth that can be used in detection of seafood.

Abstract:In this work, we systematically analyzed the secondary structure amino acid compositions of acidic and alkaline enzymes and compared them with neutral ones. We found that the propensity of the individual residues to participate in secondary structures and the consistently higher composition of neutral and tiny residues might be the general stability mechanisms for their adaptation to pH extremes. Based on this, we presented a secondary structure amino acid composition method for extracting useful features from sequence. The overall prediction accuracy evaluated by the 10-fold cross-validation reached 80.3%. Comparing our method with other feature extraction methods, the improvement of the overall prediction accuracy ranged from 9.4% to 18.7%. The random forests algorithm also outperformed other machine learning techniques with an improvement ranging from 2.7% to 21.8%.

Abstract:The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZα-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPV16L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37°C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.

Abstract:Phospholipase D (PLD) hydrolyzes phosphocholine into choline and phosphatide acid, and these metabolites play an important role in regulating cell physiology and biochemistry. To study the biological function of phospholipase D3 (PLD3) during the insulin stimulation in C2C12 myoblasts, we constructed PLD3 over-expressed cell lines (C2C12/pPLD3) and investigated the phosphorylation of Akt. The results showed that the level of phosphorylated Akt (P-Akt) was significantly increased in control C2C12 cells when insulin concentration was elevated during cell treatment, whereas the level of P-Akt in C2C12/pPLD3 cells was not changed. When extending the time of insulin treatment, P-Akt level in C2C12/pPLD3 cells was increased around 2 folds, but the total level of P-Akt in C2C12/pPLD3 was still lower than that in control group. 1-Butanol, a PLD inhibitor, could completely block Akt phosphorylation in C2C12 cells that even stimulated by insulin. However, 1-Butanol did not inhibit the Akt phosphorylation in C2C12/pPLD3 cells, but increased the phosphorylation up to 6 folds higher than control cells. The level of Akt phosphorylation in control C2C12 cells was increased significantly when stimulated by phosphatidic acid (PA), while there was no change in C2C12/pPLD3 cells with the similar treatment. When cells simulated by both PA and insulin, P-Akt level in both C2C12/pPLD3 cells and C2C12 cells were down regulated. Our observations indicated that PLD3 over expression may inhibit Akt phosphorylation and further block the transduction of insulin signaling in C2C12 cells.

Abstract:To express the domain III gene of Japanese encephalitis virus (JEV) and to learn the possibility of developing the DIII protein as a subunit vaccine, we amplified the JEV DIII gene by PCR and constructed the expression plasmid pET-JE DIII by inserting JEV DIII gene into the prokaryotic expression vector pET-32a(+). The domain III protein of the attenuated strain SA14-14-2 was expressed as a thioredoxin (Trx) fusion protein, which was unique in forming a large fraction of the soluble recombinant protein. We immunized the rabbits and mice with the purified protein, tested the antigenicity and immunogenicity of JEV DIII protein by ELISA, Western blotting, plaque reduction test and observed the protective efficacy on challenged weanling mice with JEV. Rabbits immunized with the purified JEV DIII protein generated 1:7×105 anti-JEV specific antibody titers. BALB/c mice immunized with the purified JEV E DIII protein generated 1:8.2×104 anti-JEV specific antibody titers. And the neutralized antibody titer can reach 1:256, the survival rate of the immunized weanling mice was approximately 75%. Overall, this study highlighted that recombinant JEV E DIII protein delivered in mice and rabbits can generate high antibody titers against JEV, and protect some mice challenged with JEV. These studies can provide useful information for further developing the domain III recombinant protein as subunit vaccine against JEV.

Abstract:To study the inhibitory effect and anti-cancer mechanisms of interleukin 24 (IL-24) on human osteosarcoma cel1 MG-63, we delivered IL-24 into MG-63 cells in vitro and in vivo by adenovirus. The expression level of IL-24 was detected by RT-PCR and fluorescence microscope; the growth inhibition, apoptosis rate and apoptosis body were measured by MTT, Flow cytometry and Hoechst staining respectively. Furthermore, we analyzed the expression of bcl-2, bax, caspase3 genes by RT-PCR after overexpression of IL-24. For in vivo study, we first established the MG-63 tumor model by grafting MG-63 cells in athymic nude mice; and then injected Ad-IL-24 into the tumors. Two weeks after injection, we sacrificed the mice, removed the tumors, weighed and calculated the ratios of tumor-suppression. We also detected the expressions of Bcl-2, Bax, Caspase-3 and CD34 with immumohistochemistry. Our in vitro results indicated that Ad-IL-24 was transcribed and translated in MG-63 osteosarcoma cells. More interestingly, IL-24 inhibited the growth of MG-63 cells and induced apoptosis by up-regulation of bax, caspase-3 and down-regulation of bcl-2. The in vivo data showed that IL-24 suppressed the tumor growth conspicuously through down-regulating the expression of bcl-2, and up-regulating the expression of bax, caspase-3. This study would provide evidence for the gene therapy of IL-24 on osteosarcoma.

