Objective: To investigate the effects of the cyclic stretch on rat bone marrow mesenchymal stem cells (BMSCs) migration and its molecular mechanism. Methods: The cyclic mechanical stretching apparatus were used to study the effects of the cyclic stretch on the migration of BMSCs with different parameters. Transwell and scratching method was used to detect the number of cell migration and the levels of matrix metallopoteinases-2, -9 proteins were detected by zymography. Results: The migration of BMSCs could be remarkably stimulated by appropriate cyclic stretch, and the number of cells was increased to 1.58 times compared with that of the control group at 1 Hz, 10% strain for 8 hours. Moreover, the expression level of matrix metallopoteinases-2, -9 protein was increased signific antly after the stimulation of the stretch. After treated with GM6001, the secretion of MMP-2,-9 and the migration of BMSCs stimulated by the cyclic stretch was inhibited prominently. Conclusions: Matrix metallopoteinases-2, -9 play an important role in the cours that the cyclic stretch regulated on the migration of BMSCs.
To study the effects of lentiviral vector of microRNA targeting CHD5 gene on human colon carcinoma Lovo cell. Methods: Specific small interference RNAs were designed on line software and screened with the optimize principles, synthesized and cloned into the pPRIME vetor. Recombinant lentiviral was producted by 293FT cells following co-transfection of pPRIME-TET-GFP-CHD5 with the packaging plasmids pCMV-VSV-G, pRSV-Rev and pMDLg-pRRE. Human colon carcinoma Lovo cell were infected by the recombinant lentivirus.The expression of CHD5 was confirmed by RT-PCR and Western blotting, and the proliferation of Lovo cells was evaluated by MTT assay. Results: The recombinant lentiviral vecor carried the CHD5 gene was successfully constructed by restriction enzyme digestion and sequencing, respectively.The packaged lentiviral titer was 3.1×106 TU/ml.RT-PCR and Western blot analysis revealed that pPRIME-TET-GFP-CHD5 infection inhibited CHD5 mRNA and protein expressions in Lovo cells. The proliferation of Lovo cells was increased. Conclusion: The recombinant lentiviral vector pPRIME-TET-GFP-CHD5 was constructed successfully. It can be deliver target gene to human colon carcinoma Lovo cell, and inhibit CHD5 can promoted the proliferation of colon carcinoma Lovo cell.
DNA polymerase δ (Pol δ) consists of p125, p68, p50 and p12 subunits. The smallest subunit p12 is thought to play a crucial and versatile role in modulating the functions of Pol δ during DNA replication and various DNA repair processes. In order to obtain polyclonal antibody against p12 with high specificity and sensitivity, the recombinant plasmid pGEX-5X-3-p12 was generated by PCR and subcloning of digested PCR product (p12 DNA) into the pGEX-5X-3. GST-tagged p12, expressed in BL21 cells which was transformed with pGEX-5X-3-p12 and induced by IPTG, was purified on Glutathione-Sepharose 4B beads and further purified on FPLC Mono Q column. Non-tagged p12, used as immunogen, was released by proteolysis with Factor Xa. The polyclonal antibody against p12 was produced by immunizing rabbits with highly purified non-tagged p12 protein. The collected antiserums were purified by precipitation with (NH4)2SO4 and followed by the purification on Protein A/G Plus Agarose. Western blotting analysis showed that the obtained antibody recognized endogenous p12 in good specificity and sensitivity. While a cell based immunocytochemistry assay showed a subcellular co-localization of p12 with PCNA to distinct nuclear spots which is the first time to confirm a physiological interaction of p12 with PCNA in vivo. Thus, the study provides a powerful tool for the further research on the p12 modulating Pol δ functions and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.
