Abstract:Four out of 10 patients of X-linked severe combined immunodeficiency (X-SCID) were finally developed leukemia after receiving the treatment of gene therapy delivered by γ-retroviral vectors. This is due to the vector integrated to the proximity of lmo2 etc proto-oncogene promoters, leading to the activation of onco-gene expression, which raises the concern of the bio-safety of gene therapy vectors. Lentiviral vectors, especially self-inactivating lentiviral vectors, are considered to be much safer than γ-retroviral vectors. However self-inactivating lentiviral vectors also have encountered with some unsafe factors and one of them is the problem of transcriptional “read-through”. During the past years, achievements have been made to reduce lentiviral vector transcriptional read-through, which are reviewed herein.

Abstract:Polyelectrolyte with a large number of cations or anions could precipitate the oppositely charged proteins to form polyelectrolyte-protein complexes, which then aggregated to form larger particles via electrostatic attraction or hydrophobic interaction. The precipitation was affected by the molecular weight and concentration of the polyelectrolyte as well as the ionic strength and pH of the solution. The use of precipitation is an efficient method for selective separation of proteins from crude biological mixtures in the downstream processes of bioengineering.

Abstract:Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted into shuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL- DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0×109 PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P＜0.05), respectively, when goat mammary gland epithelial cells were infected with AD-PTHrP-322 after 24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.

Abstract:Mytilin-derived-peptide-1 (MDP-1) and mytilin-derived-peptide-2 (MDP-2) are two truncated decapeptides with reversed sequence synthesized corresponding to the residues 20-29 of mytilin-1 (GenBank Accession No. FJ973154) from M. coruscus. The objective of this study is to characterize the structural basis of these two peptides for their antimicrobial activities and functional differences, and to investigate the inhibitory mechanism of MDPs on Escherichia coli and Sarcina lutea. The structures of MDP-1 and MDP-2 in solution were determined by 1H 2D NMR methods; the antibactericidal effects of MDPs on E. coli and S. lutea were observed by transmitted electron microscopy (TEM). Both MDP-1 and MDP-2 have a well-defined loop structure stabilized by two additional disulfide bridges, which resemble the-hairpin structure of mytilin-1 model. The surface profile of MDPs￠ structures was characterized by protruding charged residues surrounded by hydrophobic residues. TEM analysis showed that MDPs destroyed cytoplasmic membrane and cell wall of bacteria and the interface between the cell wall and membrane was blurred. Furthermore, some holes were observed in treated bacteria, which resulted in cell death. Structural comparison between MDP-1 and MDP-2 shows that the distribution of positively charged amino acids on the loop of MDPs is topologically different significantly, which might be the reason why MDP-2 has higher activity than MDP-1. Furthermore, TEM results suggested that the bactericidal mechanisms of MDPs against E. coli and S. lutea were similar. Both MDP-1 and MDP-2 could attach to the negatively charged bacterial wall by positively charged amino acid residues and destroy the bacteria membrane in a pore-forming manner, thus cause the contents of the cells to release and eventually cell death.

Abstract:The preliminary role of respiratory burst oxidase homolog (Rboh) in plant immune response is defined, but the exact function of OsRboh gene in rice immune response and its expression pattern is yet unclear. In order to clarify the role of OsRboh in rice immune response, we screened seven OsRboh genes from the latest rice genome annotation database. The result of tissue specific expression analysis demonstrated that OsRbohD was expressed only in spike and calli, and OsRbohE and OsRbohF were only expressed in calli. The rest of OsRboh genes were constitutively expressed in rice. In addition, the expression level of OsRboh gene family was analyzed in the rice leaves respectively treated with salicylic acid (SA), methyl jasmonic acid (MeJA) and Xanthomonas oryzae PV. oryzae (Xoo) PXO99 strain by Real-time PCR, and H2O2 content was also quantified by spectrophotometry after the three treatments. The result shows that the expression of OsRbohA, B, C and D was increased under the treatments of SA, the expression of OsRbohA, B, C and G was increased under the treatments of MeJA, and the expression of OsRbohA and OsRbohB was induced by Xoo PXO99 strain. However, the levels of expression and responsive times of these genes were different. Moreover, all three treatments led to H2O2 accumulation. These OsRboh genes have functional roles in rice native immune response.

