Objective: To investigate the neurotrophic activity of recombinant fusion protein epopeptide AB-nerve growth factor analog peptide (EpoAB-NGF9/12). Methods: The DNA fragments encoding the sequences of EpoAB-NGF9 and EpoAB-NGF12 polypeptide were amplified by PCR and cloned into the pET-42a prokaryotic expression vector. The recombinant plasmids of pET-42a-EpoAB-NGF9/12 were transformed into E. coli BL21 (DE3) and exogenous protein expression was induced by IPTG. Fusion proteins GST-EpoAB-NGF9/12 were purified by affinity chromatography. The biological activities were determined using the experiment of inducing axon outgrowth of PC12 cells and flow cytometry analysis of apoptotic rate in R2L1 cells. Results: Apparent molecular weight of fusion proteins GST-EpoAB-NGF9/12 were approximate 30kDa. Immunoblot analysis showed that the fusion proteins were immunoreactive with anti-GST antibody. Fusion proteins stimulate the differentiation and promote the axon outgrowth in PC12 cells. Cell apoptosis was induced in (31.7±0.60)% of R2L1 control cells by serum free incubation, whereas cell apoptosis rate were in (25.2±3.52)% or (25.7±1.46)% by adding GST-EpoAB-NGF9 or GST-EpoAB-NGF12 into serum free condition respectively. The result indicates that fusion proteins were enabling to prevent cell death in R2L1 cells. Conclusion: These findings in cell biology region suggest that recombinant fusion protein containing Epo-NGF peptide mimics have the neurotrophic effects similar to that of NGF.
Objective: To investigate the effects of tumor necrosis factor-alpha (TNF-α) on the osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells(BM-MSCs). Methods: BM-MSCs were cultured in osteogenic media (OS) with TNF-α using BM-MSCs in OS without TNF-α as a control group. Alkaline phosphatase staining was tested on the 7 days and Alizarin Red S (ARS) staining and quantification were tested on the 21 days. On the 7 days, mRNA were extracted from one set of the cells and Runx2 and Osterix mRNA expression were analysed by real-time RT-PCR , another set of the cells were lysed and Runx2 and Osterix protein expression were analysed by SDS-PAGE. Results: TNF-α inhibits alkaline phosphatase activity. TNF-α inhibits mineralized nodules formation. TNF-α inhibits Runx2 and Osterix mRNA and protein expression. Conclusion: TNF-α inhibition of the osteogenic differentiation of mouse BM-MSCs, in part, is through suppressing Runx2 and Osterix mRNA and protein expression which are the two key transcription factors of osteogenic differentiation.
By using two functional epitopes of HSP65 with preventive effects to autoimmune inflammatory disease, high-level expression vectors of pET28a-HSP65-P1P2P1 and pET28a-CTB-P1P2P1 were built with HSP65 and CTB as a carrier protein respectively. The fusion protein of HSP65-P1P2P1 and CTB-P1P2P1 were acquired by anion exchange column chromatography and then used to immunize the New Zealand white rabbits with atherosclerosis disease induced by high cholesterol diet-fed. The animals were nasally immunized with low-dose proteins for multiple times. The results showed that two groups of fusion proteins possessed the effectiveness of reducing atherosclerotic plaque formation. Compared with the PBS control group the aortic lesion areas of the two groups decreased by 34.9% and 27.9% separately. The study provided a good design idea for the further development of clinical anti-AS vaccine. After further optimization design for increasing their immunogenicity, it could be developed as a vaccine against atherosclerosis.
Objective:To investigate the effect of Arresten protein on proliferation and migration of human umbilical vein endothelial cell (HUVEC) and the inhibitory effect of Arresten protein on angiogenesis. Methods:Arresten protein was incubated with HUVEC in vitro: The effect on proliferation of HUVEC was examined using MTT assay. The effect on apoptosis of HUVEC was monitored by FCM. The effect on migration of HUVEC was examined by Boyden Chamber. Chicken chorioallantoic membrane (CAM) angiogenesis model was used to identify the influence of Arresten protein on neovascularization. Results:Arresten protein inhibits the proliferation and migration of HUVEC and promotes its apoptosis effectively; And the effect was dose-dependent. Compared with PBS group, the vessels of Chicken chorioallantoic membrane (CAM) in Arresten protein treated groups were obviously damaged(P<0.01). So Arresten protein can significantly inhibit angiogenesis of CAM. Conclusion:Arresten protein inhibits angiogenesis through the inhibition on HUVEC.