Objective: Prokaryotic expression,purification of the EpCAM protein,preparation and characterization of the monoclonal antibodies against the protein. Methods:The gene was cloned into pET-28a(+) plasmid. The recombinant plasmid EpCAM/PET28a was transformed into E.coli BL21 and induced with IPTG. The recombinant protein was purified with Ni-NTA resin. The monoclonal antibodies were prepared by fusion of the purified EpCAM protein immunized BALB/c mouse’s spleen cells with SP2/0 myeloma cells,positive cells were screened with indirect ELISA and the positive cells were used to immune BALB/c mice to obtain the monoclonal antibodies against EpCAM protein. The binding specificity of the purified monoclonal antibodies were analysised by Western blot and FACS. Results: The recombinant plasmid EpCAM/PET28a and purified protein were obtained, two hybridoma cell lines secreting anti-EpCAM IgG McAbs were established and named as 4B2, 2F2.FACS analysis showed that the two McAbs showed high specificity to FaDu cell line,while Western blotting analysis showed that only 4B2 can recognize the denatured EpCAM protein in FaDu cell line. Conclusions:The anti-EpCAM IgG McAbs were prepared which had high specificity to FaDu cell line, and it could be used to develop the anti-EpCAM antibody in cancer therapy.
Objective:In order to research the bioactivity of GALNT3, the truncated part of GALNT3 (huGALNT3-sol) which was deleted of the hydrophobic trans-membrane domain were obtained using Pichia pastoris expression system, and assayed the transferring GalNAc activity of recombinant huGALNT3-sol. Methods: The gene of human GALNT3-sol (1 755 bp)was amplified from pET15b/ GALNT3-sol and cloned into expression vector pPIC9k, and the recombinant plasmid was transformed into Pichia pastoris GS115 strain through electroporation. The high copy recombinant strains with high-level huGALNT3-sol production were screened out by MD plate and G418. High level of huGALNT3-sol was obtained in BMMY medium the induction of methanol, and purified from the supernatant with Ni-NAT.The identity of the recombinant protein was confirmded by SDS-PAGE and then Western blotting analysis. HPLC and MALDI-TOF-MS analysis were used to identify the bioactivity of recombinant huGALNT3-sol. Results:The recombinant Pichia pastoris which could secretory express the human GALNT3-sol protein was constructed successfully. High level of huGALNT3-sol was obtained in BMMY medium (pH 6.0) after 96 hours induction of 20℃ and 0.5% methanol, with the highest yield of 5mg/L in shake flask culture. The identity of the recombinant protein was confirmed by Western blot analysis and the huGALNT3-sol expressed in Pichia pastoris is in higher molecular weight glycoforms. The activity assay showed the recombinant huGALNT3-sol has the activity of transferring GalNAc to Ser/Thr residues in peptide. Conclusion:The human GALNT3-sol, which has the activity of transferring GalNAc to Ser/Thr residues in peptide, was successfully expressed and purified in Pichia pastoris. It provides support for the further research and application of GALNT3.
In order to high level express anti-bFGF ScFv antibody in Pichia pastoris, the gene of human anti-bFGF ScFv was subcloned, and constructed into expression vector pPICZαA. The constructed expression vector pPICZαA-ScFv was linearised and transformed to Pichia pastoris by electroporation. The transformants were induced by methanol, and the anti-bFGF ScFv was expressed. The expression products were purified by affinity chromatography of Ni-Seproase 6 FF and ion exchange chromatography of DEAE Sepharose FF. The results of SDS-PAGE and Western blotting showed that anti-bFGF ScFv was high level expressed successfully and the expression quantity was about 124mg/L. The target protein was purified from the expression products and the purity was more than 95 %. The results of ELISA showed that the purified recombinant ScFv could combine with bFGF specifically. The results of CCK8 showed that the purified recombinant ScFv could inhibit the proliferation of A549 cells in a dose-dependent manner in vitro. The results demonstrated that the anti-bFGF ScFv can be high level expressed in Pichia pastoris with good biological activity.
O-GlcNAcylation is a dynamic and reversible process, regulated by two key enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). An insect OGA gene was extracted from the fifth instar larva of the Asian corn borer Ostrinia furnacalis (Guenee).Full length OfOGA gene was 3 541bp, containing 241bp 5'-UTR, 3 165bp coding region and 132bp 3'-UTR.Open reading frame (ORF) of OfOGA was cloned and expressed in E.coli. The recombinant OfOGA consisted of 1 055 amino acids, with a theoretical molecular weight of 118 kDa, but an apparent molecular weight of 130 kDa by SDS-PAGE. Its optimal pH was 5.5, and the optimum temperature was 50℃. Recombinant expression of OfOGA may facilitate the understanding of its role in insect development and growth, and provide potential biological control target.
