Abstract:Cell-free protein synthesis (CFPS) systems based on crude cell extracts have been used in protein expression in vitro. With the researchers’ endeavor for decades, the CFPS system has been developed as an important research tool in many frontiers of fundamental and applied biology because of its clear genetic background and simplicity to control the reaction. The yield of CFPS systems derived from prokaryote or eukaryote has increased to several grams per liter with constantly decreasing cost. Nowadays grams of protein could be prepared using a large-scale cell-free system. Recently, the advantages on the expression of complicated, toxic and membrane proteins have shown the great potential of the CFPS systems. The rapid progress of this technology made us to believe that it will take an important place in biopharmaceutical industries undoubtedly.

Abstract:Transposons are the mobile and autonomic replication DNA fragments in genomes. With more understanding of the structure and function of transposons, numerous transposons have been developed to the genetics tool for gene function analysis, gene transformation and gene therapy. The low transpositional activity of the natural transposons is the main obstacles to the utilization of transposons. Recently, with the progress in bioinformatics and protein engineering methods, researchers have reconstructed and optimized natural transposases to create hyperactive transposases that catalyze the transposition with high efficiency. The resulted hyperactive transposons have been applied to gene-modification and gene-tagging. Meanwhile, transposase chimeras were created by protein fusion technology. The insertion characteristic of transposons were artificially regulated which could be utilized in gene therapy.

Abstract:When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to dedifferentiated fat (DFAT) cells. DFAT cells have many advantages compared with adipose-derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs). For example, DFAT cells are homogeneous and could be obtained from donors regardless of their age. Furthermore, DFAT cells also have the same multi-lineage potentials and low immunogenicity as ASCs. As an excellent source of seed cells for tissue engineering and stem cell transplantation, DFAT cells have better prospects in the treatment of many clinical diseases, such as bone defects, neurological diseases, ischemic heart disease and kidney disease. It is necessary to make more intensive studies of DFAT cells. This article summarizes progresses in the immunological characteristics, differentiation ability and potential clinical applications of DFAT cells.

Abstract:The purpose of this study was to study the effect of three different transfection reagents (LipofectamineTM LTX & PLUSTM, Lipofectamine 2000 and Nano-PAMAM-D) and three different testicular injection methods (rete testicular injection, seminiferous tubules injection and testicular interstitial injection) on the efficiency of production transgenic mice. After the mixtures of plasmid DNA (pEFP-C1) and transfection reagent were injected with different testicular injection methods, the sperm density, vitality, positive sperm rates and PCR positive transgenic mice rate were examined 30 days after injection. The results showed that the damage degree from slight to serious of three transfection reagents was LipofectamineTM LTX & PLUSTM, Lipofectamine 2000, and PAMAM-D. The sperm positive rates with green fluorescence of these three groups were 35.65%±0.69%, 12.86%±0.35% and 10.04%±0.20%, respectively. The PCR positive rates of transgenic newborn mice were 29.17%, 13.70% and 5.88%, respectively. Among the groups of different testicular injection methods, the damage degree from slight to serious was rete testicular injection, seminiferous tubules injection, and testicular interstitial injection, whereas the sperm positive rates with green fluorescence were 35.13 %, 15.13%, and 0%, respectively. The PCR positive rates of transgenic newborn mice among different testicular injection groups were 33.3%, 12.5%, and 0.0%. The combination of rete testicular injection and LipofectamineTM LTX & PLUSTM had the lowest toxicity and highest transgenic efficiency in the production of transgenic mice.

Abstract:To study the role of BAMBI in adipogenesis, we constructed lentivirus interfering vector targeting on porcine BAMBI, packaged and infected the porcine preadipocyte. The differentiation state of preadipocyte was detected by Oil Red O staining and Oil Red O extraction assay and the expression levels of adipogenic marker genes were detected by Real-time qPCR and Werstern bloting. Results show that BAMBI expression was significant decreased after lentivirus infection, which was repressed more than 60% by shRNA2. Moreover, knockdown BAMBI increased the lipid accumulation of porcine preadipocyte and improved the expression of PPARγ (peroxisome proliferator-activated receptorγ) and ap2 (adipocyte protein 2). In summary, these data indicated that BAMBI inhibited adipocyte differentiation by facilitating the phosphorylation of ERK1/2.