Abstract:In order to produce interspecific somatic hybrids between Brassica campestris (2n?=?20, AA) and Brassica oleracea (2n=18, CC), we isolated protoplasts from cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with 0.2 mg/L 2,4-dichorophenoxyacetic acid (2,4-D) +0.5?mg/L 6-benzylaminopurine (6-BA)+0.1 mg/L naphthaleneacetic acid (NAA)+ 0.1 mg/L Kinetin (Kin), 0.3 mol/L sucrose and 0.3 mol/L glucose were used as osmoticum. At the eight-to ten-cell stage, divided cells were transferred to Kao’s basal medium supplemented with 0.3 mol/L sucrose as carbon source and 0.1% agarose, 2 mg/L 6-BA+ 2 mg/L Zeatin (ZEA)+1 mg/L NAA+ 0.5 mg/L Kin for callus induction. After 35 days, when small calli reached 2–3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/L Zeatin (ZEA) and 2mg/L indole-3-acetic acid (IAA). After the length of the shoots reached 1–2 cm, the shoots were transferred to 1/2 MS+0.2 mg/L NAA for root induction. Morphological, cytological and molecular biological analysis methods were used for identification of somatic hybrids. The results showed that, the first cell division occurred during 2–7 days of culture. Five weeks after culture initiation, the plating efficiency attained 0.66%. Finally, the shoot regeneration frequency was 3.7%. A total of eleven regenerated plants were obtained and verified as somatic hybrids by morphological observation and flow cytometry. Cytological studies showed that all tested plants had a chromosome number of 38, the sum of both parents. Hybridity was also confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis, indicating that these regenerated plants were all true hybrids of B. campestris and B. oleracea. All amphidiploid somatic hybrids showed low pollen fertility. Pollen fertility was gradually recovered in the first and second progenies.

Abstract:Heat shock protein gp96 isolated from tumor tissues holds great promise for tumor immunotherapy. However, at present only very limited amount of gp96 protein can be isolated from tumor tissues. Here, we reconstituted the yeast-expressed gp96 (recombinant gp96, rgp96) with B16.F10 melanoma antigens in vitro to prepare new gp96 tumor vaccine on large-scale, and analyzed its induction of specific anti-tumor immunoresponses by ELISPOT, IFN-γ intracellular staining and cytotoxicity assays. Immunization with rgp96-tumor antigen complexes significantly inhibited B16 tumor growth compared with either rgp96 or tumor antigens alone and led to enhancement of tumor-specific T-cell activities, which was found similar to that of tumor tissue derived gp96. Our results therefore may provide bases for large-scale preparation of the new generation of gp96 tumor vaccines.

Abstract:Extracellular matrix (ECM) keeps cell’s shape, protects and nourishes cells; it plays a great role in cell proliferation and differentiation. Therefore, ECM is very important in cell and tissue engineering. In this study, after primary mouse osteoblasts and fibroblasts maintained at confluence in vitro were removed, their ECM coated on cell culture plate was prepared, and bone morphogenetic proteins 2 (BMP-2) was detected in the osteoblasts ECM. MC3T3-E1 preosteoblasts cells were seeded on cell culture plates covered with fibroblasts ECM and osteoblasts ECM respectively. The proliferative activity of the cells cultured on fibroblasts ECM was higher than that on osteoblasts ECM and the control group. The alkaline phosphatase activity, relative protein levels of BMP-2 and osteopontin, secreted calcium of the cells cultured on osteoblasts ECM were all the highest. The results indicate that the two different ECMs produced in vitro had different bioactivities, the fibroblasts ECM coated on cell culture plates could accelerate MC3T3-E1 cells proliferation, and the osteoblasts ECM could promote cells osteogenic differentiation.

Abstract:The instability of secondary metabolite production is a ubiquitous problem in plant cell culture. In order to understand the instability in plant cell culture, we investigated anthocyanin accumulation in suspension cultures of Vitis vinifera, as a model system, in our laboratory. Not only the anthocyanin contents but also its composition exhibited instability along with the long-term subculture. New methods were developed to indicate the instability of plant cell culture. Both the definition of instability coefficient (δ) and the application of factor scores were the first time in this field. To examine the effects of culture conditions on instability of anthocyanin biosynthesis, different subculture cycles and inoculum sizes had been investigated. Subculture cycle and inoculum size were both environmental cues driving the instability. Compared with subculture cycle, inoculum size was more effective in working on the instability of anthocyanin accumulation. Among all the conditions investigated in our study, (6.5 d, 2.00 g), (7 d, 2.00 g), (7.5 d, 2.00 g), (7 d, 1.60 g) and (7 d, 2.40 g), the condition of 7 d-subculture cycle together with 1.60 g-inoculum size was the best one to keep the stable production of anthocyanins.

Abstract:The purpose of this paper is to establish sulfhydryl site-directed PEGylation method for lysostaphin and to evaluate effects of mutagenesis and modification of amino acid residue within putative linker on enzyme activity. On the basis of structural analysis of lysostaphin, amino acid 133?154 of tentative linker between the N-terminal and C-terminal domain were chosen as the candidate residues for site-directed mutagenesis to cysteine. Subsequently, sulfhydryl site-directed PEGylation was performed by reacting PEG-maleimide reagent with the newly introduced cysteine residue of the mutant lysostaphin. The Cys-mutant and PEG-modified proteins were both purified, and their enzymatic activity were further determined. The results show that the modification method for lysostaphin was highly efficient, resulting in the single uniform PEGylated lysostaphins. The mono-PEGylated lysostaphins were separated from unmodified lysostaphins through highly efficient one step method with Ni2+-NTA column chromatography. However, both Cys-mutant and PEGylated lysostaphin only retained partial activities of the wild-type enzyme. It suggests that sulfhydryl site-directed PEGylation modification of the tentative linker between the N-terminal and C-terminal domain may affect the catalytic activity of lysostaphin.

