The antagonist of tumor necrosis factor alpha is the first choice for the treatment of multiple inflammatory autoimmune diseases. Treatment with TNFα antibodies is restricted by their side effects, particularly, the production of anti-antibodies, which seriously affect the treatment efficacy and drug metabolism. Short peptides have low immunogenicity, and compared to small molecules, they also have lower toxicity and stronger target specificity. Using M13 based 7-mer and 12-mer peptides phage display libraries to screen TNFα binding peptide ligands, and analyzed the affinity and functionality of the selected TNFα antagonist. After 3-4 rounds of screening, two 7-mer and two 12-mer peptide sequences were obtained. Binding affinity of the synthetic peptides for TNFα was determined by ELISA. 7-mer peptide with number 632 showed affinity of Kd=138nmol/L, while the 12-mer peptide with number 636 showed lower affinity of Kd=8.59μmol/L. Insight II software was used to carry out Zdock with TNFα dimer, and it was found that both the 7-mer peptide could bind to TNFα with a more stable state than the 12-mer peptide. At the cellular level, the peptide 632 were more resistant to the activity of TNFα than peptide 636. In the presence of peptide 632, the survival rate of L929 cells induced by TNFα was 3 times higher, but the 636 peptides were only 2 times. Altogether, the 7-mer peptides were more suitable than 12-mer peptide as TNFα antagonist.
Purpose:To investigate the effect of overexpressed miR-155 on the osteogenic differentiation of mesenchymal stem cells C3H10T1/2 cells induced by BMP9, and the related mechanisms contained in this process. Methods:① Used adenovirus BMP9 (BMP9) to induced the osteogenic differentiation of C3H10T1/2 cells, tested the expression of miR-155 by quantitative PCR (qPCR), detected the expression of Runx2 and ALP use RT-PCR. ② Cotransfected BMP9 and miR-155 into C3H10T1/2 cells, measured the expression of miR-155 by qPCR, ALP activity and ALP staining evaluated the early osteogenesis ability of C3H10T1/2 cells. ③ Cotransfected BMP9 and miR-155 into C3H10T1/2 cells, used Alizarin red S staining to estimate the osteogenesis ability in the later stage of differentiation in 14d. ④ Treated C3H10T1/2 cells with BMP9 and miR-155, tested osteogenesis-related genes by qPCR, include Runx2, OSX, COL1A1, ALP, OCN and OPN. ⑤ Treated C3H10T1/2 cells with BMP9 and miR-155, detected the expression of p-Smad1/5/8, OCN and OPN on protein levels used Western blot. ⑥ qPCR and Western blot examined the expression of HIF1α and VEGF on mRNA and protein levels respectively. ⑦ Confirmed the target gene of miR-155 by luciferase reporter assay. Results:During the process of osteogenic differentiation of C3H10T1/2 cells induced by BMP9, overexpressed miR-155 decreased ALP activity, ALP staining and Alizarin red S staining. Repressed the expression of bone-related genes, such as Runx2, OSX, COL1A1, ALP, OCN and OPN, also. On the protein levels, overexpressed miR-155 inhibited the expression of p-Smad1/5/8, OCN and OPN, the expression of HIF1α and VEGF were decreased on mRNA and protein levels, too. Through luciferase reporter assay, confirmed that HIF1α is one of the target genes of miR-155, miR-155 decreased the expression of HIF1α on protein level, not on mRNA level, yet. Conclusion:Overexpressed miR-155 could inhibit the osteogenic differentiation induced by BMP9 in mesenchymal stem cells C3H10T1/2 cells, it may through Smad/BMP signaling pathway and its target gene HIF1α to play the inhibitory effect in this process.
The full length cDNA sequence of V-type proton ATPase subunit a3 gene was cloned from sunflower P50 by RACE. The sequencing result showed that full length cDNA(2 873bp) contain the 2 469bp open reading frame (ORF), encoding a 822 amino acids, 109bp 5'-untranslated region and 295bp 3'-untranslated region. It was a hydrophobic transmembrane protein and the predicted protein's molecular weight was 204.55kDa, isoelectric point was 6.29, GenBank accession number was KU315054. The protein had highly similar conserved region between sunflower and other 10 kinds of plants. The genetic relationship of sunflower was closest to Cynara cardunculus var. scolymus. RT-qPCR showed that the gene was up-regulated under different concentrations of NaCl, ABA and PEG in sunflower with different expression patterns in different conditions and organs. The research suggested that the V-type proton ATPase subunit a3 gene of sunflower responded to abiotic stresses, which laid the foundation for strengthening its utilization.