Abstract:To obtain active protein of pIL-18 expression in Lactococcus lactis, and to observe its biological activity, the total RNA was extracted as template from peripheral blood mononuclear cells. Porcine interleukin 18 (pIL-18) was amplified by RT-PCR. The resulting fragment was cloned into pAMJ399 L. lactis vector, and then transformed to L. lactis MG1363 cells by electroporation. Expression of pIL-18 protein was detected by SDS-PAGE and Western-blot. Bioactivity of the product was tested by pig spleen lymphocyte proliferation test and cytopathogenic effect inhibition assay. The result of Western blot and bioactivity test shows that the molecular weight of pIL-18 protein was 19 kDa. The react line was observed in both supernatant and precipitated of the recombinant bacteria pAMJ399-pIL18/MG1363. The expressed pIL-18 can promote the proliferation of pig spleen lymphocyte, and significantly inhibit virus multiplication. As conclusion, porcine interleukin-18 was successfully expressed in L. Lactis, and the product was biologically active.

Abstract:In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Mtabolically engineered E. coli has the potential to further improve the production of DHS.

Abstract:We constructed several recombinant Escherichia coli strains to transform phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS system) and compared the characteristics of growth and metabolism of the mutants. We knocked-out the key genes ptsI and ptsG in PTS system by using Red homologous recombination in E. coli and meanwhile we also knocked-in the glucose facilitator gene glf from Zymomonas mobilis in the E. coli chromosome. Recombinant E. coli strains were constructed and the effects of cell growth, glucose consumption and acetic acid accumulation were also evaluated in all recombinant strains. The deletion of gene ptsG and ptsI inactivated some PTS system functions and inhibited the growth ability of the cell. Expressing the gene glf can help recombinant E. coli strains re-absorb the glucose through Glf-Glk (glucose facilitator-glucokinase) pathway as it can use ATP to phosphorylate glucose and transport into cell. This pathway can improve the availability of glucose and also reduce the accumulation of acetic acid; it can also broaden the carbon flux in the metabolism pathway.

Abstract:In order to study the effect of phytohormone on growth and isoflavones contents of Pueraria phaseoloides hairy roots, we cultured the hairy roots with different concentrations of 6-benzylaminopurine (6-BA) alone or in combination with α-naphthaleneacetic acid (NAA). Then we determined the effects of 6-BA alone or in combination with NAA on the growth and the contents of isoflavones compounds and levels of antioxidase activities of hairy roots by spectrophotometry. The results show that 6-BA inhibited the growth, and decreased biomass and total isoflavones compounds of P. phaseoloides hairy roots. Furthermore, the inhibition was increased with the concentrations of 6-BA. Compared with the controls, different concentrations of 6-BA in combination with NAA 2.0 mg/L could inhibit the growth of hairy roots and decrease the content of total isoflavone compounds, and also significantly enhanced the contents of soluble protein and levels of peroxidase (POD) activities, but decreased the activities of superoxide dismutase (SOD). DNA ladders detected by agarose gel electrophoresis can be observed after hairy roots of P. phaseoloides were cultured with 6-BA alone for 30 days, but can appear on the 20th day after culture with 6-BA in combination with NAA 2.0 mg/L. This result indicates that 6-BA or 6-BA in combination with NAA can both stimulate appearance of programmed cell death (PCD), and NAA may play a synergistic role on PCD.

Abstract:In order to study T-bet function in mouse cells, a novel retroviral vector expressing mouse T-bet and reporter gene Thy1.1 was constructed. Retrovirus particles were then produced by transfection of the recombinant retroviral plasmid into a packaging cell line Platinum-E. The recombinant retrovirus played considerable infection ability. T-bet expression was then identified by FACS after infection of CD4+ primary T cells from T-bet knockout mouse with recombinant retrovirus. To determine if exogenous expressing T-bet has normal function, we checked the expression level of T-bet target gene, Ifng. IFN-γ expression was upregulated in the T-bet knockout T cells infected with recombinant retrovirus. In conclusion, we successfully constructed an effective mouse T-bet recombinant retroviral vector.

Abstract:Limulus Factor C, a serine protease zymogen from the amoebocytes of the limulus, has high affinity for endotoxin. When Factor C is activated by endotoxin, it hydrolyses artificial tripeptide substrate and measurable products are released, so it can be used as an alternative reagent for endotoxin analysis. Factor C gene of Tachypleus tridentatus was obtained through RT-PCR and the recombinant protein was expressed by Bac-to-Bac/BmNPV baculovirus expression system in silkworm larvae. The activity of Factor C was detected with diluted serum of silkworm larvae , and the sensitivity of endotoxin detected was 0.2 EU/mL when the serum was diluted at 1:500. The silkworm larvae expressed limulus Factor C could be used to develop a new low-cost endotoxin test reagent.

