Objective: To construct a prokaryotic expression plasmid of human TNFα with SUMO,obtain recombinant protein by expression and purification,which is expected to provide basis for the further research and utilization of TNFα.Methods: The gene encoding mature TNFα protein was amplified from plasmid pET32a-hTNFα by PCR and cloned into pET28a with SUMO tag on the upstream.The fusion protein SUMO-hTNFα was expressed in BL21(DE3) engineering bacteria,then was purified by Ni-NTA Resin purification system.The purified production was digested by SUMO protease. One more purification in order to obtain mature TNFα protein.The biological activity was measured in a cytotoxicity assay using L929 cells by CCK-8 test. Results: The prokaryotic expression plasmid pET28a-SUMO-hTNFα was successfully constructed.The results identified by enzyme digestion and nucleotide sequencing corresponded exactly well as expected. The fusion protein SUMO-hTNFα expressed in a soluble form in BL21(DE3).Fusion protein was purified by Ni-NTA Resin purification system. Mature hTNFα protein was obtain after digestion by Sumo protease and purification. ED50 of recombinant hTNFα is 12.8μg/ml. Conclusion: The prokaryotic expression plasmid pET28a-SUMO-hTNFα was successfully constructed. TNFα protein was expressed and obtained by digestion and purification. These established a foundation of further research and utilization of hTNFα.
Objective: To construct an eukaryotic expressing plasmid of lipid transfer protein from Peganum harmala, and investigate its inhibitory effects on the growth of murine melanoma(B16) cells in vitro and in vivo. Methods: The eukaryotic expressing plasmid of PhLTP(pcDNA3.1-PhLTP) was constructed by DNA recombination technique. The recombination plasmid and empty vector were transfected into B16 cells, respectively. The effect of pcDNA3.1-PhLTP on the growth of B16 cells was measured by MTT assay. B16 cells were inoculated subcutaneously of C57BL/6J mice, which randomly divided into 4 groups. Tumor volume and weigh were measured, and the tumor inhibition rates were calculated. The change of the mouse spleen and liver were observed under microscope. The expressions of PhLTP, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor(bFGF) in tumor tissues were detected by immunohistochemistry. Results: The proliferation of B16 cells were inhibited after transfection with pcDNA3.1-PhLTP for 72 h(P< 0.01). Growth rate of tumor in mice model was also significantly inhibited in pcDNA3.1-PhLTP therapy group as compared with the control group and empty vector group (P< 0.05). The tumor tissue cells had different levels of necrosis in recombinant plasmid group. The liver, lungs and other organs in mice haven't significant pathological damage. The PhLTP could be expressed in recombinant plasmid group. And the positive expression indexes of VEGF and bFGF in recombinant plasmid group were significantly lower than ones of VEGF and bFGF in control group and empty vector group (P< 0.01). Conclusion: The recombinant eukaryotic expression vector pcDNA3.1-PhLTP is constructed successfully, and pcDNA3.1-PhLTP can inhibit the growth of B16 cells in vitro and in vivo. It is suggestted that the recombinant plasmid pcDNA3.1-PhLTP may have potential applications in the treatment of melanoma.
Objective: To observe the therapeutic effect of human amnion mesenchymal stem cells transplantation labeled with 5, 6, 2-carboxy fluorescein diacetate succinimidy ester(CFSE)on mice model with liver damage induced by carbon tetrachloride. Methods: The amnion was mechanically separated from the placenta obtained by cesarean section after 38~40 week-pregnancy. The amniotic mesenchymal stem cells were obtained by using two-step digestion with trypsin and collagenase type I, then the flow cytometry and immunofluorescent staining techniques were adopted to identify the surface molecular markers and stem cell characteristics. Hepatic damage model groups were established through carbon tetrachloride and oil inducing, 20 μl/g doses of the mixture were given to mice by abdominal injection. Mouse models of hepatic damage were randomly divided into two groups. The control group was injected with the same volume of PBS via the tail vein; the treatment group was injected with human amnion mesenchymal stem cells suspension labeled with CFSE, and the number of the stem cells was 1×106. At 4 weeks after transplantation, animals were sacrificed. Liver tissues were obtained for the histological observation and the eyeball blood was collected to detect the liver function indicators. Results: The human amniotic mesenchymal stem cells obtained by using two-step digestion with trypsin and collagenase type I were high-purity. At 1 weeks after transplantation, the immunofluorescent staining of frozen slice showed that human amnion mesenchymal stem cell colonization could be seen in the mice liver tissues of the cell transplantation group. The labeled human amnion mesenchymal stem cell showed strong green fluorescence. At 4 weeks after cell transplantation, compared with the model group, levels of serum aspartate aminotransferase, alanine aminotransferase in the cell transplantation group were significantly decreased, while the total albumin level was increased significantly. The liver cell inflammatory necrosis, steatosis and liver fibrosis were improved significantly. After 4 weeks, the immunofluorescent staining of frozen slice result indicated that the expressions of human serum albumin could be observed in the mice liver tissue of the cell transplantation group, but no expression could be seen in the model group. It is visible that human amnion mesenchymal stem cells can improve liver function and pathological damage of liver damage mice in a certain extent. The findings may provide useful experimental datas for cell positioned transplantation with treatment liver disease.