The growth and secondary metabolites production of Anoectochilus formosanus Hayata PLBs were increased by manipulating various culture factors and variable process. The results indicates that MS basal medium with S-3307 1.0mg/L, 6-BA 0.5mg/L and 3% sucrose was found to be equally suitable for biomass accumulation (214.45g/L FW and 18.23g/L DW). Whereas the accumulation of total flavonoids, total phenolics and polysaccharides (5.43mg/g DW, 2.87mg/g DW and 243.23mg/g DW) was maximized at MS medium (NH4+∶NO3- ratio is 60∶0mmol/L) supplement with S-3307 1.0mg/L, 6-BA 3.0mg/L and 5% sucrose. The growth pattern was also investigated in suspension culture of PLBs. The maximum biomass production (225.98 g/L FW and 18.53 g/L DW), total flavonoids (5.09mg/g DW) and total phenolics (2.04mg/g DW) were obtained after 7 weeks. However, the polysaccharides production reached its peak (229.36mg/g DW) 5 weeks after suspension culture.
4-Hydroxyphenylglycine aminotransferase which can synthesize D-phenylglycine transaminase is produced by Pseudomonas. The hpgt gene was synthesized through the codon optimization technology. The recombinant prokaryotic plasmid pCDF-hpgt was obtained. The plasmid was transformed into the competent cell E. coli BL21 (DE3). The recombinant His-HpgT protein was obtained after the optimized expression and purified by nickel chelate affinity chromatography method. The enzyme activity of the forward and reverse reactions was measured and the activity of the forward reaction reached 749mU/mg which was lower than the reverse, 2 257mU/mg. Also the optimized temperature and pH were measured, with the result of 35℃and 8.0. Other kinetic parameters and the mechanism analysis of enzyme characteristics were explained. The enzyme affinity to phenylglycine was higher than the glutamate which obtained by the Michaelis-Menten equation; and the reaction was inhibited by the lower concentration of phenylglyoxylic acid.
Objective: The present study was to evaluate the safety of Bacillus amyloliquefaciens microcapsules used for aquaculture. Methods: According to Chemicals—Alga growth inhibition test (GB/T21805-2008), Water quality—Determination of the acute toxicity of substance to Daphnia (Daphnia magna straus) (GB/T13266-91), Water quality—Determination of the acute toxicity of substance to freshwater fish (Brachydanio rerio Hamilton-Buchanan) (GB/T13267-91) and Clinical experiment technical practice for fishery drugs, the growth inhibition of the B. amyloliquefaciens microcapsule on Chlorella sp. and its acute toxicity to Daphnia, zebra fish and grass carps were observed, and its influence on main physicochemical factors of aquaculture water was also assayed. Results: The growth of Chlorella sp. was promoted with B. amyloliquefaciens microcapsules at the final concentrations of 0.2~2 000mg/L, its IC50 to Chlorella sp. was 2 000mg/L, and its LD50 to Daphnia, zebra fish and grass carps were all >2 000mg/L (or mg/kg body weight). In addition, in the period of 14 days after the input of the B. amyloliquefaciens microcapsule into the farming water at 0.2~2 000mg/L, the contents of the ammonia and sulfide as well as the pH values were gradually reduced, only the nitrite nitrogen concentration first rose slightly and followed by a slow decrease, and the changes of the physiochemical factors related negatively to the concentration of the B. amyloliquefaciens microcapsules. Conclusion: The B. amyloliquefaciens microcapsule was actually nontoxic, its effects on the ammonia, nitrite nitrogen, sulfide and pH in the fish faming water were under the control of their safe concentrations to aquatic animals, such as the goby fry, Pelteobagrus fulvidraco, Esox lucius, Procambarus clarkia. The present study provided important scientific basis on the safe use of B. amyloliquefaciens microcapsules in aquaculture.
