It was chosen the Bac-To-Bac® Baculovirus Expression System to express DEK protein, and the protein was purified under native condition. First, the full-length DEK gene was amplified from pMD18-T-DEK, and then inserted into plasmid pFastBacI of the Bac-To-Bac® Baculovirus expression system to form plasmid pFastBacI-DEK. Recombinant shuttle vector named Bacmid-DEK was obtained in E.coli DH10Bac through transformation. After transfection with Cellfectin® II Reagent, recombinant Baculovirus (AcNPV-DEK) was developed in Sf9 cells. Then this viral stock was used to infect new Sf9 cells to generate a high-titer baculoviral stock. After that, the baculoviral stock can be used to infect Sf9 cells for DEK expression. The DEK protein was purified under native conditions using Ni-NTA agarose. The specific 50 kDa band was showed by SDS-PAGE and Western blotting, which indicated that DEK protein was expressed in Sf9 cells and the highly purified His-DEK proteins were obtained. Using the purified His-DEK, the Electrophoretic mobility shift assay (EMSA)was performed to test the binding activity of His-DEK to DNA molecules. The results showed that His-DEK and His-CDB (expressed by E.coli BL21 strain) could bind DNA molecules, especially, preferring the supercoiled form in vitro; meanwhile, the DNA binding activity of His-DEK was higher than His-CDB. It is reported that phosphorylation of DEK protein could inhibit the binding of DEK to DNA. The expressed His-DEK protein in Sf9 cells was phosphorylated, so the His-DEK protein was dephosphorylated using λ-PPase. The result of EMSA showed that the DNA binding activity of dephosphorylated His-DEK protein increased than the phosphorylated protein.
Objective: To identify domains of PINK1 interacting with α-synuclein. Methods: HEK293T cells were transfected with plasmid encoding human pCMV-Myc-α-synuclein together with human 3xFlag-tagged wild type PINK1 or several deletion mutants(G309D;ΔN35; ΔC145; 156~509; Δ156~581;). After detecting the protein expression by Western blot, the functional domains of hPINK1 interacting with human α-synuclein were detected by co-immunoprecipitation. The co-localization of different domains of hPINK1 and human α-synuclein was detected by immunocytochemistry. Result: Co-IP and immunocytochemistry have proved that the PINK1 kinase domain interacted with α-synuclein. Conclusion: The kinase domain of PINK1 interacting with α-synuclein.
The human cystathionine β-synthase(CBS) gene was ligated into vector pGEX-4T-1.The recombinant pGEX-4T-1-CBS was transformed into E.coli Rosetta (DE3), and the recombinant E.coli Rosetta (pGEX4T-1-CBS) strain which highly express CBS gene was constructed. After the recombinant E.coli was grown at 37℃ to an A600 of 0.4~0.6, induced with IPTG at a final concentration of 0.1mmol/L for 16h at 30℃. The productivity of the soluble CBS reached 28mg/L. The supernatant of the disrupted cells by sonication was directly loaded on GSTrap Fast Flow, and the GST-CBS fusion protein was absorbed on the column. The GST-tagged CBS fusion protein bound to the column was treated with thrombin(10 unit of thrombin/mg protein) at 22℃ for 12-16h in the presence of 3% glycerol and 0.1% CHAPS. An easy one-step protocol to purify recombinant human CBS and in 54.8% overall yield had been used. From a 1L culture, some 15.2 mg of purified CBS protein at 95% purity as judged by SDS-PAGE could be abtained, and the purified enzyme had a specific activity of 143 unit/mg protein. S-adenosylmethionine(AdoMet) activates the CBS enzyme by as much as 5.1-fold in the presence of 1 mmol/L AdoMet with a specific activity of 735 unit/mg protein. Additionally, the recombinant E.coli Rosetta (pETDuet-1-CBS1-413) strain which highly express truncated CBS(CBS1-413) gene was constructed. Using a HisTrap Fast Flow affinity chromatography, the purity of recombinant CBS1-413 lacking the C-terminal regulatory domain reached 95% by one-step purification with the specific activity of 965 unit/mg. The productivity of the soluble CBS reached 12.8mg/L and in 74.3% overall yield. In addition, The expression and purification of recombinant cystathionine β-lyase (CBL) in E.coli were described. The present study established a novel method, which relies on CBL as coupling enzyme, for detemination of CBS activity based on the color reaction between pyruvate and 2,4-dinitrophenylhydrazine.
