The cleaved amino-terminal fragment of human amyloid precursor protein(N-APP) binds death receptor 6(DR6) and triggers a caspase-dependent self-destruction process, which was suggested to contribute to Alzheimer's disease. To investigate the N-APP-DR6-induced degeneration pathway at the molecular level, producing abundant and purified recombinant DR6 ectodomain and identification binding sites of NAPP and DR6 ectodomain are critical. Here, the recombinant DR6 ectodomain from the methylotrophic yeast Pichia pastoris were successfully expressed and purified with a high yield of 90 mg/L. In order to identify the protein-protein interaction between DR6 and NAPP in vitro, prokaryotic expression plasmid pGST-DR6(aa42-349) was constructed, and transformed into E. coli, the expression of fusion protein GST-DR6 was achieved by induction. Meanwhile, NAPP was purified. GST pull-down assay showed that DR6 and NAPP could interact with each other in vitro.
Objective:To construct a universal influenza virus vaccines(Flu@uV), a universal influenza virus vaccine containing HA and M2e of influenza A virus was disigned and constructed. Hepatitis B virus core protein(HBc-VLP) was used as the delivery vector and adjuvant in the Flu@uV. The universal influenza antigen proteins were expressed in E. coli BL21(DE3) and purified for preliminary activity assay. Method:Using the synthesized gene sequence as a template, recombinant plasmids expressing HA-M2e-HBc, M2e-HBc, HBc, 3M2e-HBc and 3HA-3M2e-HBc were successful constructed. Their expression in E. coli was tested through SDS-PAGE, Western blot and electron microscopy. Mices were co-inoculated by purified protein with Freund's adjuvant. Flow cytometry was used to analyze peripheral blood of immunized mice. Expression of the corresponding DNA vaccine in human embryonic kidney cells(HEK293T) was preliminary validated by fluorescence analysis and Western blot. Result:Four kinds of HA-M2e-HBc, M2e-HBc, HBc and 3M2e-HBc proteins were successfully expressed and purified. Protein nanoparticles about 30 nm could be observed through electron microscope. Flow cytometry analysis of the peripheral blood showed that HBc and 3M2e-HBc increased the immunity of mice, and HA-M2e-HBc and M2e-HBc had no effect on that. DNA universal influenza virus vaccines were successfully expressed in eukaryotic cells through fluorescence detection and Western blot. Conclusions:A virus-like particles containing HBc, HA, and M2e of influenza A virus was successfully constructed, it laying a base for the development of universal influenza virus vaccine.
Objective:To construct a kind of expression vector which is cotrolled by survivin promoter, making a survivin promotor driving HSV-TK/GCV suicide gene system, and then detect its apoptosis inducing effect on HepG2 and HL-7702 cells. Methods:The study group synthesized a plasmid PBI-SUR-TK which contains TK gene, and transfect it into HepG2 and HL-7702 cells by liposome Lipofectanfine 2000.The HSV-TK gene expression by RT-PCR and the proteinlevel by Western blotting were detected. After adding GCV, CCK8 and flow cytometry were used to analyse the cell proliferation and apoptosis. Results:Contrasted with the control group, the HepG2 experimental group expressed more TK gene products, less proliferation and more apoptosis. There was no obvious difference between the HL-7702 groups. Conclusion:The HSV-TK/GCV suicide gene system driven by survivin promoter possibly has therapeutical effects on hepatoma.
Objective:(1) To study if there is a crosstalk between bone morphogenetic protein 9(BMP9) and microRNA-21(miR-21);(2) to investigate the effect of miR-21 on BMP9-induced osteogenic differentiation of mesenchymal stem cells line C3H10T1/2.Methods:(1) Using Ad-BMP9 infecting C3H10 T1/2 cells, the expression of miR-21 were detected by Real-time-PCR. After transfecting miR-21 mimic 1 day and 5 days, the expression of BMP were tested by Real-time-PCR.(2) In vitro, after transfecting miR-21, BMP9-CM treating C3H10T1/2, ALP staining and ALP activity were detected and the calcium deposition were detected by Alizarin Red S staining.(3)The expression of BMP9 downstream factors ALP and OCN were detected by Real-time-PCR.(4) The protein expression of p-Smad1/5 were detected by Western blot. Results:BMP9 can decrease the expression of miR-21 temporarily and miR-21 also decreases the expression of BMP9 temporarily. After transfecting miR-21 and treating C3H10T1/2 cell with BMP9-CM, the early osteogenic differentiation marker ALP activity and ALP staining and later osteogenic differentiation marker calcium deposition were enhanced. What's more, the expression of ostengenic factors ALP and OCN also increase during miR-21 combined with BMP9-CM. The protein expression of p-Smad1/5 were upregulation during the same process. Conclusion:There is a crosswalk between BMP9 and miR-21. MiR-21 synergizes with BMP9 promoting mesenchymal stem cells C3H10T1/2 osteogenic differentiation by increasing BMP9/Smad signaling pathway activated.
