Abstract:Stem cells can be differentiated into many kinds of somatic cells under defined culture conditions. In addition, the homing possess can be partially imitated by co-culture of stem cells with mature somatic cells. Regarding the importance of clinical application of adipose-derived stem cells (ADSCs), our review first introduced the sources and signs of ADSCs, and then the current knowledge of ADSCs co-culture technology, including drug and chemical induced culture, two-dimensional (2D) and three-dimensional (3D) co-culture, mechanisms of ADSCs differentiation, and application development in recent years in details. Finally, we also addressed prospects of ADSCs.

Abstract:As the pathogenic bacterial virulence and avirulence factors, transcription activator like (TAL) effectors of Xanthomonas can resulted in the host diseases or resistance responses. TAL effectors can specifically bind the target DNA of host plant with a novel protein-DNA binding pattern in which two amino acids recognize one nucleotide. The complexities of TAL-DNA binding have the feasibility in use of gene therapy through homologous recombination and site-specific mutation. By using the molecular recognition code between TAL-effectors and host target genes, we can exploit both the susceptible and resistance genes; broad spectrum resistance induced by multiple TAL effectors could also be manipulated. Deeper insight in the area of protein-DNA binding mechanism will benefit the application in the biomedical engineering and agricultural engineering. This article reviews the findings and functions of TAL effectors, the binding specificity and recognition code between TAL-effectors and host target genes. The possible applications and future prospects of the molecular recognition code have been discussed.

Abstract:Secondary metabolites of filamentous fungi are important sources of new drugs, and their biosynthetic processes are regulated by numerous factors. Recent studies indicate that many filamentous fungal secondary metabolites are regulated by epigenetic modifications, which not only affect the titers of secondary metabolites, but also activate the cryptic gene clusters. This review summarizes recent advances of epigenetic application in filamentous fungal secondary metabolite biosynthesis, especially the types of fungal epigenetic modification and epigenetic remodeling of the fungal secondary metabolism. The application of epigenetic theory in filamentous fungi is becoming a new strategy for fungal strain improvement and a powerful method to obtain novel natural products.

Abstract:To evaluate the effectiveness of rabies vaccination, we developed the SPA-ELISA method to detect the antibodies against rabies virus (RV) using the main antigenic determinant of nucleoprotein (RV N1) as antigen. The complete Nucleoprotein (N) gene and the partial N1 gene (1 000?1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. Then we transformed the recombinant plasmids into Escherichia coli BL21(DE3) strain and expressed them by adding 1 mmol/L of IPTG (isopropyl-β-D-thiogalactopyranoside). SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete nucleoprotein, the partial protein (RV N1) was expressed at a much higher level in E. coli BL21(DE3). The antigenic specificity of the partial N1 protein was confirmed by Western blotting. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2 mg/L. The optimal concentration of serum samples and SPA-HRP was 1:100 and 1:4 000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results show that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.

Abstract:According to the amino acid sequence of des-pGlu1-Brazzein, 4 pairs of oligonucleotide with cosmic site were synthesized by using yeasty biased codons. After linkage and PCR, the 179 bp code area of des-pGlu1-Brazzein was obtained and inserted into pPIC9K, which resulted in the recombinant expression vector pPIC9K-Bra. By digestion with Sal I, the lined pPIC9K-Bra was transformed into Pichia pastoris GS115 by electric shock. The results of expression indicted that the secreted target protein accounted for 51.6% of total protein in the supernatant and showed biological activity after purification.