Effect of purA gene overexpression on adenosine accumulation was studied in industrial strain Bacillus subtilis XGL(Xan-+Deam-+8-AGr+SGr). PurA gene was cloned from XGL's chromosome by PCR and ligated into the shuttle vector pBE43 which contained P43 promoter. Recombinant plasmid pBE43∷purA was transformed into XGL by electroporation. Reverse transcription PCR and quantitative PCR were used for measuring the transcription of purA. Fermentation parameters were determined in 5 L fermenter. Expression analysis showed that the purA in XGL-SY expressed as 9 times many as that in XGL. Cell growth was dragged a little. However, the production of adenosine was 10.8% higher than before. In conclusion, the overexpression of purA can promote further accumulation of adenosine, which made a foundation to further genetic engineering strain construction.
Streptococcus pneumoniae is a major pathogen of bacterial pneumonia. PsaA is a surface metal-binding lipoprotein and genetically conserved in all kinds of Streptococcus pneumoniae. psaA gene was amplified by polymerase chain reaction (PCR) from Streptococcus pneumoniae D39 and cloned into the expression vector PBAD/HisA containing 6His tag. The recombinant plasmid was transformed into Escherichia coli Top-10 to express the fusion protein after induction with L (+) arabinose. The recombinant protein was further purified by a nickel affinity column and the 6His tags were removed with recombinant enterokinase. Inductively coupled plasmamass spectrometry (ICP-MS) analysis revealed that the purified PsaA binds Zn2+ in a ratio of 1:1. Circular dichroism showed that zinc binding did not induce changes of the α-helix and β-sheet content in PsaA. Fluorescence spectra determined the Zn2+-binding affinity and the dissociation constant(Kd) which was calculated to be 1.19058 μmol. These results are useful for the further study on the metal-binding properties of PsaA protein in vitro, bacterial metal transport and virulence mechanism of Streptococcus pneumoni.
Objective: To investigate the effects of tissue factor pathway inhibitor-2 (TFPI-2) gene on the proliferation, apoptosis and expression of alpha fetoprotein of hepatocarcinoma cell line Hep3B. Methods: The recombinant vector of PCDNA3.1-TFPI-2 was transfected into Hep3B cells with liposome, and the cells were selected by G418. The expressions of TFPI-2 mRNA and protein were detected by RT-PCR and Western blot respectively. The influence of TFPI-2 on the proliferation of Hep3B cells via growth curve was assayed by CCK-8 method. The colony-forming unit assay was used to measure single cell self-replication ability. The expression of AFP mRNA was detected by RT-PCR and the AFP secretion of Hep3B cell was measured by electro-chemiluminescence (ECL) immunoassay. Furthermore, the apoptosis was tested by flow cytometry. Results: Expressions of mRNA and protein of TFPI-2 were identified in cells transfected by PCDNA3.1-TFPI-2. The growth rate and self-replication ability of Hep3B cells were significantly lower than those of the two control groups. The inhibition ratio of AFP mRNA expression was 16.51%, and the AFP secretion of Hep3B cells transfected with PCDNA3.1-TFPI-2 was significantly lower than the control groups (P<0.01). The rate of early apoptosis was significantly increased (24.03%±7.28% vs 8.77%±3.66%). Conclusions: The expression of TFPI-2 can simultaneously inhibit the growth and expression of AFP of hepatocarcinoma cells as well as induce early apoptosis.
Human CD24 is a candidate biomarker specifically expresses in many types of malignant tumor cells. In order to obtain its polyclonal antibody for following-up studies, the encoding sequence of mature human CD24 polypeptides without glycosylation (hCD24N) was amplified and cloned into pGEX-KGV, which is a modified prokaryotic expression vector containing a N-terminus GST fusion tag and another internal Strap tag II. Prokaryotic expression plasmid pGEX-KGV-hCD24N was constructed and expressed in the strain of E. coli BL21 (DE3) using lactose induction. Fusion protein, named GST-StraptagII-hCD24N, mainly expressed in its soluble forms, and existed in the supernatant after cell lysis. Using GST affinity chromatographic separation, this target protein was purified and used to immunize rabbit to produce rabbit anti-hCD24N polyclonal antibody. An indirect ELISA assay was further used to measure the anti-hCD24N titer of prepared antibody. Moreover, the specificity against native glycosylated CD24 molecular which is expressed in the positive human cancer cells was also confirmed by Western blot and immunofluorescence, and compared with commercial antibody. The results showed that the prepared polyclonal antibody had a high titer to recombinant hCD24N, and could react with native human CD24 and its positive human malignant cancer cells specifically, which provides a favorable tool for further studies on application of CD24 as a candidate antigen for cancer prevention and treatment. Moreover, the GST-StraptagII-hCD24N fusion protein could also be used as a target for screening novel specific ligand against CD24 and associated positive cancer cells. In conclusion, the prepared anti-CD24N polyclonal antibody provides the basis for tumor biology research and related diagnostic reagents development which are targeting CD24 molecular.