Abstract:In order to express PebC1 in Pichia pastoris, the pebC1 sequence was amplified from genome Botrytis cinerea BC-4-2-2-1 by PCR and subcloned into the Pichua pastoris expression vector pPIC9K to generate pPIC9K-pebC1. The recombinant plasmid was linearized by Bgl II and transformed into Pichia pastoris GS115 by electroporation. Recombinant Pichia pastoris GS115/pPIC9K-pebC1 was screened by MD and G418-YPD plates and further confirmed by PCR. The protein expression was induced by methanol and analyzed by SDS-PAGE. SDS-PAGE analysis showed a special band about 39 kDa and western blotting indicated a good antigenicity of the expressed protein. Bioassay results showed that the recombinant protein PebC1 can induce resistance to gray mould disease of cucumber and Arabidopsi thaliana.

Abstract:We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.

Abstract:This study focused on the contamination mechanism and regeneration strategies of sulfopropyl ion exchange resin (SP Sepharose FF) during the separation of recombinant human lactoferrin from transgenic bovine milk. We analyzed primary constituents’ contents in chromatorgraphic material and fractions. The results showed that the lipid in milk can clog the column or adhere to the resin through hydrophobic interaction, leading to an increase in column pressure. Some casein molecules were found to adsorb onto the resin through electrostatic interaction, therefore the adsorption capacity was decreased. There was no direct interaction between lactose and the resin in the chromatorgraphic process. Increased continuous chromatographic cycles and prolonged time interval between protein purification and column regeneration could enhance the undesirable interaction between the contaminants and resin, thus lowering the regeneration efficiency. NaOH was found to be effective in the removal of lipid and casein molecules from the column. Furthermore, normal microstructure and chromatographic performance of the ion exchanger was recovered after this cleaning procedure.

Abstract:To generate active recombinant human β-secreatase (BACE1) for studying its interaction with its inhibitors, we constructed two recombinant plasmids, pPIC9K-MetBACE22 (bearing pro-bace1 gene) and pPIC9K-MetBACE46 (bearing bace1 gene). These two plasmids were then transformed into Pichia pastoris GS115 by electroporation to obtain the recombinant strains 9k-B22 and 9k-B46. After induction in buffered methanol complex medium, we found the supernatant activity of 9k-B22 significantly higher than that of 9k-B46. The culture filtrate of 9k-B22 was concentrated, and then purified by HisTrap affinity column. The purified proteins, showing good BACE1 protease activity, were found to be a mixture of glycoproteins because they can be stained by periodic acid-Schiff reagent. After this mixture was treated with Endo Hf (a recombinant protein of endoglycosidase H), we found two new adjacent bands around 50 kDa on SDS-PAGE. These two bands were cut and subjected to peptide mass fingerprint analysis, and identified as proBACE1 and BACE1 proteins. Enzyme assays revealed that the activities of both BACE1 proteins in glycosylated and deglycosylated form were lower than that of commercial BACE1 (expressed in HEK-293), inferring glycosylation and the type of glycosylation are crucial to the activity. However, we found no apparent difference in the inhibition of those all above three enzyme forms by one known BACE1 inhibitor. This observation demonstrated that the glycosylation of BACE1 by Pichia pastoris does not affect its interaction with this inhibitor. After optimization of culture conditions, the production of BACE1 in Pichia pastoris was enhanced to about 1 mg/L. This work enables us to further investigate the interaction of BACE1 and its inhibitors, and assists in discovering and optimizing BACE1 inhibitors as anti-Alzheimer’s disease agents.

Abstract:To investigate the effect of hSCGF-α on human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs), we obtained hSCGF-α using genetic engineering. hSCGF-α gene was amplified from hUCMSCs cDNA using two-step PCR and was inserted into pET-28a(+) plasmid vector. Induced by IPTG at 20 degrees Celsius for 24 h, the fusion protein expressed in E.coli BL21(DE3) was mainly existing in soluble form. The recombinant hSCGF-α was purified using NI-NTA affinity chromatography and the purity was up to 90%. The colony forming test revealed that combined use hSCGF-α and rmGM-CSF (recombinant murine GM-colony stimulating factor, rmGM-CSF) had granulocyte/macrophage (GM) promoting effects on murine bone marrow GM progenitor. In addition, the results indicated that hSCGF-α and rhGM-CSF had stimulatory effect on hUCMSCs and their synergetic effect was the strongest.