Abstract:We constructed and identified cardiac-specific α-myosin heavy chain (α-MHC) promoter-driven expression vector. α-MHC promoter was amplified by PCR by using mouse genomic DNA as template and inserted into pGEM?-T Easy vector. The inserted fragment was released by enzyme digestion, and then the cytomegalo virus (CMV) promoter in pcDNA3.1(+)-EGFP-hygro vector was replaced by the α-MHC promoter to construct α-MHC-EGFP expression vector. After identification with enzyme digestion, α-MHC-EGFP was transfected into mouse primary cardiomyocytes by electroporation. Green fluorescence could be observed in transfected cardiomyocytes, but not in transfected non-cadiomyocytes. α-MHC-EGFP expression vector was specifically expressed in cardiomyocytes, and could be used to purify embryonic stem cell-derived cardiomyocytes.

Abstract:We chose Gaussia luciferase (Gluc), a secreted luciferase gene as reporter to real-time detect and dynamically monitor hydrodynamic injection gene expression. First, we constructed an expression vector pAAV2neo-Gluc. Then Huh7 and HepG2 cells were transfected with pAAV2neo-Gluc and the activity of Gluc in the supernatant and cell lysates were assayed. Results showed that the Gluc activity in the supernatant was about 100 higher than that in cell lysates, indicating the expressed Gluc existing mainly as a secreted form as reported. Live bioluminescence imaging of mice hydrodynamic injected pAAV2neo-Gluc showed whole body distribution, while the pAAV2neo-Fluc primarily located in the liver. Then we injected different doses of pAAV2neo-Gluc into mice by tail-vein hydrodynamic injection, took minor amount of blood from mice tails at different time points and measured the luciferase activity to investigate dynamic changes of Gluc expression and secretion in vivo. The results suggested that the time courses of Gluc expression were highly consistent among each dose groups. The luciferase activity in blood could be detected as early as 2 h after injection, reached the peak at about 10 h and gradually decreased from then on. The expression level of Gluc was positively correlated with the dose of injected plasmid DNA. To further detect the assay sensitivity of the ex vivo Gluc measurement method, we investigated three additional groups of mice injected with lower doses of 0.001 μg, 0.01 μg and 0.1 μg pAAV2neo-Gluc respectively. Results revealed that activity of Gluc in blood could be detected even at dose as low as 0.001 μg DNA, suggesting the assay sensitivity was extremely high. In conclusion, a real-time ex vivo detection method of dynamically monitoring of gene expression in vivo by hydrodynamic injection can be a valuable means for the study of gene expression regulation in vivo.

Abstract:In order to improve the transduction efficiency of insect baculovirus in mammalian cells, we constructed two recombinant baculoviruses, AcRed-tat and AcRed. Both viruses expressed red fluorescence protein gene (dsRed) as a reporter in mammalian cell lines. AcRed-tat also contained the coding sequence of HIV-1 Tat transduction peptide fused with viral major capsid protein gene vp39 and enhanced green fluorescence gene (egfp) driven by virus polyhedrin promoter. It expressed the Tat fusion protein in infected insect cells, which was incorporated into the nucleocapsids of progeny virus. As a control, AcRed had the fusion gene of vp39 and egfp driven by polyhedrin promoter. Flow cytometry analysis demonstrated that although similar level of red fluorescence was produced in HEK23 cells transduced by the two recombinant viruses, significantly higher red fluorescence level was seen in CHO and Vero cells transduced by AcRed-tat than that by AcRed. These results suggested that Tat transduction peptide might improve the baculovirus-mediated gene expression in some mammalian cells. Our work provided a new approach to improve baculovirus as a gene delivery vector for mammalian cells.

