Objective:To establish a miR-106b transgenic mouse and investigate its effect on the development of Alzheimer’s disease (AD). Methods:The expression vector of miR -106b was constructed. The transgenic mouse was produced by microinjection and genotype was detected by PCR. The expression levels of miR-106b were detected by real time RT-PCR. The protein levels of TGFBR2 were detected by Western blot. Results:Five founders of miR-106b transgenic mice were established and one high-level expression line was identified. Compared with the wild type mice, the expression of TGFBR2 was increased in miR-106b transgenic mice. Conclusion:miR-106b transgenic mouse has been established and it can be used to investigate the function of miR-106b on the development of Alzheimer’s disease.
The two human stem cell factor (hSCF) mimetic peptides (CS2 and LS2) fused with the c-jun leucine zipper respectively were expressed in E.coli and the bio-activity of the fusion proteins was identified. The three expression plasmids pET30a-CSJ, pET30a-LSJ and pET30a-Jun, which contain fusion proteins (CSJ and LSJ) and c-jun gene respectively, were constructed by using overlapping PCR. Results showed that the CSJ, LSJ and c-Jun(CJ )were expressed about 20% of total protein in E.coli BL21. After purified by using affinity Ni-NTA and Sephadex G-50 column chromatography, the purity of the CSJ, LSJ and CJ were over 95% identified by using SDS-PAGE analysis. Their molecular mass was 7336.0 8, 7991.54 and 6672.74 respectively determined by mass spectrometry. The bioactivity of the fusion proteins was evaluated by using cell proliferation assay with MTT in UT-7 cell. Compared to CS2 and LS2, the bioactivities of CSJ and LSJ were dramatically improved and it is about 1000 fold higher than that of CS2 and LS2.
Objective: Construct recombinant adenoviral vector co-expressing angiogenin-1(Ang-1) and vascular endothelial growth factor 165(VEGF165) mediated by IRES and polyA-promoter,then compare the expression of Ang-1/VEGF165 mediated by IRES and polyA-promoter as well as in the downstream and upstream of polyA-promoter/IRES likewise their vascular inducibility,to provid experimental evidence for constructing superior recombinant vector co-expressing double gene or multiple gene.Methods: The VEGF165 and Ang-1 fragments were amplified by PCR using pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmids as templates.The VEGF165 and Ang-1 fragments were subcloned into pAdTrack-CMV-IRES and pAdTrack-CMV-PolyA-promoter transfer vector,and then identified by PCR,double endonuclease digestion,and DNA sequencing.The pTrack-CMV-Ang-1-polyA-promoter-VEGF165、pTrack-CMV-VEGF165-polyA-promoter-Ang-1、pTrack-CMV-VEGF165-IRES-Ang-1 gene recombinated transfer vectors were cotransformed into the bacteria BJ5183 competent cells with pAdEasy-1 backbone vector for homologous recombination,then they were linearized with PacI digestion and transfected into the human embryonic kidney 293 (293A) cells, leading to formation of the recombinant adenoviruses Ad-Ang-1-polyA-promoter-VEGF165、 Ad-VEGF165-polyA-promoter-Ang-1 and Ad-VEGF165-IRES-Ang-1.The transcription of VEGF165 and Ang-1 were identified by RT-PCR,and the expression of VEGF165 and Ang-1 in different adenoviral vector were then identified by enzyme-labeled immunosorbent assay(ELISA),then compared the expression level of Ang-1 and VEGF165 gene in IRES and polyA-promoter mediated vector as well as in the downstream and upstream of the same adenoviral vector.Injected Ad-Ang-1-polyA-promoter-VEGF165,Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,Ad-Ang-1-IRES-VEGF165 to the rabbit corneal limbal and detected the new vessels areae, then compared their vascular inducibility.Results: The sequencing of Ang-1 and VEGF165 were correct,three kinds of recombinant adenoviral vector were all successfully constructed and obtained,and their potency were up to 2~5×1010pfu/ml, the transcription of VEGF165 and Ang-1 were also significant by RT-PCR.Moreover VEGF165 and Ang-1 gene expressing in the WI-38 cells was confirmed by ELISA,also found that the IRES-mediated Ang-1 and VEGF165 gene, either upstream or downstream,its expression level was lower than the same location in polyA-promoter-mediated genes,degraded about 60%~70%, moreover either the IRES vector or polyA-promoter vector,the expression of downstream genes volume was significantly lower than the upstream gene volume, degraded about 30%~40%.At the same time the animal experiment of vasiformation in rabbit cornea suggestted that the vascular inducibility of Ad-VEGF165-PolyA-promoter-Ang-1 and Ad-VEGF165-IRES-Ang-1 were better,and the former was stronger than the latter.Conclusion:In the adenoviral expression vector,Ang-1 and VEGF165 gene could both successfully expressed in cell,and both had vascular inducibility.The expression level and vascular inducibility of polyA-promoter-mediated double gene was higher and strongger than the IRES-mediated double gene,at the same time the expression level and vascular inducibility of the gene volume in the downstream of polyA-promoter/IRES were lower than the genes volume in the upstream.