Objective: The purpose is confirming and acquiring of high titer anti-CD133 antibodies from immunized camel, and providing the basis for preparing anti-CD133 nanobody. Methods: The gene segment coding of CD133 extracellular region were PCR amplified and ligated into pET28a plasmid and construction of a prokaryotic expression vector. The recombinant CD133 protein was expressed by IPTG induction and purified with Ni-affinity chromatography. A male Bactrian camel and two New Zealand rabbits were immunized with purified rCD133 antigen respectively. The enzyme-linked immunosorbent (ELISA) and Western blotting were applied to assay the titer of polyclonal antibody and the specific binding to CD133 protein. Results: the ELISA results revealed that the titer of anti-CD133 antibody raised from camel reached about 1:106 after the 5th immunization, and the titer of anti-CD133 antibody raised from rabbit reached about 1:5×105 after the 4th immunization.The anti-CD133 antisera could bind rCD133 specifically in Western blotting. Conclusion: The high titer anti-CD133 polyclonal antibodies were raised in camel compared with rabbit and it lay a good foundation for the future experiment.
CcpA, encoded by the ccpA gene, is the global mediator of carbon catabolite repression (CCR) in gram-positive bacteria. Increasing evidences suggest that CcpA not only participate in CCR, but also regulate the expression of virulence genes in pathogenic bacteria directly or indirectly. To investigate the effect of CcpA on virulence of Listeria monocytogenes, CcpA deletion strain was constructed using homologous recombination. The LD50 of the wild strain EGDe and EGDeΔccpA for BLAB/c mice was measured, the amount of bacteria in liver and spleen was counted, and pathological changes of liver and spleen were observed. The results showed that compared to EGDe, the LD50 of EGDeΔccpA reduced by 10 times, and lesion caused by EGDeΔccpA in liver and spleen of mice was more severe, indicating that deletion of CcpA enhances bacterial virulence in L. monocytogenes, CcpA might regulate L. monocytogenes, virulence gene expression directly or indirectly.
Aspergillus niger is widely used in industrial enzyme production for its excellent protein expression and secretion capacity. The differences of physiological behaviors and metabolic flux distribution between Aspergillus niger mutant and wild strains under same cultivation conditions are investigated, so as to determine limited factors in glucoamylase production. Based on kinetic analysis, it is confirmed that the mutant strain gets a higher maximum specific growth rate (+30%), a lower by-product productivity (－90%) and a higher substrate uptake efficiency (+30%), which implies significant differences in carbon distribution and substrate usage efficiency between these two strains. By applying Flux Balance Analysis (FBA), it is found that supplies of reducing power and ribose are main factors which effect cell growth. What's more, precursor amino acids is confirmed to be the main limited factor in glucoamylase production. These conclusions provide significances for subsequent bioprocess optimization and strain gene modification.