Abstract:We have recombined genes VvSUC11, VvSUC12 from Vitis vinifera L., and root-specific promoters of sweet potato storage protein gene from Ipomoea batatas L. Lam., named as SP1 and SP2. We have constructed a vector pCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12 using pCAMBIA2301 as an original vector. VvSUC11 and VvSUC12 were under the control of root-specific promoters of sweet potato storage protein gene. We transformed the vector into KWS-9103 breeding line of Beta vulgaris L. with Agrobacterium-mediated transformation. We have established the optimal genetic transformation protocol of sugar beet as following: the explants pre-cultured for 4 days were immersed in Agrobacterium suspension of OD600=0.5, supplemented with 0.005% Silwet L-77, and followed by a 4-day culture on medium containing cefotaxime, then the buds were selected on medium containing kanamycin and cefotaxime. The percentage of kanamycin- resistant buds was as high as 42%. Results of PCR and RT-PCR proved that the target genes had integrated into sugar beet genome and expressed. It will lay a foundation for further studying their function in Beta vulgaris.

Abstract:The aim of this study was to reveal the protection role and the related mechanism of cytoglobin on the oxidation induced hepatic stellate cell damage. We applied siRNA to interfere the endogenous cytoglobin gene, used recombinant cytoglobin protein to treat the completely activated human hepatic stellate cell line LX-2 and the incompletely activated primary rat hepatic stellate cells, or over-expressed cytoglobin protein in LX-2 cells. We used two different oxidative-stress related models, the hydrogen peroxide model and the iron-overload model in our experiments and investigated the proliferation status and the intracellular superoxide level of the cells. The results showed that endogenous cytoglobin exerted significant protective effects on hydrogen peroxide or iron-overload induced LX-2 cell damage, confirming that upregulation of cytoglobin was the protective response of activated hepatic stellate cells to oxidative stress. Recombinant cytoglobin protein could protect LX-2 cells from oxidation induced damage, and prevent primary rat hepatic stellate cells from excessive proliferation and injury. The cytoplasmic reactive oxygen species (ROS) scavenging capacity of the recombinant cytoglobin protein was not as good as its capacity in scavenging ROS outside the cells, likely owing to the lack of active transporting mechanisms. Intracellular over-expression of cytoglobin protein could exert significant protective effect on LX-2 cells treated with hydrogen peroxide or iron-overload. Our results would accelerate the exploitation of new anti-fibrotic targets.

Abstract:In order to probe the biological function of O-GlcNAc and the pathogenesis of associated diseases, it is essential to prepare a potent and specific O-G1cNAcase (OGA) antibody. Based on protein sequence analysis, we found N terminal 1?350 amino acids of OGA (sOGA) has high antigenicity and hydrophilicity and then constructed it into plasmid pET28a vector. First, we optimized the expression of sOGA in Escherichia coli BL21(DE3) (0.05 mmol/L IPTG, 10 hours) and purified it with the Ni-NTA affinity chromatography and size exclusion chromatography respectively. SDS-PAGE verified the molecular weight (45 kDa) and the purity (>95%) of sOGA and the purified protein was subjected to immunize New Zealand rabbits. Finally, we obtained OGA polyclonal antibody by affinity purifying the antiserum with CNBr-activated Sepharose 4B beads. Western blotting and ELISA assay showed that this antibody could recognize three OGA isoforms with high specificity and the sensitivity was 0.11 ng/mL (the titer was 1:80 000). These results indicated the prepared polyclonal antibody of OGA can be used for the biological function study of OGA.

Abstract:Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3α. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3α (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.

Abstract:In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene.

Abstract:The aim of this research is to find an effective cardiomyocyte-induced method derived from porcine amniotic fluid stem cells (pAFS). For cardiac differentiation, the cells were formed embryoid bodies (EBs) firstly, then cultured in induced-medium including 5-azacytidine (5-aza) and vitamin C (Vc). We detected the specific markers of cardiomyocyte by immunocytochemistry, RT-PCR and transmission electron microscope. The results showed that some embryoid bodies beat rhythmically after 10 days of induction. Furthermore, analysis of t test revealed that the percentage of beating cardiomyocyte-like cell clusters was highest (33%) when induction using 0.1 mmol/L Vc and 5 μmol/L 5-aza. Immunocytochemistry analysis demonstrated that cardiomyocyte-like cell clusters expressed α-actin, Tnni3. RT-PCR analysis also illustrated that TbX5, Gata4, α-MHC and Tnni3 were expressed positive in cardiomyocyte-like cell clusters. Especially, we observed basic structures of myocardium, such as myofilament, glycogen granule and so on by transmission electron microscope. In conclusion, 5-azacytidine and vitamin C could promote differentiation of pAFS into myocardium.