Objective:To construct shuttle expressing plasmids of H37Rv eis gene, and identify biological activity of recombinants in Mycobacterium smegmatis mc2155(M.S).Methods: eis gene was amplified from Mycobacterium tuberculosis genomic DNA and clonded into an Escherichia coli-Mycobacteria shuttle plasmid pMV261 to construct a recombinant plasmid pMV-eis. The pMV-eis was performed successfully by PCR, enzyme digestion analysis and sequencing. pMV-eis then was transformed into M.S by electroporation. After cultivation positive colonies were picked out and the plasmid was amplified by PCR and enzyme digestion analysis again.The expression of Eis protein in the M.S was proved by SDS-PAGE and Western blot. Results: Sequencing and homologous analysis veryfied the cloned eis gene, and its nucleotide and deduced aminoacid sequences was equaled to the related gene in GenBank.The growth curve of the recombinant Mycobacterium smegmatis(rM.S) was consistent with that of the wild-type strain, suggesting the absence of Eis protein toxicity against M.S.SDS-PAGE and Western blot result showed that relative molecular mass of expression product was about 42kDa. Conclusion:The shuttle plasmids pMV-eis has been constructed successfully. and the recombinant plasmid has biological activity in rM.S, which lays the foundation for further research about of Eis function.
Thiamin monophosphate kinase (ThiL) catalyzes the ATP-dependent phosphorylation of thiamin monophosphate (TMP) to form thiamin pyrophosphate (TPP), the active form of vitamin B1. ThiL is a member of a small ATP binding superfamily. The gene of ThiL from Shigella flexneri 2a(strain 301)was constructed into the expression vector and over expressed in the E.coli. Then it was purified through two steps of chromatography leading to the high purity of the protein. The purified protein was screened for crystallization. The hit condition was optimized and gave rise to the single crystals for X-ray diffraction, which is the fundamental step for its structure determination, illumination of its catalytic mechanism, and corresponding drug design.