Temperature is an important factor affecting biomass productivity in large-scale cultivation of Arthrospira. To improve Arthrospira production, it is crucial to screen strains with high productivity and excellent temperature-tolerance ability. Two strains Arthrospira sp. DICP-D (D) and Arthrospira sp. DICP-F (F) were isolated and identified from full-scale Arthrospira cultivation raceway pond. Although strain D and strain F displayed significant difference in morphology, they shared closest phylogenetic relationship among the existing Arthrospira strains with accessible 16S rRNA gene information. Under normal conditions, strain D and strain F harbor almost the same biomass compositions, and they also accumulated identical carbohydrate content in cell under nitrogen stress or low temperature stress conditions. However, the biomass productivity in strain D was 33% to 230% higher than that in strain F under the same cultivation conditions tested. Strain D showed 1.1 times better tolerance ability towards high temperature stress than strain F. The biomass productivity in strain D under 41℃ and 15℃ could be maintained at 73% and 61%, respectively, of that under normal temperature, demonstrating that strain D had good tolerance ability towards temperature stress. Strain D held more efficient photoprotection mechanism than strain F did, which endowed it better ability to acclimate to stressful conditions. The excellent biomass productivity and stress tolerance ability in strain D could make it applicable with great potential in outdoor large-scale Arthrospira cultivation.
The ComX pheromone precursor peptide of Bacillus amyloliquefaciens Q-426 quorum sensing system was successfully synthesized by Fmoc solid-phase synthesis method, and then isolated and purified by semi preparative chromatography (RP-HPLC) and liquid chromatography (HPLC-MS). A new method for the determination of free amino groups was established, and the effects of swelling process and reaction temperature on the degree of substitution were also investigated. The results showed that the traditional Kaiser reagent for the detection of free amino Fmoc in solid-phase synthesis can be replaced by the NNA reagent (ninhydrin + N-butanol + acetic acid solution). And the resins have the highest substitution degree after they were alternate swelling by DCM and DMF for 1h at 35℃. The product yield and the purity of the crude precursor peptide of ComX pheromone is 98% and 58.60% respectively. And the purity of the precursor peptide of ComX pheromone is 99% after being purified by semi preparative high performance liquid. Antibacterial experiments showed that the precursor peptide of ComX pheromone had obvious biological activity.
Fluorescence resonance energy transfer (FRET) microscopy is widely used for the research of protein interactions in living cells. With the development of fluorescence activated cell sorting (FACS), FACS-based FRET not only could be well suited to study protein interactions in living cells but also could be measure and quantify FRET. However, may be due to the reason of flow cytometry is expensive and the special requirements of luminescent spectra by FRET, this technology was so far only applied to a few special scientific questions. To overcome these limitations developed a new FRET pair EGFP-mCherry and try to detect the FRET signal in the Accuri C6 flow cytometry, and then verify that the new FRET pair EGFP-mCherry was working by using two proteins p53 and MDM2 protein which there is an interaction has been clear. A new FERT pair EGFP-mCherry offers the precious possibility to detect FRET signal by using the Accuri C6 flow cytometry be successfully constructed, and not only promote the development of the FACS-FRET technique but also is expected to contribute to the identification of novel therapeutic target for treatment of human diseases.
Glucagon like peptide-1 (GLP-1) is a potential drug for the treatment of type II diabetes. However, it has a short half-life in vivo and lot-to-lot expression instability, which limit its clinical application. In our early study, the modified fusion protein NGGH[6×His-tag+Ek+2×GLP-1(A2G)+HSA] was successfully expressed in CHO cells using vector pMH3. The eukaryotic expression plasmid pcDNA3.1/NGGH was constructed and transfected to CHO cells by electroporation. After G418 resistant pressure screening, single cell clone was selected by a new Celigo Imaging Cytometer to obtain stable and high-expression cell clones. The results of Western blot showed that the fusion protein has both GLP-1 and HSA immunogenicity. After batch culture, a stable and high-expression clone was obtained and the yield was 58mg/L. Recombinant cells were cultured in 5L AP20 torrent bioreactor. The result showed that the optimal conditions were pH6.8~7.4 and DO controlled in two-phase way. In these conditions, the yield of NGGH protein rearched 148mg/L.