Abstract:The stable isotope labeling by amino acids in culture (SILAC) based quantitative proteomics serves as a gold standard because of the high accuracy and throughput for protein identifications and quantification. In this study, we discussed the application of SILAC technology in mammal model, and developed quantitative internal standard for comparative proteomics of disease model. The C57BL/6 mice fed by special diet containing the 13C6-Lysine and bred F2 generation. We identified and analyzed total proteins of 9 mice tissues of F2 generation, including brain, lung, heart, stomach, intestine, liver, spleen, kidney, and muscle. Quantitative analysis information could evaluate the mice and different tissues’ labeling efficiency. Liver was the most efficient, brain the least, and the labeling efficiency were 96.34%±0.90% and 92.62%±1.98% respectively. The average of the labeling efficiency of F2 generation was 95.80%±0.64%, which met the international standard (≥95%) for SILAC quantitative proteomics effective study. SILAC technology was successfully extended to mammalian model system, which will provide powerful tools for the mechanism study of the pathophysiology process with mouse model.

Abstract:We prepared protoplasts from Salvia miltiorrhiza Bunge suspension culture cells. Then, the protoplasts’ vitality and functions were tested by fluorescein diacetate staining method and Fluo-3/AM flourescent probe. The optimal condition of protoplast isolation was Cellulase R-10 1.5%, Pectinase Y-23 0.3%, Macerozyme R-10 0.5%, 40 r/min 12 h, 600×g 5 min, and the protoplasts yield was 1.1×106 cells/g FW, the vitality was more than 95% by using fluorescein diacetate staining method. It has been confirmed that calcium fluorescent probe Fluo-3/AM can be successfully loaded into protoplasts.

Abstract:Resveratrol is a natural phytoalexin with special pharmacological and health functions. Stilbene synthase (STS) is a key and rate-limiting enzyme in the biosynthesis of resveratrol that is present only in a limited number of plants. The content of resveratrol from Polygonum cuspidatum is more than 1 000 times higher than grapes and peanuts. We speculate that the catalytic ability of different STS may be one of the reasons causing differences in the content of resveratrol. To verify the above speculation, Vitis vinifera stilbene synthase gene (VvSTS) was amplified according to overlap PCR protocol with genomic DNA as template. VvSTS and PcSTS (PcPKS5) were analyzed through heterologous expression in Escherichia coli. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins was about 43 kDa. Enzyme reaction and product analysis showed that the two products were resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of PcPKS5 was 2.4 times of the VvSTS. Our findings confirms that STS from certain plants has much higher catalytic capability.

Abstract:Antithrombin III (AT Ⅲ) is the most important anti-clotting substance. Recombinant human antithrombin Ⅲ (rhAT Ⅲ) expressed in transgenic goat milk attracts more and more attention. Develop an effective purification route for rhAT ⅢI is vital to its industrial production. An efficient purification method was developed for the rapid purification of rhAT Ⅲ by isoelectric precipitation and heparin affinity chromatography.?First, casein was effectively removed by isoelectric precipitation. rhAT Ⅲ was further purified by heparin affinity chromatography.?In the process of heparin affinity chromatography, the effects of pH and temperature on the stability of rhAT Ⅲ were studied, and the effects of operating conditions, elution gradient, flow rate and sample loaded, on the purification efficiency were also studied.?Under the optimized conditions, the protein recovery of rhAT Ⅲ was about 90% with purity over 99%, while its activity recovery was about 50%. Such a purification process?is?very simple and effective, and it would provide a valuable reference for the further scaling-up of industrial production.

Abstract:Photoautotrophic cultivation with heterotrophic cells as seeds (heterotrophic cells / photoautotrophic cultivation) is an effective way for the development of microalgal biofuel, but its development potential from the point of process optimization has not been investigated in literatures. To evaluate this, the optimizations of medium and culture conditions for Chlorella ellipsoidea were studied. In the heterotrophic stage, the biomass concentration reached 11.04 g/L with the optimized medium in flask, which were 28.0% higher than that with the original medium, and the biomass concentration reached 73.89 g/L in 5-L fermenter. In the photoautotrophic stage, the culture medium and conditions were studied in a 2-L column photobioreactor. The maximum biomass concentration, lipid content and lipid productivity reached 1.62 g/L, 36.34% and 6.1 mg/(L·h) under the optimal photoautotrophic conditions. The lipids were mainly composed of C16-C18 fatty acids, which were raw material suitable for biodiesel. After optimization, heterotrophic cells / photoautotrophic cultivation can significantly improve the capacity of biofuel production by Chlorella ellipsoidea, this method is also expected to be an efficient way for the cultivation of other microalgae that can grow heterotrophically.