Objective:To achieve the mutant stains of Escherichia coli K12 which up-expressed OmpW located in outer membrane by vector pLLP-OmpA. Methods:The complete sequence of ompW from E. coli K12 was cloned by PCR. The cloned DNA fragments were linked with the plasmid to construct the vector pLLP-OmpA-ompW. The recombinant vector was then transferred into E. coli K12 to obtain the engineering strain. Western bolt analysis with primary antibody against OmpW was used to verify whether the highly expressed OmpW of the strain located in outer membrane or not. The polyclonal antibody was prepared by immunizing mice with OmpW protein extracted from strain of pET-28a-ompW. Results: The recombinant expression vector of OmpW was successfully constructed and the high expression strains were also obtained after transformed into the host. The strains which the high-expression of OmpW located in the outer membrane were further confirmed by Western blot analysis. Conclusion: The strain of high-expression proteins location in outer membrane was achieved for the first time. The result will contribute to investigation the function of OmpW.
Sialyl lewis X (Slex), one of the common glyco ligands of the selectin family, restrains inflammation reaction by combining with the inflammatory cells competitively. Clone and expression of the key enzymes in the biosynthesis process of Slex could make its biosynthesis in vitro, and some related mastitis therapy research possible. Beta 1, 4 galactosyltransferase (GT) was the very one of the key enzymes in the biosynthesis process. In order to understand some related physicochemical property of GT gene, the gene sequence was analyzed by using approaches of bioinformatics online. The synthetic CDS of this gene was inserted into a recombinant plasmid pMD18-T, and subcloned to the expression plasmid pPIC9K later. The linear expression plasmid pPIC9K-GT was integrated to the genome of P. pastoris GS115 by electrotransformation. After inducible expression, the soluble target protein was detected by SDS-PAGE. It was proved that this gene could be expressed successfully in P. pastoris GS115. The phenol red method was used to determine the activity of the unpurified enzyme, and the specific activity was 16.4U/ml. This might be the foundation work for the further study.
Objective: To construct recombinant retrovirus vectors carrying human Trim22 gene, which could be stably expressed in U937 cell line, and to study the effects of Trim22 on pro-inflammatory cytokines production in LPS-stimulated macrophage-like cells. Methods: Trim22 gene was amplified with PCR and subcloned into retroviral vector (MSCV2.2 IRES-GFP). The recombinant vector was confirmed by restriction enzyme digestion, colony PCR and sequencing. The retroviral vector together with GAG-POL and VSV-G helper vectors were co-transfected into the packaging cells 293T by Lipofectamine. The supernatant of 293T cells was used to infect U937 cells and GFP positive U937 cells stably expressing Trim22 were sorted by flow cytometry. Eventually, PMA was used to differentiate U937 cells into macrophages and then LPS-induced pro-inflammatory cytokine levels were measured by ELISA. Results: Trim22 gene was successfully constructed into retrovirus vectors MSCV2.2 IRES-GFP, and the cloning site and reading frame were confirmed by enzyme digestion, colony PCR and sequencing. U937 cells stably expressing Trim22 and GFP were obtained by flow cytometry and were revealed by RT-PCR. Ectopic expression of Trim22 severely attenuated TNFα, IL6 production in LPS-stimulated macrophages derived from U937 cells. Conclusion: Trim22 gene retroviral vector was successfully constructed, and Trim22 could down-regulate LPS-mediated TNF-α, IL6 expression in macrophages-like cells.