MPL-2, a pituitary adenylate cyclase-activating polypeptide (PACAP) derivative was prepared by gene recombination engineering technique and the effect of anti-type 2 diabetes mellitus was studied in db/db mice. The results showed that the molecular weight of MPL-2 was 3 902Da, and the purity was about 97% with its yeid up to 29.3mg/(per liter of fermentation product). The in vivo glucose tolerance test on db/db mice showed that MPL-2 can effectively promote the secretion of insulin in the first phase (5-15min), and improve glucose tolerance in mice significantly. Furthermore, chronic administration of MPL-2 by daily injection for 8 weeks significantly improves lipid profiles and also greatly increases insulin sensitivity in db/db mice,which decreased the blood glucose of the mice to 63.52% of the initial value at 60min in the insulin tolerance test. At the meanwhile, the body weight, fasting blood glucose, food and water intake of db/db mice were also decreased by MPL-2, which was respectively lower 21.98%, 21.46%, 22.20% and 60.07% than that of NS group. It showed that MPL-2 was more effective than BAY55-9837. In conclusion, the recombinant MPL-2 could effectively improve the glucose tolerance, insulin sensitivity and lipid level of db/db mice, and significantly decreased body weight, fasting blood glucose, food intake and water consumption, thus play a important role in the biological treatment of type 2 diabetes. Experimental basis for the research and development of MPL-2 can also be provided.
As one of polyol, xylitol has been extensively used in the food, pharmaceutical and other light industries. Presently, xylitol is produced by the reduction of D-xylose derived from hemicellulosic hydrolysate in the presence of Raney nickel catalysts. In the process of extracting xylose, a large amount of xylose mother liquor as by-product will be generated. Xylose mother liquor contains in a certain concentration of glucose, xylose, arabinose and other carbon sources, and a small amount of furfural, tetrahydrofuran and other substances. Production of biochemical from xylose mother liquor via microbial transformation can improve economic value of xylose mother liquor and reduce the environmental pollution. Candida tropicalis can not only use glucose, but also has a highly efficient xylose metabolic pathway. Two allelic genes of xylitol dehydrogenase gene from C. tropicalis was deleted by genetic technique, and the resulting mutant strain was obtained, which can not use xylitol as carbon source. The fermentation performance of the mutant in xylose mother liquor medium was evaluated. The fermentation conditions of xylitol fermentation process was optimized. The optimal fermentation conditions were as follows:300g/L xylose mother liquor, 5g/L corn steep liquor, fermentation temperature 35℃, initial pH 5.0, inoculate volume 15%, under 200r/min for 140h. Using the optimized fermentation process, xylitol yield reached 83.01g/L.
In order to screen the biocontrol strain with antagonistic effects on Didymella bryoniae and enhance the inhibitory efficacy of the fermentation broth, antagonistic fungi was screened using the dual culture assay and shaking flask culture experiment. Subsequently, biocontrol strain was identified according to morphological character and 18S rDNA sequence analysis, and observation of parasitic effect of antagonistic strain on Didymella bryoniae was made by scanning electron microscope. The fermentation conditions were optimized according to single factor test and response surface methodology. In addition, the biocontrol efficacy of Bjerkandera adusta against Didymella bryoniae in plants was determined in greenhouse. The results reveal that the strain M1 has the highest inhibitory rates to Didymella bryoniae, reaching up to 59.56% and 49.56% in the dual culture assay and in shaking flask culture experiment separately. The morphological characteristics of M1 are consistent with those of Bjerkandera adusta and 18S rDNA sequence phylogenetic analysis reveal that M1 appears a sister lineage to Bjerkandera adusta and gathers together in a phylogenetic tree. So the strain M1 is identified as Bjerkandera adusta. Additionally, M1 is able to penetrate Didymella bryoniae mycelium directly. After optimized, the fermentation conditions are C/N of 7.1, pH of 7.4, the bottling quantity of 44%, fermentation time of 19d, the rotate speed of 180r/min, culture temperature of 29℃with the inhibitory rate of the fermentation broth increased from 49.56% to 52.64%. The biocontrol efficiency of Bjerkandera adusta against Didymella bryoniae is up to 74.3% in greenhouse, which is higher than that of treatment with carbendazim. These results demonstrate that as a biocontrol fungus, Bjerkandera adusta has an antagonistic effect on Didymella bryoniae, and also has potential value to develop into antiseptic agent in the field application.