Curcumin, obtained from the rhizomes of Curcuma longa L., Zingiberaceae (turmeric), are the most widely used phytoconstituent in food industry and recently for its therapeutic activity. It has very wide spectrum of therapeutic use like in inflammation, psoriasis and various tumors. But its highly lipophilic nature and very poor bioavailability hampers its therapeutic usefulness. The synthesized cationic poly(butyl) cyanoacrylate (PBCA) nanoparticles are coated with chitosan encapsulated formulation of curcumin-nanocurcumin. The particle size and zeta potential of prepared nanocurcumin was about 250nm and + 37.3 mV. The TEM study revealed the spherical nature of the prepared nanoparticles along with confirmation of particle size. MTT was used to assay the biologic activities of nanocurcumin and its anti-proliferative effect. Human hepatocellular carcinoma (HepG2) cells were treated with different concentration of nanocurcumin, curcumin and empty PBCA nanoparticles for 24h. MTT test showed that nanocurcumin was cytotoxic to HepG2 cells, the number of the apoptosis cell line increased. The inhibitory effect of nanocurcumin on cell growth was in a dose-dependent manner. Cell apoptosis percentage was gradually increased along with nanocurmumin concentration rising. The apoptosis rate for 5, 10, 20, 30, 40 and 50μg/ml is about 13.65%, 33.11%, 43.45%, 67.93%, 77.79% and 91.5% respectively.It shows obvious statistical difference against normal HepG2 cells.While the empty PBCA nanoparticles exhibit a low cytotoxicity to HepG2 cells. The morphologic alteration of HepG2 cells after treatment of nanocurcumin was observed under fluorescent microscope. When treated with nanocurcumin for 4h or longer time at 30μg/ml, HepG2 cells turned to circle, fell down from wall, and proliferated slowly. According to flow cytometry, after treatment with nanocurcumin, HepG2 cells were observed to block cell cycle in G2／M phase. Nanocurcumin has been shown to inhibit angiogenic biomarkers, vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) expression. Nanocurcumin’s mechanisms of action on liver cancer cells mirror that of free curcumin. Therefore, this kind of nanocurcumin could be used as a candidate for hepatocellular carcinoma (HCC) in the future. Nanocurcumin also provides an opportunity to expand the clinical application of this efficient agent by enabling ready aqueous dispersion. Future studies utilizing nanocurcumin are warranted in pre-clinical in vivo models of cancer and other diseases.
In Beijing, there has emerged an external invasion plant, Solanum rostratum dating from the beginning of this century. This plant originated from the North America. It is poisonous all over the body. There exists a sort of neurotoxin, solanine in Solanum rostratum. On the other hand, solanine is also a valuable material that, for example, is effective for inhibiting tumors. In order to utilize waste materials by obtaining pharmacologically and economically valuable products such as solanine from Solanum rostratum as well as becoming familiar with its toxic degree and tackling with this external invasion plant. It was designed to assess the tumor suppressing effect of Solanum rostratum by detecting the inhibiting effect of the whole-plant extract on the membrane ATPase activity of mouse tumor cell H22, with the functional effects of solanine and traditional chemotherapeutic cyclophosphamide as controls. The result revealed, in the nature of Beijing, the growing Solanum rostratum arising from invading expressed a tumor cell membrane ATPase activity inhibiting effect around as high as that of 3.63 mg/kg solanine, and around as high as or slightly higher than that of potato budlet equivalent. Therefore, it is possibly opportune to utilize waste material during renovating the ecology destroyed by the invading Solanum rostratum through obtaining medical components like solanine. It would be an economically efficient reaction, in the meanwhile emancipating the productive forces of potato and other valuable Solanum plants.
The α-cgt gene was obtained from Paenibacillus macerans by PCR, and then it was cloned into Escherichia coli-Bacillus subtilis shuttle vector pGJ103 and transformed into B. subtilis WB600. After cultivation for 48 h with shake flask in 1.5% maltose initial medium, the α-CGTase activity of recombinant B. subtilis was 6.1U/ml. In addition, the experiment optimized the culture conditions of the recombinant B. subtilis strain in shaking flask by means of a single factor synthesis and Box-Behnken design (BBD). The analysis predicted the concentrations of maltose, corn starch and yeast extract were at 15 g/L, 13 g/L, and 20 g/L, respectively. In this condition, the experimental result of 17.6 U/ml could be obtained when the cells were cultured for 36 h in shaking flasks. The CGTase activity reached 20 U/ml at 30 h of culture in a 5 L bioreactor (hydrolysis activity was 1.4×104 IU/ml).
T4 lysozyme pilot-scale fermentation was studied. T4 lysozyme was induced and expressed by Pichia pastoris in the 200L fermentor. The yield of target protein was 1.69g /L with 192U/mg enzyme activity in the broth. The enzyme protein was freeze-dried. The weight of enzyme powder was 211.1g. T4 lysozyme content was 68.9%, and activity was 182U/mg in enzyme powder. The total yield was 66%. The T4 lysozyme pilot-scale process line was established successfully.