Two FT-like genes named PtFT1 and PtFT2 were cloned by RT-PCR from Populus tomentosa. Sequencing results indicated that the length of both PtFT1 and PtFT2 is 525bp encoding 174 amino acids. Blast analysis showed that PtFT1 and PtFT2 respectively share over or more than 75% homology in amino acid sequence with Arabidopsis(Arabidopsis thaliana) FT(BAA77838.1), grape(Vitis vinifera) FT(ABI99465.1) and other FT homologues. The deduced amino acid sequence contained one conserved motifs and two critical residues. Phylogenetic tree analysis suggested both PtFT1 and PtFT2 were classified into the FT-clade. Furthermore, the expression patterns of PtFT1 and PtFT2 in different tissues were examined using real-time quantitative RT-PCR. It showed that the constitutive and stable transcripts of PtFT1 and PtFT2 were detected in each tissue, but the expression levels between PtFT1 and PtFT2 were different. The expressions of PtFT1 and PtFT2 in male and female floral bud at early stage on July 5th were apparently higher than at late stage on March 10th in the next year. In addition, the data indicated that the expression of PtFT1 and PtFT2 in Populus tomentosa responsed to daylength, PtFT1 and PtFT2 transcripts became abundant under long days. The results suggested that PtFT1 and PtFT2 might function in photoperiod pathway about regulating flowering in Populus tomentosa. This will contribute to understanding the molecular mechanism of PtFT1 and PtFT2 in light-dependent pathways and also will benefit to regulation of flowering.
Cassava is very attractive in bio-butanol production for its natures of cheap, high productivity, and no competition with the foods for arable land. Cassava was used as the raw material to produce bio-butanol in a 7L static/anaerobic fermentor. The results indicated that, performance of cassava-based bio-butanol production was much lower than that of corn-based production in both traditional and extractive fermentations. The major problems include severe delay or complete failure in phases shifting from acidogenic phase into solventogenic phase, long fermentation time and low butanol productivity. The experimental results showed that adding 2.5g/L yeast extract when phases-shifting delay appeared, could stimulate solvents conversion from organic acids and shorten the phase-shifting time for about 10-30 hours. Under this condition and with traditional and extractive fermentation mode, final butanol concentration reached 12.95g/L and 29.81g/L respectively, and butanol productivity almost approached the equivalent levels when using corn as fermentation source.
Autophagy has been reported to contribute to wound repair and regeneration, but in vivo, it is infeasible to detect autophagy of human patient's skin. HaCaT cell line is spontaneously immortalized human keratinocytes from human skin. To establish a stable GFP-LC3-expressed HaCaT cell line.The pcDNA3.1-GFP-LC3 plasmid was constructed and transfected into HaCaT cell with transfection reagent.The stable transfectants were screened by G418.The GFP-LC3 protein expression was analyzed by Western blot.The fluorescent signals were detected by inverted fluorescence microscope.Rapamycin-induced autophagy was detected by confocal microscope, Western blot and transmission electronic microscope (TEM).Selected by G418,3 transfected cell lines showed high expression level of GFP-LC3,as demonstrated by Western blot analysis.More than 95% cells showed positive fluorescent signals under inverted fluorescence microscope.The formation of autophagosomes and the increases in the conversion of LC3-I to LC3-II was observed in the constructed cells when treated with Rapamycin.Rapamycin-induced autophagy in HaCaT-LC3 cell line was detected by TEM.A HaCaT cell line stably expressing GFP-LC3(named HaCaT-LC3)was constructed successfully. The HaCaT-LC3 cell line may act as a cellular model to study the autophagy in epidermic cell exactly.
Lentiviral is a genus of retroviridae, which has minor immune response and can deliver and express large exogenous gene fragment for longer time. Therefore, it is widely used as an ideal vector for delivering the target gene. One type of lentivirus vector containing three types of reporter gene, e.g. mCherry, luciferase and eGFP was constructed. Mcherry and eGFP can be used for cell line screening in vitro because of its intuitive imaging, while luciferase can be used for detecting the growth and metastasis of the tumor cells on animal models in vivo sensitively. The constructed vector was transfected to the malignant glioblastoma U87MG successfully, and the corresponding animal model was developed on nude mice by planting the transfected cells. It provided a model for the evaluation of therapeutic effect on glioblastoma in vivo, and demonstrated a reference way for other tumor cell lines and models which carried reporter genes.