Withthe growing development of therapeutic monoclonal antibodies in the clinical treatment, the market share and research cost of antibody drugs are increasing by years. In addition to the novel antibody drug development, the antibody engineering on efficacy and safety has been more strongly desired.Among theseengineering technologies, antibody half-life related engineeringhas been popularly used in improving antibody pharmacokinetic properties in recent years. Several kinds of antibody half-life modification methods are briefly described, as well as to their current status in clinical research.
Glycomics is emerging after genomics and proteomics research, glycosylation is an important post-translation modification of proteins which plays an significant role in many biological processes, abnormal glycosylation is closely related to the occurrence of some diseases development, therefore, glycosylation research has become a hot research topic. Deglycosylation can affect the activity, secretion and other physical and chemical properties of glycoprotein, and deglycosylation is also the main technical means of glycosylation research, various methods of deglycosylation, the advantages and disadvantages of various methods and the applicable scope as well as the detection methods of the deglycosylation were summarized, and provided a reference for the study of structure-activity macromolecules.
Neural stem cells (NSCs) are stem cells with the ability of self-renewal and multiple differentiation potential. Under certain circumstances,NSCs are able to differentiate into neurons,oligodendrocytes and astrocytes,participating in functional recovery of neural system, which is known as neurogenesis. It has always been considered that neurogenesis occurs predominantly in embryonic period but not in adulthood of mammalian. However,recent studies have shown that neurogenesis occurs throughout lifetime in the central nervous system of mammalian, meanwhile it can be regulated via a variety of signaling pathways. Here the review focus on current researches of neurogenesis in adult mammals.
FGF22 (fibroblast growth factor 22, FGF22), the member of fibroblast growth factors (FGFs) family, has a wide range of biological activities and its effects mainly focused on the brain or skin. Previous studies have found FGF22 play a important role in the process of epilepsy, skin cancer, depression, brain development and the formation of synapses. Therefore, FGF22 maybe provides a potential way for the treatment of this disease. However, the mechanisms of FGF22 in this disease are not fully understood, further investigations are needed to clarify these issues.
Hyaluronic acid is a linear high molecular weight mucopolysaccharide with its molecular weight determining its rheological properties, physiological role and applications. Traditional studies have made significant efforts to improve the yield of hyaluronic acid by optimizing the fermentation. In recent years, the research focus has gradually shifted to how to improve the molecular weight of hyaluronic acid products. At present, the high molecular weight hyaluronic acid has good viscoelasticity, moisture retention and adhesion, and the application of the medicine is irreplaceable by the middle or low molecular weight hyaluronic acid.The mechanism of hyaluronic acid molecular weight regulation and the research progress of the production of high molecular weight hyaluronic acid by microbial fermentation were reviewed, and its development direction was prospected.
In recent years, due to their widespread usage and abuse, pyrethroid insecticide residues in environment and food have resulted in the adverse effects on human health. The microbial biodegradation of pyrethroid insecticides has become increasingly popular in environment bioremediation due to their safety and high efficiency. The aim is to present the types and characteristics of degrading microbial, the degrading-enzymes and the degradation mechanism. Meanwhile, recent and most applicable progress are presented and future trends are discussed.
Coagulation factor is a kind of special drugs, which is the treatment drug for hemophilia and has become an important part of blood products. There are more than 20 kinds of recombinant coagulation factor listed in the global market and the market sales are 7.854 billion dollars in 2015. The sales of recombinant coagulation factors of Baxalta ranked first in the world, reaching 2.84 billion dollars. The issued institution of coagulation factors in domestic is good. With the abolishment of the maximum retail price of blood products by National Development and Reform Commission, the prices of all kinds of products have different ranges of growth. Among them, the growth rate of human fibrinogen is the highest, reaching 189%. There is great potential for domestic coagulation factor market. But some factors hinder the development of the market, such as products supply shortage and lacking of R&D efforts and so on. Reforming of the industry management system, improving the technology of plasma separation, strengthening the research and development of recombinant products is necessary now.