The purine nucleoside phosphorylas (PNP,EC.2.4.2.1) genes deoD and punA which amplified from the Bacillus subtilis168 genome by polymerase chain reaction were identified, cloned and expressed in E. coli XL-Blue, respectively. Recombinant purine nucleoside phosphorylases PNP702 and PNP816 were purified by Ni+-NTA column, and several characteristics were determined. The results revealed that PNP702 and PNP816 both have the same optimal temperature (60℃) and pH (7~8). Enzymic kinetics experiment showed that the catalytic efficiency (Kcat/Km) of PNP816 is 11.12 times higher than that of PNP702 toward inosine. Compared with PNP816, PNP702 has broader substrate specificity. The engineering strain XL-Blue (pPNP816) has much higher catalytic activity than the XL-Blue (pPNP702) in enzymatic synthesizing of the nucleoside antiviral drugs ribavirin, indicating that the low molecular weight homologous trimer PNP derived from microorganisms has the same or higher value in the microbial enzymatic synthesis of nucleoside drugs and intermediates.
A universal method of the high efficiency and low background T vector construction was developed. The gateway cassette fragment containing the ccdB killer gene, used as medium DNA, was ligated to the vector backbone pGEM-T easy, and then the recombinant plasmid was digested by XcmI restriction enzyme to produce the T vector. The recombinant plasmid was confirmed by digestion pattern, PCR identification and sequencing, and the T vector was proved to be high efficiency and low background. This T vector had not only many advantages of pGEM-T easy vector, but also had very high efficiency and low background for molecular cloning. What’s more, several restriction enzymes introduced and gateway technology with LR recombination reaction provided convenience for insert fragment subcloning.
A lot of proteases are synthesized for cell growth and metabolism by Streptomyces avermitilis during growing in the complex medium. However, the 2-DE protein sample preparation for intracellular proteomics analysis of S. avermitilis became difficult because of the hydrolysis of protein by massive proteases when cells were disrupted. An effective protease inhibitor mixture was obtained by single factor and orthogonal optimum design based on the knowledge of intracellular protease composition of S. avermitilis. By verifying expriments, it was validated that the protease inhibitor mixture can effectively protect large molecular weight proteins from enzymolysis during sample preparation.
Objective: To establish a rapid, sensitive and specific real-time PCR method for detection of Clostridium perfringens (C.perf) and application in clinic. Method: The C.perf-specific primers and probe were designed through a specific primer designing program based on C.perf-specific sequence, DNA of secretion and liquor puris were tested with this assay. To confirm sensitivity, speedability and specificity of this method compared with bacterial culture. Results: The best forward and reverse primers concentration were 0.45μmol/L and 0.15μmol/L respectively, the best probe concentration was 0.3μmol/L.This technique had good sensitivity,had no consensual reaction with other 21 bacterium species.The result of PCR accorded with that of the culture. Conclution:. The Taqman PCR assay, especially in the war, can be used for the rapid, sensitive and specific detection and enumeration of Clostridium perfringens from secretion and liquor puris with a short enrichment.
Objective:To establish an BL21 expression system of Francisella tularemia-specific antigen (FopA-L and FopA-S), then obtain their highly active recombinant protein and the polyclonal antibodies which can be applied for Francisella monitoring, diagnosis and treatment.Methods:Constructing a plasmid expression vector pET100 FopA-L and FopA-S, then transforming FopA plasmid into E. coli BL21 cells, induced by IPTG, purifing protein by chelate nickel ion-NTA (Ni-NTA) affinity chromatography, making polyclonal antibody by recombinant FopA-L and FopA-S proteins immune rabbits and antibody were determined by Western blot, ELISA and GICA. Results:Constructed expression vectors pET100 of FopA-L and FopA-S, the BL21 cell lines obtained can highly express target proteins, antibody specifically combined with them which can be used in Francisella detecting were prepared successfully, with a titer of more than 1 ∶100000 and highly specific. Conclusion:Preparation the FopA-L and FopA-S antigen and antibody laid the foundation for the establishment of rapid detection method of Francisella.