Objective: To improve the expression of antifungal peptide CGA-N46, the polycistronic expression of CGA-N46 were studied. Methods: The recombinant cloning plasmids pT-CAN46, pT-3CAN46, pT-5CAN46 and pT-8CAN46 were constructed firstly, in which one copy, three copies, five copies and eight copies of heterogeneous gene fragment which was pET-30a rbs sequence-initiation codon-CGA-N46 encoding sequence-termination codon was recombined with cloning plasmid pEASY-Blunt respectively. And then the recombinant expression plasmids pET-CAN46, pET-3CAN46, pET-5CAN46 and pET-8CAN46 were constructed, in which one cistron, three cistrons, five cistrons and eight cistrons of CGA-N46 gene were recombined with expression plasmid pET-30a at the down stream of rbs sequence of it respectively. The recombinant expression plasmids were transformed into the competent strains of E. coli Rosetta. The expression efficiency of CGA-N46 was researched. Results: Under the induction of IPTG, CGA-N46 gene cistrons expressed the monomers of CGA-N46 and transferred into the periplasm space of host strains, The percentage of each expression cassete of CGA-N46 in total protein was 3.4%, 5.0%, 12.4%, 17.1%. The results showed that the production of CGA-N46 gene increased with the icreasing of its cistrons in expression vector, the production of 8 cistrons was the most. Conclusions: Polycistronic expression can overcome the shortage of fusion expression and tandem expression. The technology of construction of polycistronic expression plasmid simplified the traditional gene engineering construction method by using the same end of endonucleases Nhe I, Spe I and Xba I, and sets up a sound foundation for efficient gene engineering expression of peptides.
Single factor test and response surface analysis were used for optimization of Ganodetma lucidum-Astragalus membranaceus polysaccharide solid fermentation. Optimized solid fermentation conditions were as follows: herbs solid fermentation medium, initial pH6.5, inoculum volume 10.16ml/100g, fermentation temperature 30.65℃, relative humidity 59.54%, fermentation period 28d. Under the above optimum conditions, Ganodetma lucidum-Astragalus membranaceus polysaccharide(GAPS) content was up to 9.12 ± 0.09mg/g and increased by 13.15% than unoptimization(8.06±0.07 mg/g), which provided a theoretical experimental basis for pharmacodynamics research and industrialization development.
Single-chain Fv Fragment (ScFv) has been introduced into the study of inhibiting Hepatitis B virus (HBV) replication. Nowadays, ScFvs bound to HBV pre-S1, core antigen, DNA polymerase and X protein have been developed, respectively. ScFv can make the coupled molecule with therapeutic effects localized at the target site. Thus, affinity with the antigen, internalization property and structural stability of ScFv are the main factors limiting the utilization of ScFv.
MicroRNA(miRNA) is a class of small RNAs with the length of 19~24nt, which usually regulated the degradation or inhibiting translation of target genes at the post-transcriptional level. MiRNA molecules are highly conserved in evolution, a growing number of miRNA molecules that involved in eukaryotic growth and development, physical activity, cell proliferation, differentiation, apoptosis, complex disease control and other functions have been found. The important prospects of miRNAs in the regulation of gene expression through introducing origins, synthesis, modification, characteristics of cells, and eukaryotic cells regulation of miRNAs with the latest developments and research methods are provided.
Mesenchymal stem cells have been the topic of cell-replaced therapy in recent years.There are similar functional features in hAMSC and BM-MSC,such as low immunogenicity, immunoregulation, multilineage differentiation capacity and hematopoietic support. In addition, hAMSC has several special advantages,such as relatively easy isolation and purification, better proliferation,more resourses and no ethical disputes. The ability of hAMSC to successfully engraft and survive long-term in various organs and tissues, makes sure its significant impact in the fields of cell-replaced therapy and regenerative medicine.
Numerous preclinical and clinical studies have demonstrated the promising efficacy of gene therapy. However, precise regulation of therapeutic gene expression in vivo is still a challenge. Most of the gene regulation systems commonly used now are based on the control of transcription, but their clinical applications are limited because of their reliance on chimeric transcriptional transactivators and specialized promoter elements. More recently, several RNA-only strategies that control gene expression in mammalian cells and mice through RNA self-cleavage, RNA interference, modulation of mRNA translation initiation or termination, etc, and whose activity can be regulated by a small molecular ligand, have been developed. The extent of gene expression induction is substantial, the induction is rapid, and the expression regulation can be achieved both in vitro and in vivo. Those modular, tunable systems may overcome some of the limitations of transcription-based gene regulation systems. So they should have important clinical application in the setting of gene therapy protocols.
A brief analysis about National High Technology Research and Development Program(863) Bioinformatics & Computational biology topics in "Eleventh Five-Year Plan" was given. The details about topics plan, institutions carrying out the topics and representative research results were presented, these informations maybe be useful to scientific and technical workers.