As one of common cancers in urinary system, the morbidity of renal tumor is increasing year by year. Firstly, the genes differentially expressed were screened based on Affymetrix hgu133b microarray data. The algorithm of Weighted Gene Co-expression Network Analysis was used to construct the co-expression network of the genes differentially expressed in renal tumor. Secondly, the module that is closely related with the tumor and hub genes were selected by analyzing the correlation patterns of the genes differentially expressed between normal and tumor tissues in kidney. Finally, the Gene Ontology enrichment of the hub genes was analyzed. Cell aging is one mechanism of suppressing tumorigenesis. The results indicated hub genes PLA2R1 and TBX3 are related to cell aging and could have an important effect on the tumor formation. The results corresponded with the investigation that gene PLA2R1 suppresses tumorigenesis in the kidney by promoting cellular senescence.
Doublesex gene dsx controls the somatic sexual development by sex-specifically alternative splicing. The expression of Bmdsx was analysed and a novel male-specific splice form BmdsxM3 was also found, but whether it has biological function is unknown. Here, two transgenic lines T1 and T2 were constructed to ectopically express BmdsxM3 in females. As a result, sex reversal and development abnormality all failed to turn up, but the expression levels of SP1 and Vg were downregulated in the fat body of transgenic females, and the expression level of PBP was upregulated in the antenna of transgenic females. All the results indicated that the novel splice form BmdsxM3 had the role of regulation of downstream target gene expression, and it took part in the somatic sexual development in silkworm, Bombyx mori.
Bacteria hemoglobin could improve the cell's oxygen utility and enhance the cell growth rate under low oxygen environment. To improve the biomass of E. coli and supply a novel type of host cell for bioengineering, the effect of four types of promoter controlled Campylobacter jejuni hemoglobin on the growth of E. coli cells was comparatively analyzed. The C. jejuni hemoglobin chb was initially synthesized and then it was expressed under the control of inducible promoter PT7 and Pvgh, constitutive promoter P2 and PspoI-II. The growth curves of these four recombinant E. coli cell in flask and capped bottle were depicted and founded that the chb gene has been actively expressed in these cells and could significantly improve the growth of cells, which was further testified by the experiment conducted in bioreactor. The relationship between cell density, cell fresh weight and CO differential spectrum value was comparatively analyzed, and the characteristics of these four type of promoter controlled recombinants on the hemoglobin inducible expression and growth regulation were discussed, and a selection on these host cells under different cultivation conditions such as microaerobic and aerobic environments was proposed.
Jiangxi is one of the main producing area of citrus fruits. Green and blue mold are the cause of major diseases of post-harvest decay of citrus fruits. Chemical fungicides have generated pathogen resistance and residual. A macrofungal strain Gymnopus sp. 0612-9 with activity of antagonistic the pathogens was found. In order to investigate the metabolic culture of producing active substance and its action against Green and blue mold, the anti-fungal activity was determined by the method of mycelium growth rate and microscopic observation. The medium composition and culture conditions were optimized by the single factor test, Plackett-Burman, steepest ascent test and the response surface Box-Behnken design. The results showed the liquid medium composition and culture conditions are potato 279.94g/L, glucose 30g/L, bran 8.87g/L, MgSO4·7H2O 0.47g/L, KH2PO4 2g/L; 10 days of fermentation period, inoculation volume 5ml/100ml, shaking speed 180r/min, culture temperature 26℃, liquid medium volume 100ml/250ml with the anti-pathogen activity of the fermentation broth increased from 42.26% to 55.77%. The EC50 value against Penicillium italicum and Penicillium digitatum are 69.25μg/ml and 56.70μg/ml, respectively. The minimum fungicidal concentration(MFC) is 250μg/ml, which was lower than the positive control of imazalil of 500μg/ml and it has some toxic effects on the mycelium of the pathogens. The strain of Gymnopus sp. has potential value to develop into antiseptic agent of citrus fruits.