An algorithm for classification of gene expression data based on Fiedler Vector was proposed. Firstly, the Laplacian matrix of complete graph is constructed on all the different types of gene expression data. Then, the Fiedler Vector is obtained by the singular value decomposition of this Laplacian matrix. Finally, the samples are divided into two classes by utilizing the signs of the Fiedler Vector components. The effectiveness of this algorithm has been proven by simulation experiment and real data experiment.
PCR-based chromosome walking is mainly applied to isolate flanking sequence of known sequence. It is convenient for gene cloning, obtaining regulatory elements and filling in gaps of whole genome sequencing. These methods can be classified into two sorts: dependent-ligation PCR and independent-ligation PCR. The technologies of PCR-based chromosome walking technologies in recent years and compares principles and procedures of these methods were outlined. The advantages and disadvantages of dependent-ligation PCR and independent-ligation PCR were summarized respectively, and their efficiency and usefulness were evaluated for investigator to select the appropriate method.
Cell bioprinting can be defined as an advanced technology that fabricates 3D functional multi-cellular tissue in vitro. Due to its interdisciplinary characteristics, high resolution, cell bioprinting has been of considerable importance and popularity in recently. It is a powerful technology for tissue engineering and biological studies. Rather than a single approach, cell bioprinting includes a set of techniques (e.g. inkjet bioprinting, laser based techniques) that transfer biologically materials onto a substrate. Cell bioprinting also provides a new concept that how to construct more naturally 3D tissues or organs in vitro. The principle of these techniques was introduced, An overview of cell bioprinting in biological engineering applications, such as building 3D micro tissue, investigating cell biology, fabricating cell based microarray was also presented.
The development of a safe and effective human immunodeficiency virus-1 (HIV-1) vaccine is a critically important global health priority. Despite recent advances in our understanding of HIV-1 pathogenesis and immunology, For HIV-1, obstacles to eliciting protective neutralizing antibodies (NAbs) have often seemed insurmountable. Traditional vaccine strategies will not provide protection against HIV-1. However, recent studies show that antibodies in the sera of some HIV-1–infected individuals can neutralize diverse HIV-1 isolates. Detailed analyses of these sera provide new insights into the viral epitopes targeted by broadly reactive NAbs. The findings suggest that the natural NAb response to HIV-1 can inform future vaccine design. High-resolution structural information can reveal the atomic level architecture of the region of env bound to a Nab, and this information can be used to design better immunogens.
Nowadays, peptide/protein drugs are administrated by injective formulations to ensure their bioavailability. While non-injection formulations, particularly oral delivery method, are desiderata because they exhibit great advantages such as easier administration, better patient compliance and lower cost. However, the bioavailability is usually very low if the peptide/protein drugs are used directly in the non-injection systems. And it is necessary to develop drug delivery systems with designed functions, such as adding different proportions of the protease inhibitors and/or absorption enhancers to improve drug bioavailability. Cyclodextrins(CDs) and their derivatives can form complexes with various molecules and have the capability to facilitate mucosa permeability, thus are widely applied in the non-injection administration systems of peptide/protein drugs. recent applications of CDs and their derivatives in non-injection administration systems of peptide/protein drugs were reviewed.
Bacteriophage is the most abundant bacterial virus in nature. As the natural bacterium killer, it displays exceptionally more advantages than antibiotics to the treatment of bacterial infections especially multi-drug resistant bacterium. The historical and updated research progress in the treatment of bacterial infections employing live bacteriophage and bacteriophage-derived lysin was summarized. The major obstacles in phage therapy and some feasible resolutions were discussed. It is expected that phage therapy will attract renewed interest increasingly and will play more important role in the coming post-antibiotic era.
The progress in the production of exopolysaccharides by Klebsiella sp. is discussed, including composition, function and fermentation process of the exopolysaccharides. And the problems in the process of exopolysaccharides production are also discussed. It is pointed out that the research emphasis should be placed on using the mathematic tools to optimize the fermentation process and modifying downstream recovery process. Then the comparison with their counterparts and exoploitation of the potential function of the exopolysaccharides will be focused on.