Abstract:We constructed transgenic chicken bioreactor vector, driven by chicken ovalbumin promoter, lentiviral vector and cytomegalovirus (CMV) promoter control vector encoding green fluorescent protein (GFP) and luciferase (Luc) as reporter genes. The three vectors were used to transfect or infect chicken primary oviduct epithelial cells, embryo fibroblasts cells, mouse 3T3-L1 preadipocytes cells and bovine mammary epithelial cells. High efficient and specific expression vector for transgenic chicken bioreactor was determined by detecting fluorescence and luciferase activity. Reporter gene analysis showed that chicken ovalbumin promoter expression vector was not cell type-specific in these four different cells. Additionally, luciferase reporter analysis illustrated that the chicken ovalbumin promoter activity was over 100 times lower than that of the CMV promoter in four different cells. Both of these two reporter genes were expressed in those four different cells infected by lentiviral expression vectors. Similarly, the GFP reached the similar expression level in cells infected by lentivirus and cells transfected with CMV promoter plasmid vectors when the multiplicity of infection was 20. In conclusion, the transgenic chicken bioreactor vector under the control of chicken ovalbumin promoter was not highly efficient and cell type-specific. However, the efficient expression and extensiveness of lentiviral vector could be used for studying chicken oviduct bioreactor.

Abstract:Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 mg/(106 cells·d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.

Abstract:We established a rapid detection method of New Delhi Metallo-β-Lactamase Gene (NDM-1) based on Loop-mediated Isothermal Amplification (LAMP). With the application of LAMP, we designed four sets of LAMP premiers, using NDM-1 gene as the target sequence, and selected the set of optimal primers. Meanwhile, we established optimal reaction systems and conditions to carry out the sensitivity and specificity experiments. The experiment results showed that the whole detection process took only one hour and could be observed visually. In the experiment of sensitivity, NDM-1 gene had a detection limit of 6 copies in each reaction. In the experiment of specificity, we detected NDM-1 gene in 4 pathogen strains (Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae), and the total DNA from intestinal microbes and the total DNA from soil microbes. We had not detected the amplification reactions. The detection method established could rapidly detect NDM-1 gene and visualize the experiment result. The method is easy to operate and has high sensitivity and specificity and thus has great application value in basic research laboratories, emergent detection and spot detection.

Abstract:We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFa activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFα’s cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

Abstract:To efficiently produce non-specific nuclease (NU) of Serratia marcescens through recombinant overexpression approach and to characterize the purified NU. The nuclease gene was amplified from the genomic DNA of Serratia marcescens by PCR and fused into vector pMAL-c4X with maltose binding protein (MBP) tag. The recombinant vector verified by DNA sequencing was transformed into Escherichia coli BL21. The expressed MBP-NU was purified through the amylose resin and its catalytic characters were analyzed. The results showed the NU gene had 97% identities with the reported S. marcescens nuclease gene and intracellularly expressed in E. coli BL21. The optimal expression conditions were 37 °C, 0.75 mmol/L IPTG with 1.5 h induction. The purified MBP-NU exhibited non-specific nuclease activity, able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Its optimal temperature was 37 °C and optimal pH 8.0. From 1 L culture broth 10.8 mg NU could be purified with a specific activity of 1.11×106 U/mg. The catalytic activity of NU was not inhibited by reagents such as EDTA (0.5 mmol/L), PMSF (1 mmol/L) and KCl (150 mmol/L) commonly used in protein purification.