ε-Lysine acylase from Streptomyces mobaraensis(Sm-ELA) can catalyzes hydrolysis of Nε-lauroyl-L-lysine in water with ε-lysine and lauric acid as the substrates. It's an energy-saving and environmentally friendly way that avoids the high temperature and organic solvent by using the chemical method. Two recombinant plasmids which are pET28a-SmELA and pTrcOmpXK122SmELA were constructed and a normal expression of intracellular and the cell surface were achieved respectively. The recombinant enzymes were used in catalytic synthesis of Nε-lauroyl-L-lysine and the catalytic efficiency was preliminarily compared. Nε-lauroyl-L-lysine was synthesized from 50 mmol/L ε-lysine and 10 mmol/L lauric acid in an aqueous buffer solution at 37℃. The maximum yield was 31. 1% after 24 h of reaction for 10 mmol/L lauric acid.
Purpose:To overcome the defect of nerve growth factor(NGF) in the treatment of sciatic nerve injury. A polycation-poly(argininylaspartate diglyceride ethylene)(PEAD) was designed to substitute the heparin-binding sequence of the FGF receptor and form a ternary complex containing the polycation, heparin and NGF, which provides experimental evidence for the clinical treatment of peripheral nerve injury. Methods:Experiment young adult male Wistar rats aged 8 weeks(200~220 g) were selected and divided randomly into 3 groups:Saline group, Free NGF group and NGF coacervate group with 8 rats in each group. The common sciatic nerve was exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris and moderate contusion injuries were performed using a vascular clip, the skin was then closed with 5-0 stitches. Following surgery, each group was injected equivalent volume saline, free NGF(80 ng/day), or the NGF coacervate containing equivalent NGF in the injury sites of right limb through a 1 ml syringe. At 30 days after surgery, the regenerated rat sciatic nerve was evaluated by footprint analysis, immunohistochemistry and histologic assessment. Experimental data were processed using the statistical software SPSS 13. 0.Results:The behavior, pathological structure and protein expression treated by NGF or NGF coacervate exhibited better than that saline group. Moreover, the therapeutic effect of NGF coacervate group was better than free NGF group. Conclusion:The new type of coacervate loaded with NGF could evident effects on the repair and regeneration of peripheral nerve injury, this may lay the groundwork for future translational studies of NGF coacervate for peripherial nervous system(PNS) diseases, especially those related to sciatic nerve injury.
Trehalose synthase can be used to transform the production of trehalose in one step, and its substrate specificity is higher, the production process is simple, and can not be influenced by the concentration of substrate maltose, which is the first choice for industrial production of trehalose. To obtain the surface display vector of Pichia pastoris with the ability of producing trehalose synthase, trehalose synthase gene(tres, 2064 bp) was amplified by PCR from genome of Pseudomonas putide P06(gi=1042893, NCBI), then linked to the plasmid pPICZαA to get the recombinant plasmid pPICZαA-tres. Pir series protein Pir1p which is covalently linked cell wall of Saccharomyces cerevisiae, was used as the anchoring protein, and the pir1p(847 bp) was amplified by PCR technique, then linked to recombinant plasmid pPICZαA-tres, and the recombinant plasmid pPICZA-tres-pir1p was obtained. The recombinant plasmid was transferred into Pichia pastoris GS115 by electroporation, and the protein was directed to the cell wall by a-factor signal peptide and then was displayed on the surface of Pichia pastoris. The positive clones were selected by Zeocin resistance screening. Centrifuging, crushing and using laminarinase hydrolysis the fermentation products, SDS-polyacrylamide gel electrophoresis analysis showed obvious fusion protein bands. The trehalose synthase successfully anchored in Pichia pastoris. The Pichia pastoris strains was hang up using pH 7.5 buffer suspension and the concentration of substrate for 30% of the maltose in 30℃ to 60℃ water bath roling 2 h. Reaction products were analyzed by HPLC and the enzymatic activity can be detected. In the optimized condition, pH 7.5, 50℃, the activity of trehalose synthase reached 300.65U/g. The enzyme activity was stable in the range of 40℃ to 50℃, holding 60 min, the residual activity was more than 75%. The optimum pH was 7.5, and in alkaline environment enzyme activities remained stable.