To enhance the functionality of dehalogenase from Pseudomonas sp. CGMCC 3267, Used error-prone PCR to create mutants with improved specific activity. Through error-prone PCR and high-throughput screening method, a desirable mutant DehII-B2 was obtained. Compare to initial strain, the specific activity of DehII-B2 was increased 3.9-fold. Three-dimensional structures of DehII-S and DehII-B2 were constructed by homology modeling, and molecular docking of dehalogenase and S-2-CPA. The results suggested that the mutant site is Ala7, the binding energy of DehII-B2 is reduced 1.4kcal/mol than DehII-S, and the distance of active site Asp10 and the α carbon atom of substrate is shorten 0.321 6nm, thus accelerate the enzyme reaction rate and increased enzyme specific activity. At present, the specific activity of DehII-B2 was higher than all enzyme in our lab. According to the results, the optimum pH and pH-stability were not changed, but the optimum temperature and thermo-stability of DehII-B2 were slightly increased than those of initial strain. The preliminary application of DehII-B2 was studied, and the best result was obtained at 40℃, 60mmol/L 2-CPA reacting for 10h.
Objective to construct high yields recombinant E. coli strains producing phenyl lactic acid(PLA). Methods Analyze the X-ray structure of active site amino acid residues of D-LDH from Aquifex aeolicus, and compare it with mutant D-LDH of the homologous model. The optimized 3D structures of the mutant enzyme were elected by analyzing the space conformation of the amino acid residues in substrate binding domain of the mutant D-LDH. The genes of enzyme were site-directed mutated, cloned and expressed. Results F49A or Y297S mutants were finally elected for single-site mutation models, F49A/Y297S mutant was elected for double -site mutations, and three recombinant E. coli strains were proved for PLA production. Conclusion The method of mutation visualization is an effective method for construction of high yield gene engineering bacterium producing PLA.
Natural and artificially engineered inteins have been more widely used in the field of protein engineering. However, inteins are often inactive when cloned into a heterologous host protein, and also need the presence of the native exteins, which would be left in the target protein. Inteins should overcome these limitations. In order to improve splicing activity of Ter DnaE-3 (Trichodesmium erythraeum) mini-intein in heterologous host, random mutagenesis was based on error PCR by change the concentration of dNTP、Mg2+、Mn2+, and then mutants were screened in kanamycin resistance-dependent system. After directed evolution, mutant 5th increased splicing activity from ~20% to ~85%, and mutant 9th can avoid occurring cleavage compared with other mutants by western blot analysis. The relationship of amino acid mutations and splicing activity showed that amino acids involved in the formation of α-helix structure may influence intein cleavage reaction, and amino acids involved in the formation of β-sheet structure may provide some help for intein structure. The resulting improved inteins showed high activity in heterologous host, which verify the kanamycin resistance-dependent system feasibility and expand intein application.
Purpose:Chitosan immobilized zinc ion affinity chromatography matrixes were prepared,and its properties were studied.Methods:Immobilized metal affinity chromatography (IMAC) matrixes were successfully prepared with cross-linked chitosan prepared by reverse phase suspension polymerization,and 3-Chloro-1,2-epoxypropane as activated agent, ethylenediamine as chelating ligand and Zn2+ as center ions.Its effective grain size and uniformity coefficient,moisture content,rate of lostmass, amino content,skeletaldensity,bulk density and porosity were characterized.Time,concentration of ZnCl2 solution,temperature,pH of Zn2+ immobilized conditions were optimized to determine the amount of Zn2+ immobilized.After purifying with salting out,aldehyde dehydrogenase containing a histidine-tag was used to examine the affinity of chitosan immobilized Zn2+affinity chromatography matrix.Results:the effective grain size,uniformity coefficient,moisture content,rate of lostmass,amino content,skeletaldensity,bulk density and porosity of the prepared matrix is 105μm,1.46,58.03%,85.43%,9.20 mmol/g, 1.2178 g/ml, 0.8432 g/ml and 36.40% respectively.The optimum conditions of Zn2+ immobilized were:time 3h,ZnCl2 concentration 0.1 mol/L,temperature 28℃,pH 5.5.And the amount of Zn2+ immobilized is 3.35 mmol/g under these optimum conditions.The affinity of chitosan immobilized zinc ion affinity chromatography matrix for aldehyde dehydrogenase is 4.14 IU/g(dry weight).Conclusions: The high-efficiency chitosan immobilized zinc ion affinity chromatography matrixes were prepared,and it can be used for quickly separating and purifying the recombinant protein containing a His-tag.