Human angiostatin is composed of 1-4 kringles of plasminogen, which can inhibit the angiogenesis of tumor, the tumor growth and metastasis through preventing tumor's blood and nutrition from supplying. The complete encoding cDNA of human angiostatin Kringles 1-3 was isolated from human embryo liver with PCR and inserted to pPIC9K vector. Then, K1-3 was expressed in secretory P.pastoris expression system by induction of methanol and the expression yield of K1-3 in 5 L fermenter was 41.2 mg/L. The supernatant of cultivation liquid was collected, then concentrated and dialyzed. Recombinant human K1-3 was purified by affinity chromatography with Lysine Sepharose 4B. As a result, HPLC showed purity of K1-3 was more than 98% and the recovery yield was more than 95%. Recombinant human K1-3 inhibited significantly the migration of human microvascular endothelial cells and half inhibitory concentration was 1.86μg/ml. Research showed that K1-3 suppressed specifically the angiogenesis on the CAMs with 95% inhibition.
Corynebacterium glutamicum SYPS-062, a strain isolated from soil sample can directly product L-serine from sugar substances. The sdaA gene encoding L-serine dehydratase (EC 4.3.1.17, L-SerDH) from C.glutamicum SYPS-062 was amplified and sequenced. The plasmid pET-28a- sdaA was constructed and transfromed into E.coli BL21 (DE3). SDS-PAGE showed that the gene sdaA was expressed successfully in recombinant E.coli BL21.Then L-SerDH was purified by Ni-NTA affinity chmmatography. The results showed a single band about 46 kDa on SDS-PAGE gel, and the specified activity was about 0.57 U/mg. However, compared the sdaA genes between C.glutamicum SYPS-062 and ATCC13032, it was appeared that the two sdaA cannot lead to the differences of activity of L-SerDH. To further investigate the mechanism of L-serine accumulation by SYPS-062. A sdaA deleted mutant of C.glutamicum SYPS-062 was successfully constructed. The resulting plasmid,pK18mobsacB-△sdaA was constructed and introduced into C.glutamicum SYPS-062 by electroporation .The Recombinant bacteria C.glutamicum SYPS-062△sdaA was conformed by PCR. The deletion of gene sdaA in C.glutamicum SYPS-062 yielded 15.13% increase of L-serine production with slow growth.
In order to confirm the application feasibility of overproduction of glutamic acid in C. glutamicum with H+-ATP inactivation in the construction of genetically modified bacteria. The sequence encoding H+-ATPase γ subunit in C.glutamicum ATCC13032 was partially deleted by crossover PCR, and the mutant, in which H+-atp gene was inactivated, was obtained by insertion inactivation. The glutamic acid production and growth rate of the mutant were tested. The results showed that the maximum production of glutamic acid of the mutant was 51.6 g/L in 100 g/L glucose culture medium, with 42.9 % increase in contrast to the wild parent strain, and that growth rate of the mutant in measurement of the growth curves was lower than wild parent strain. The results suggest that H+-atp gene inactivated in C.glutamicum increases glutamic acid production, and slightly reduces growth rate of the bacteria.
To develop an robust strategy for the tandem repeat co-expression of multiple genes in one plasmid, the point mutations were introduced into commercial expression vector pET-22b by the overlap PCR. After the mutation, the restriction enzyme site XbaⅠ was moved to the upstream of T7 promoter and a SpeⅠ site was introduced to the downstream of T7 terminator. The whole expression cassette including the T7 promoter, ribosome binding sites, multiple cloning site, T7 terminator were covered by the XbaⅠ-SpeⅠrestriction fragment. Restriction digestion and sequencing indicate that the mutated expression vector pET-m22b was constructed successfully. The four genes of different sources were choose for the coepxression by the pET-m22b. The SDS-PAGE shown that the four genes were highly co-expressed and without any affection on each other. The results indicated an efficient co-expression vector suitable for the tandem combination expression of multiple genes in single plasmid was constructed, which will make the co-expression of many genes more easy and will also greatly facilitate the expression and purification of multi-subunit protein complex, the rapid heterogenesis re-construction and optimization of biochemical pathways etc.