In order to obtain hybrid antimicrobial peptides which have low haemolytic activity and strong antibacterial activity, six hybrid antimicrobial peptides were designed depend on Musca domestica Cec Md and Rana Chensinensis as well as preference condon of Pichia yeast, named CC22, CC28, CC29, CC30, CC34(1) and CC34. The target genes of six hybrid antimicrobial peptides were synthesized successfully by SOE-PCR and, were cloned into the expression vector pGAPZαA of Pichia pastoris. Which were transformed into Pichia pastoris SMD1168 by electrotransformation, and the positive transformants which containing hybrid antimicrobial peptides gene were selected using high concentrations of Zeocin. When the screened recombinant gene engineering bacteria were cultured 72 hours in YPD medium, the expression products were identified by Tricine-SDS-PAGE and purified by HPLC. The biological activity measurement results showed that the minimal inhibitory concentration(MIC) of CC29 to Escherichia coli and Salmonella paratyphus were 25μg/ml; CC34(1) had lower inhibitionon and the MIC to Escherichia coli was 100μg/ml; the MIC of CC34 to Salmonella gallinarum and Staphylococcus aureus were 50μg/ml; six hybrid antimicrobial peptides had no antimicrobial effect on intestine bacteria. As for hemolytic activity, all of the antimicrobial peptides were low, CC29 was the lowest, CC34(1) and CC34 was the higher in which showed the antibacterial activities of them. Considering the antibacterial activity, CC29 and CC34 were more effective on it, so two new hybrid antimicrobial peptides were obtained which had both more effective antibacterial activity and lower hemolytic activity.
Compared with the antibody drugs, the non-immune globulin scaffolds with a small molecular weight, do not need to be modified after translation, usually lack disulfide bonds, and can undergo straightforward multimerization. In recent years, the development of non-immune globulin scaffolds have reached more than 20 different types, such as Adhirons, Alphabodies, Centyrins, Pronectins, Repebodies, Affimers, and Obodies. 102 proteins have been specifically targeted by 139 different non-Ig scaffold binders. These non-immune globulin scaffolds are used for the treatment and diagnosis of cancer and inflammatory diseases, and there are more than 10 types of non-immune globulin scaffolds have been used in clinical trials. Recently, non-immune globulin scaffolds have also been used in research as structure determination chaperones, which are used in the study for intracellular monitoring of post-translational modifications or as an alternative to antibodiesfor microscopy, flow cytometry, and Western blotting, and so on.
Cecropins, a series of linear 31~39-residue cationic α-helical antimicrobial peptides, display extensive bioactivities against bacteria, fungus, virus and neoplastic cells. The mechanism of cecropins which is different from traditional antibiotics gives them the advantage that they tend to be not easily develop drug resistance, which indicate that cecropins may be potential potent novel antimicrobial agents to resolve multi-drug resistence problems. However, several problems of cecropins concerning antimicrobial activity, selectivity, toxicity, and stability remain to be solved. Moreover, natural cecropins are not suitable for mass production due to their complicated extraction process and huge costs as well as high toxicity to prokaryote limit the use of engineering bacteria. Molecular design methods to solve these problems are reviewed.
Plant-parasitic nematodes secreted numerous effectors during infection and parasitism stages. These effectors play various roles within the plant cells, facilitating growth and development of nematodes. Functional analysis of effector proteins is important to understand the interactions between nematodes and hosts, which will provide the scientific foundation for the new nematode control strategy. The main methods used in function analysis of effectors from plant-parasitic nematodes were summarized.
L-lactic acid is one of the key metabolites of microorganism, and has an extensive application value. To date, microbial origins L-lactic acid contribute to the main source of this compound, whereas it is restricted by precise fermentation control, relative low bacterial product tolerance and high substrate requirements, and therefore resulted in L-lactic acid supply deficiency and a higher price. Due to the advantages of Saccharomyces cerevisiae in the production of valuable products with cheap substrate, along with the development of molecular biology technology, more attention has been attracted on the metabolic engineering of S. cerevisiae for higher production of L-lactic acid. Recent research progresses in the production of L-lactic acid in Saccharomyces cerevisiae, including the accumulation of L-lactic acid, the improving of lactate production and cell productivity were presented. Finally, the limitation of current progress and proposed the future research needs for microbial production of L-lactic acid were also discussed.
As a new type of renewable energy, cellulose butanol with well gasoline compatibility, low vapour pressure, high safety factor, high energy density and well antidetonating, it has been widely concerned as an alternative of fossil fuel. At present, there are still some technical bottlenecks in the industrial production of cellulose butanol, and the technology economy is also poor. Therefore, the development of the cellulose butanol industrial is hindered. But in the future, with the support from the government policies, and with the conditions of rich agricultural resources and huge market demand, cellulose butanol as a new energy has a very broad prospects. In order to better promote the industrial development of cellulose butanol, the development status was summarized and the existing problems were analyzed. The prospects of cellulose butanol industry were predicted from the aspects of policy, resources and market. At last, the proposal was raised to promote the development of the cellulose butanol industrial in the future.