According to the known thermostable mannanase ManAd3 sequence and the codon bias of yeast, the optimized mannanase ManA sequence (Accession No. KJ806637) was designed and synthesized. The ManA gene was cloned into the pPIC9k vector, and then expressed in Pichia pastoris GS115 to obtain a recombinant strain ManA-GS115. The fermentation product of mannanase ManA was electrophoresis pure by SDS-PAGE identification with the molecular weight about 30 kDa. The enzymatic properties were shown that the optimal reaction temperature and pH value were 75℃ and pH6.0 respectively, and the specific activity was up to 3 200 IU/mg. Moreover, the residual activity was above 90% after 30 min treatment under 75℃, pH 6.0. With high expression, excellent thermostability, and the good thermostable tolerance under acidic condition, the mannanase ManA could be potentially applied in brewing, food, feed, textile and pharmaceuticals fields.
A predicted chitosanase was cloned from a fosmid genomic library of Streptomyces olivaceus strain FXJ 7.023 by PCR. The coding fragment of 885 bp encodes of 295 amino acids was fused to the expression vector pET32a (+) and transformed into Escherischia coli strains BL21 (DE3) plysS. A novel fusion protein with molecular weight of 50.3 kDa was obtained by induced the engineered strain with 1mmol/L IPTG under 18℃ and purified with the affinity chromatography of Ni2+-NTA. The recombinant chitosanase showed multifunctional catalytic activity of hydrolyze colloidal chitosan and carboxymethylcellulose with the maximal catalytic activity of 3.673U/mg and 1.302U/mg respectively. Due to it has multifunctional catalytic activity, such protein may have a potential application for the recycling of the carbohydrates and sugars in the waste.
Lipase(EC 3.1.1.3) is a important biocatalyzer used in a variety of industrial fields. High-level lipase producing strains is the prerequisite for industrial bioapplication of lipase. De novo design, gene synthesis and high-throughput screening strategies were used to realize the overexpression of Yarrowia lipolytica lipase YLL in Pichia pastoris. By de novo design and gene synthesis of YLL lipase gene, the expression level of synthesized lipase gene was improved 1.26 fold than the original YLL gene. After that, a high throughput screening strategy including olive oil Rhodamine B plates phenotype check, 96 well plates and flask fermentation was used to acquire the over expression Pichia recombinant SILVER. In the 14 L fermentor, Pichia recombinant strain SILVER achieved the highest activity of 40 500 U/ml broths, and the protein content reached 2.52 g/L broths, which set the foundation for industrial application of YLL lipase.
The Cephalosporin C fermentation was improved through mutations and optimization of the seed medium. High-producing strains Cephalosporium acremonium FC-1-4 and FC-4-2 were obtained with 60Co irradiation and Uv+LiCl treatment, the production were 26% and 54.5% higher than that of control. Meanwhile, the optimal seed medium component were 2.62% glucose, 3.70% corn steep liquor, 0.15% MgSO4 via Plackett-Burman and central composite design(CCD). The 34.7% and 13.2% increase of Cephalosporin C production was achieved with the Cephalosporium acremonium FC-1-4 and FC-4-2 respectively. The obtained high-producing strains and optimal seed medium have crucial industrial utilization in the future.
Objective: A novel approach was developed to selective extraction of glycoproteins by hyperpolymer-assisted hydrazide functionalized microsparticles(HHMs). Methods: Firstly, synthesizing hyperbranched poly-glycerols (HPG) by ring-opening multibranching polymerization of glycidol using dioxane as an emulsifying agent following converted them to aldehyde-functional hyperbranched polyglycerol (HPG-ALD). Upon immobilization on the microspheres, aldehyde group of HPG-ALD were convert to hydrazide group via a reductive alkylation reaction. Conclusion: After characterized by scanning electron microscope (SEM) and Thermo Gravimetric Analysis (TGA), it can be confirmed that HHMs were successfully synthesized and effectively used for the low-abundance glycoproteins/glycopeptides enrichment.
TAL (transcription activator-like) effectors secreted by plant pathogenic bacteria of genus Xanthomonasis capable of binding to specific genomic dsDNA. TAL is composed of modular architectures of DNA binding domains encompassing a tandem array of several almost identical repeat sequences. dTALEs (designed TALEs) restructured with TAL effector and other nucleotide acid-binding domains (such as nuclease, activator and suppressor) can specifically target and modify specific genomic DNA sequence, and play a pivotal role in genetic engineering. The repeat variable diresidues (RVDs) in each TAL repeat is exclusively responsible for recognition of single DNA base. TALENs consisting of TAL and restriction enzyme Fok I can contact with the specifical genomic DNA site and undergo the cleavage of dsDNA. The resulting double-stranded breaks usually are remedied through HR (homologous recombination) and NHEJ (non homologous end joining) and elicit corresponding gene mutations. TALENs are capable of generating highly efficient mutation in many model organisms. In virtue of modular design of the DNA-binding domain from TALEN system, TALENs can be developed into high-throughput platforms for gene modification and regulation and have broad-spectrum applications. Herein, an overview of the state-of-the-art structural advance of the TAL effectors, the design strategy for TALENs and applications and perspectives of TAL effector in genome-targeting modification and so on were provided.