Cell-free (CF) protein expression system can be effectively used for membrane protein and other toxic protein expression. In recent two decades, CF system has received wide range of attention and the yield was significantly improved. The extract activity is the critical factor that affects the efficiency of protein synthesis system. If a simple method could be established to evaluate the activity of the extract, lots of time and cost would be saved up. Glucose-6-phosphate dehydrogenase (G-6-PDH) was the key regulatory enzyme in the pentose phosphate pathway in the glucose metabolism,therefore the activity of G-6-PDH could be utilized as an indicator to assess the extract activity.A convenient and feasible method was established , which use the activity of G-6-PDH in the extract to evaluate the extract activity. Three kinds cell disruption methods were compared, which included mechanical crushing, high pressure crushing and sonication,and the optimum conditions of each method were obtained. The mechanical crushing method got the most active extract by using 0.1mm-diameter glass beads at 5 000r/min for 6 times. When the disruption pressure was 1 300 bar, the activity of the extract reached the highest point. The sonication method got the best result at 60% power level, 30 cycles. The results indicated that the activity of the extract obtained by mechanical crushing and sonication methods were a little higher than the high pressure disruption.
To date, low extraction efficiency and cytoplasmic contamination is a great problem that apoplast protein extraction faces. To effectively extract apoplast proteins, using 12 day-old 93-11 rice seedlings, the extraction efficiency and cytoplasmic contamination ratio of the apoplast protein extracts by three different extraction buffers (Buffer A, Buffer B and Buffer C) were compared. The three buffers all contain 0.1mol/L Tris-HCl pH 7.6 and 1mmol/L PMSF, however, Buffer A also contains 0.2mol/L KCl ; Buffer B also contains 0.2mol/L CaCl2; and Buffer C also contains 0.1mol/L KCl and 0.1mol/L CaCl2. The results showed that the protein yield of buffer A reached (0.49±0.07)mg/g FW(leaf)and (0.83±0.06)mg/g FW (root), respectively, 122.7% (leaf) and 102.4% (root) greater than Buffer B, and 53.1% (leaf) and 59.6% (root) greater than Buffer C. The glucose-6-phosphate dehydrogenase (G6PDH) enzyme assay showed that the cytoplasmic contamination ratios of these apoplast protein extracts were very low, below 1%, which could be considered as negligible contamination. Therefore, by optimizing the extraction buffer, an effective extraction method for apoplast proteins was established, which could be applied to the plant apoplast proteomic studies.
Iron is an essential trace element of life, ferroportin (Fpn) is the intestinal absorption of iron release of important cell proteins. Newly discovered antimicrobial peptide hepcidin secreted by the liver can regulate the important role of intestinal iron absorption, but still play a role in the lack of Fpn and experimental basis for hepcidin. So fluorescence resonance energy transfer technology (fluorescence resonance energy transfer, FRET) on the role of hepcidin and Fpn relationship between the depth study. First of all, were hepcidin-CFP fusion protein expression vector and identification; then with YFP, Fpn-YFP gene in animal cell expression vector, expression, and FRET detection. The results confirmed that hepcidin and Fpn direct interaction between, and found that two proteins interact in the cytoplasm after the distribution of hepcidin also. The results for the treatment of disorders of iron metabolism to provide a new and important theoretical basis for therapeutic strategies.
Rapid identification of mustard materials in plant raw material products and plant-based foods is essential for effective control of a potential source of Allergen pathogens. A convenient Real-Time fluorescence polymerase chain reaction (PCR)-based assay which allowed detection and identification of a mustard -specific Housekeeping gene sin AI DNA sequence in foodstuffs and food was developed. The experiment results show that: the primers and probes could specific identify three kinds of mustard with 21 kinds of samples. Sensitivities results show that: 1 mg/kg for mustard of foods could be detected. Moreover, the four commercial samples (including mustard seeds) and deep processing of mustard allergen reference material (glucose) were detected and the detection results show that mustard allergen components were detected well.