Abstract:Human hepatitis B virus surface antigen (HBsAg) binding protein(SBP) shows a specific binding ability to HBV surface antigen HBsAg. Previous work proved an ability of SBP to enhance the immune response of HBsAg vaccine. To investigate the function and mechanism of this protein, we constructed SBP-expression strains with Pichia pastoris expression system. We screened these strains and have got an expression strain with high protein expression quantity. Fermentation product was collected and purified to gain a large amount of purified protein. Identification of purified SBP with SDS-PAGE, High performance liquid chromatography, Western blotting and mass spectrometry suggested that the protein was highly purified and with a good integrity. ELISA test of purified SBP showed a significant binding ability to HBsAg, suggesting a good protein activity. This work offers a solid foundation to the research of SBP function and mechanism of immune enhancement.

Abstract:Anti-HBcAg monoclonal antibodies from mouse ascites were purified by using immobilized metal ion affinity chromatography. We optimized the conditions of sample loading and elution. The results showed that when the pH stepwise elution was used, the best solution for sample loading was 20 mmol/L phosphate buffer containing 0.5 mol/L sodium chloride at pH 8.0 and the mAb was eluted at pH 5.0. The purity of obtained mAb was more than 85% and recovery reached 80%. When the adsorbed proteins were eluted by using gradient elution of an imidazole, the best solution for loading condition was 20 mmol/L phosphate buffer containing 5 mmol/L imidazole at pH 8.0. The purity and recovery of antibody were up to 95%.

Abstract:We constructed the recombinant adenovirus expressing the glycoprotein G2 of Hantaan virus. Firstly we obtained coding gene fragment of G2 by PCR, and subsequently inserted the gene of interest into the Adenoviral pShuttle vector pAd5-CMV. Then we co-transfected the recombinant pShuttle vector and adenovirus skeleton plasmid into HEK293 cells by Calcium phosphate precipitation method. After the recombinant adenovirus were packaged and amplified in HEK293 cells, we observed the expression of reporter gene eGFP by fluorescent microscopy, and we obtained the recombinant adenovirus containing Hantaan virus glycoprotein G2. The recombinant adenoviruses were used to infect Hela cells and the expressed protein was detected by Indirect Immuno-fluorescence and Western blotting. The construct was confirmed at several levels: first restriction enzyme analysis demonstrated that the recombinant adenovirus vector was constructed correctly, second RT-PCR showed that the G2 gene could transcribe correctly in Hela cells. Then Fluorescent microscopy proved the expression of eGFP in the infected Hela cells. Finally, Indirect Immuno-fluorescence and Western-blot confirmed the expression of interested protein identified by anti-G2 monoclonal antibody. In conclusions, this study successfully constructed the recombinant adenovirus containing Hantaan virus envelope glycoprotein G2, meanwhile obtained the G2 protein, it may lay solid foundation for the structure study of G2 protein and the new vaccine of Hantaan virus.

Abstract:To study the inhibitory effect of a recombinant adenoviral vector carrying human IL-24 gene on SGC-7901 human gastric cancer cell. We infected the SGC-7901 gastric cancer cells with Ad blank adenovirus at various multiplicity of infection (MOIs) to find the optimal infective dose. The SGC-7901 tumor cells were infected with Ad-IL-24 at the optimal MOI in the following experiments. Adenovirus-mediated IL-24 transcription expression in SGC-7901cells was examined by RT-PCR. The growth-suppressing effect of Ad-IL-24 on SGC-7901 tumor cells was assessed by MTT assay. Apoptosis and cell cycle of SGC-7901 tumor cells infected with Ad-IL-24 was evaluated by flow cytometer (FCM), respectively. The karyomorphology of apoptotic SGC-7901 tumor cells was examined using Hoechst33258 staining under fluorescence microscopy. The expression of apoptosis-related genes was future determined by semi-quantification RT-PCR; We demonstrated that the MOI of 100 was the optimal infective dose in the study on adenovirus-mediated IL-24 gene transfer into SGC-7901 gastric cancer cell; IL-24 gene mediated by adenovirus could successfully transcribe in SGC-7901 tumor cells; Ad-IL-24 could significantly inhibit SGC-7901 tumor cell growth and induce apoptosis, it also can up-regulate the express of bax, caspase-3 and p53 whilst down-regulate the bcl-2 expression. Thus, adenovirus-mediated IL-24 expression had marked anti-tumor effect in suppressing SGC-7901 human gastric cancer cell growth and inducing apoptosis, which may be closely associated with its up-regulation of bax/bcl-2, caspase-3 and p53.