With the progress of molecular biology, there is a growing number of molecular targeted cancer drugs have been used in clinical to treat of malignant tumors. Molecular targeted therapy is also known as "biological missile", and has become the fourth treatment methods in addition to surgery, radiotherapy and chemotherapy. Compared with traditional chemotherapy drugs, the advantages of molecular targeted drugs are specificity, efficacy and less side effects. Molecular targeted drugs can be divided into two classes according to the nature: monoclonal antibodies such as cetuximab and small molecule inhibitors with single target or multiple targets such as gefitinib. The epidermal growth factor receptor (EGFR) has been found that it contributes to tumor cell survival, proliferation, and motility. Overexpression and mutation of the EGFR gene are the most common mechanisms by which EGFR exerts influence on tumorigenesis. Therefore, EGFR has been successfully targeted for cancer therapy. The latest clinical research of the molecular targeted drugs have been listed targeted epidermal growth factor receptor (EGFR).
As part of the innate immunity system of host organism, antimicrobial peptides (AMPs) possess a wide spectrum of antimicrobial activity against eubacteria, fungi and eukaryotic parasites. AMPs are considered as one of potential alternates to the classical antibiotics in medicine, agriculture and food industry. The pathogenic microorganisms have correspondingly developed a defense system against the actions of AMPs during the co-evolution between the hosts and the pathogens. Recent discoveries on the resistance mechanism of pathogenic microorganism against AMPs, including sensing and gene regulation, modification of cell wall and/or plasma membrane, degradation of secreted proteases, as well as efflux pump by transporter proteins are discussed. Further, the perspectives of future research on AMP productions were proposed.
Barley is one of the major cereal crops in the world. Due to its diploid nature, barley can be considered an important material for Triticeae genomic studies. With the application of techniques and methods employed in genomics, such as a large number of mapped molecular markers, BAC libraries, mutant collections and DNA arrays, the sequencing of barley genome has deepen increasingly. More and more information about the barley genome pave the way for a comprehensive analysis of structural and functional genomics of barley as well as understanding of gene expression networks linked to agronomically important traits. Progress in genomics and molecular breeding of barley were summarized, including ESTs system, physical map construction, functional genomics and barley marker-assisted selection, which provides a theoretical basis for further improving molecular breeding and expounding the structural and functional performance of barley genome.
Xylose utilization in high efficiency is a critical factor for the economic performance of lignocellulosic biomass biorefinry, and it is necessary for building of this industrial system. However, the novel ideas and thoughts for xylose utilization should be developed in that the bioconversion of xylose is now facing serious technical bottlenecks. Xylonic acid fermentation was brought forward as a new way for bioconversion of xylose from lignocellulosic biomass based on systemic analysis of its status and industry advancement. Controlling and elimination of inhibitors was illuminated as the primary problem of science and technology in xylonic acid fermentation, and then the approach to this challenge was put forward by a combined study of its cellular physiology and biochemistry, metabolism flux analysis and molecule biology with a multi-lever and multi-scale method. Furthermore, the tech-integration idea was brought up for industrial production of xylonic acid from xylose in a systemic view, involving development in steps of strains screening, material pretreatment, inhibitors controlling and removing and xylonic acid effective fermentation.
As a promising chiral synthesis tool, amidases have received growing attention with application in production of optically pure carboxyl acids and related derivatives. Enantioselectivity of amidase plays a vital role in amidase catalyzed reactions. Effects of microbial amidase sources and reaction factors such as substrate, temperature, pH as well as cosolvent on amidase enantioselectivity were summarized. It is helpful in improving amidase enantioselectivity and application of amidase in production of valuable optically pure compounds.
The bioeconomy has increasingly close and important relationships with biological resources, low carbon emission, food security, nutrition and healthy, bioenergy, biomaterials, redefinition of the role of agriculture, land use, biodiversity and ecosystem services (BES), R&D, and policy making. The bioeconomy developed in Australia has been analyzed from the understanding of "the bioeconomy", the bioeconomy framework dissected, and the opportunities to grow Australia’s economy. By comparison with the developments of bioeconomy in other countries and regions, several revelations which may be referenced by China have been given as below: usher in revolutions in many areas by proposing the development of broad bioeconomy, pay great attention to the sustainability of bioeconomy and the redefinition of the role of agriculture, take bioeconomy as a platform to a sustainable future through overstepping the concept phrase of bioeconomy, and adopt the model of "whole of biomass" properly for the development of bioeconomy.