Four sets of primers were designed according to 16S rRNA, spvC, invB, fimA genes of Salmonella typhimurium, which were used to amplify genomic DNA of Salmonella and non Salmonellas by multiplex polymerase chain reaction (m-PCR). Specificity results showed that only Salmonella strains had specifically amplified the target fragments, but none others. Sensitivity of m-PCR assay for pure cell cultures of Salmonella typhimurium was 6.3×102cfu/ml. However the detection limits of this method for artificially contaminated cooked chicken, milk powder, and beef were 17cfu/g, 11cfu/g, 13.6cfu/g following 8h of enrichment, respectively. This multiplex PCR assay successfully identifed main virulence genes of Salmonella typhimurium and may be applied to rapid and sensitive detection of Salmonella typhimurium in food when combined with an enrichement step.
The differentiation of stem cells into pancreatic β cells is a promising and novel strategy for the treatment of diabetes. This method would not only avoid the complications of insulin injections, but also reduce treatment delays due to shortages of donors for pancreatic islet transplantion. The cell types shown to have at least some potential for differentiation into insulin-secreting cells include pancreatic stem progenitor cells, pancreatic duct cells, acinar cells, liver/intestinal cells, embryonic stem cells and induced pluripotent stem (iPS) cells. The methods described most often for differentiating these cells into pancreatic β cells in vitro include variations of the D'Amour protocol and the Lumelsky protocol.Recent progresses in the directed differentiation of stem cells into pancreatic β cells was summarized.
Fibroblast growth factor-8 (fibroblast Growth Factor 8, FGF8) is one of the fibroblast growth factor (FGFs) family members. FGF8 is expressed in a lot of organizations in human embryo. And it is an important role in the formation of many organizations.the expression of FGF8 is strictly restricted in normal adult body. But FGF8 is over expressed in some kind of inflammatory cells, and especially in hormone-dependent cancers. It also plays an important role in the occurrence and development of hormone-dependent cancers. Application of FGF8 antibody in hormone-dependent cancer is an inevitable trend in clinical treatment.
The key step of cellular stress response is transcriptional activation of stress response gene including that transcription factors interact with cis-acting element. Gal4 protein is an evolutionary conserved transcription factor in the process of galactose metabolism. N-terminal DNA binding domain of yeast Gal4 protein binds to the Upstream Activating Sequence and C-terminal activation domain interacts with general transcriptional factors, recruit RNA polymerase Ⅱ to TATA-box. This process is regulated not only by regulating factors Gal80 and Gal3, but also relies on Gal4 dimerization.The role of the yeast Gal4 plays on the transcriptional activation of galactose metabolism genes is illustrated.
The progress of Genetically Modified Organisms (GMOs) detection techniques has been reviewed, and their characteristics were compared, including the sequence characteristics of GMOs, qualitative and quantitative detection techniques, their principles, characteristics, application and recent progress of the PCR-based detection methods, the isothermal nucleic acid amplification techniques , microarray-based technology, high-throughput sequencing and other new nucleic acid amplification techniques.
The structure and fluorescence mechanism of green fluorescent proteins (GFP) have been well understood after decades of study. Based on these knowledge, scientists discovered and developed a range of novel fluorescent proteins (FPs) with a wide emission spectrum covering from 424~655 nm. With the colorful palette of FPs, new technologies such as fluorescence complementation (FC) fluorescence resonance energy transfer (FRET) and super-resolution imaging have been developed and act as powerful tools in biological and medical studies. The latest progress about the structure, chromophore maturation and fluorescence mechanism of GFP, as well as the extensive FP families and the new technologies based on them were provided.
Cellulosome plays important roles in lignocellulose degradation.The cellulosome not only secreted enzymes degrade lignocellulose,but also can assemble multi-enzyme complexes which has an effective catalytic activity. The basic sructure and function of cellulosome was described,summarizes the applications progrsses in bioethanol,and analyzed the perspectives and challenges.
The history of bio-pharmaceutical industrial parks was reviewed, followed by the brief interpretations of the status in the main countries and areas around the world.The information of Chinese bio-pharmaceutical industrial parks was presented in detail.The existing problems in the bio-pharmaceutical industrial parks development process was analyzed,suggestions to Chinese bio-pharmaceutical industrial parks development was made.