DNA methylation and microRNAs (miRNAs) interact and regulate each other in cells, which co-regulate the expression of its downstream target genes, and play important functions in the physiological and pathological processes of cell growth, metabolism, immune, tumor and cardiovascular diseases, etc. The research situation of DNA methylation and miRNAs was summarized briefly. Then, DNA methylation changes under the regulation of miRNAs were analyzed; changes in miRNAs expression and activity under the regulation of DNA methylation were discussed. The feedback regulatory relationships between DNA methylation and miRNAs were also reviewed. Finally, the application prospects of DNA methylation and miRNAs were also discussed briefly. Studies on the regulatory network between DNA methylation and miRNAs, can supply references for further study and application of epigenetics.
Red recombination technology is developing rapidly, and has been widely used in Escherichia coli gene modification, such as deletions, insertions and substitutions. Compared with the traditional scarred recombination, the scarless recombination technology based on Red recombination is able to make more precise, rapid and efficient modification of targeted genes without the introduction of any exogenous sequences in the genome. From the basic mechanism of Red recombination,the principles and operation strategies about scarless recombination for Escherichia coli in recent years were reviewed, and also the advantages and disadvantages were analyzed. Finally, the applications of scarless recombination technology were introduced.
As metabolites in TCA cycle, C4 dicarboxylatic acids such as malic acid, fumaric acid and succinic acid, are widely used in food, pharmaceutical and chemical industries, the potential total annual demand for C4 dicarboxylatic acids is very huge. C4 dicarboxylatic acids production by microbial fermentation is attracting increasing attention. Some microorganism isolated from nature can produce large amounts of C4 dicarboxylatic acids, however, these processes are suffered many weakness. In contrast, the model microorganisms harbor many advantages for C4 dicarboxylatic acids production and become focus of study at home and abroad. Recent research progress in the production of C4 dicarboxylatic acids by microbial fermentation, including production of C4 dicarboxylatic acids by natural producing microorganism and its mutants, metabolic engineering of type strain for C4 dicarboxylatic acids production, and metabolism regulation of fermentation process were reviewed. Finally, the problems to be solved and future research direction was also proposed.
Bacillus subtilis spore display, a technique that presents proteins or peptides on the surface of the spore of Bacillus subtilis, enables high-throughput screening and has become an essential tool in bimolecular engineering. The major advantage of spores over the other published systems is their synthesis within the cytoplasm of the bacterial cell. Therefore, any heterologous protein to be anchored on the outside does not have to cross any membrane. Furthermore,spores are extremely resistant against high temperature, irradiation and many chemicals, and can be stored for many years at room temperature. With the advantage of complete genome information and security, Bacillus subtilis spore display now is attracting more and more attention. The following describes the recent progress of surface of Bacillus subtilis in the produce of vaccines and the immobillzed enzyme, and present a brief overview about how to improve the yield of the target protein surface display.
Tissue organ three-dimensional construction is that seed cells are combined with scaffold material to obtain the designed tissue or organ, which is a main content of tissue engineering and can represent technological merits of tissue engineering, such as the construction of blood vessel and trachea. For the flaws of traditional tissue engineering, Shimizu firstly put forward the concept of in situ tissue engineering, which applies the basic principle of tissue engineering to develop new tissue to repair defects by means of the transplanted seed cells induced by every method or the migration, proliferation and differentiation of the local cells of endogenic defect tissues. The distinctive characteristic of in situ tissue engineering is that bioreactor, an in vitro cell culture apparatus, is independent. In situ tissue engineering is a beneficial supplement of traditional ex vivo tissue engineering, which still has vast potentials for future development.
