Recent study reported that reconstitution expression of GPx7 in tumour cell led to great suppressive effect on tumour growth in mouse xenograft model. Consequently, we carried out an administration of GPx7 through subcutaneous injection to investigate effect of GPx7 on tumour growth in mouse xenograft model. The coding domain sequence of human GPx7 without putative signal peptide (amino acid 1-19) was amplified from previously constructed plasmid pcDNA3.1(-)-hGPx7 and was subcloned into prokaryotic expression pET24A vector. The GPx7 purification process included two chromatography steps in which cation exchange and size exclusion chromatography was adopted. The resulting pure product showed weak and limited GPx activity in vivo. Purified GPx7 was injected subcutaneously through scruff of the neck of xenograft mouse model. The result showed that after GPx7 injection for 14 consecutive days, GPx7-injecting group showed to have a slower tumour growth compared with control (P<0.05) and the inhibition rate is 29.26%. The result indicated that two purification steps which started from cation exchanger and followed by size exclusion are practical and effective, meanwhile, GPx7 also showed to have tumour suppressive effect in vivo.
Aberrant protein glycosylation plays major roles in neurodegenerative disease, including PD. Glycoproteomics showed that the glycosylation of sodium channel β4 was significantly increased in human brain tissue. β4-specific antibodies reacted in immunoblot assays with ~38 kDa band from the membrane fractions isolated from neonatal PD transgenic mice but not expressed in the neonatal wild-type mice. The molecular weight of ~38 kDa immunoreactive protein is in close agreement with previously reported, suggesting heavy glycosylation of this protein in adult wild-type and neonatal PD transgenic brain tissues. Enzymatic deglycosylation of the membrane preparations converted the 38 kDa band into a faster migrating protein, which was consistent with heavy glycosylation of this protein. The glycosylated state of β4 was developmentally regulated and was altered in disease state. We also expressed β4-wild type and deglycosylated mutant β4-MUT plasmids in HEK-293 and Neuro2A cells. These results lay a foundation of studying β4 subunit in the pathogenesis of PD. Further studies will focus on the effects of the oligosaccharides on the sodium channel activities and neuritic degeneration.
Lytic polysaccharide monooxygenases (LPMO) is a recently discovered new type of oxidase that catalyzes oxidative cleavage of recalcitrant polysaccharides (such as cellulose and chitin), andits oxidative cleavage are advantages for degradation byglycoside hydrolasesystem to obtain soluble oligosaccharides. To get more LPMOs, the Amir_5334 gene coding LPMO was cloned successfully from Actinosynnema mirum DSM 43827 by PCR. The gene was inserted into the pET28a(+) vector that contained maltose binding protein (MBP-tag) and transformed into E.coli BL21(DE3)cell for induced expression. The MBP-Am5 fusion protein was purified by Ni-column affinity chromatography and cleavaged by Factor Xa, and obtained the nature Am5 finally. The predicted molecular weight of nativeAm5 is about 26 kDa, and its isoelectric point is 8.3.The analysis of Am5 sequence shows that the sequence identityis very low, compared with other LPMOs in AA10 family.The characterization of Am5 shows that Am5 is a LPMO that could act on chitin but not cellulose. On the other hand, Am5 can degrade chitin with chitinase synergistically, and the hydrolysis efficiency increased by 60%. The affinity of Am5 demonstrated Am5 combined with chitin more strongly than cellulose. All of above indicates that Am5 may be a LPMO acted on chitin with high efficiency and selectivity.
Objective:The aim of the study is to investigate the expression and enzymatic properties in E.coli and oxidation resistance in yeast of ascorbate peroxidase (LmAPX) cloned from Lycium chinense Mill., which will provide the theory foundation of antioxidant stress for future studying.Method: LmAPX was transformed into heterologous expression system E.coli BL21 and wild type yeast strain W303. The recombinant protein was purified by Ni2+ affinity chromatography and the enzyme activities at different temperature and pH were detected. The enzyme kinetics constant Km and Vmax were calculated by double-reciprocal plot. Results: The results showed that the optimal pH and temperature for this enzyme were 6.5 and 40℃, respectively.At fixed concentration of AsA, the Km and Vmax values of the enzyme for H2O2 were 0.17±0.02 mmol/L and 11.78±1.88 mmol/min·mg. And at fixed concentration of H2O2, the Km and Vmax values for AsA were 2.19±0.40 mmol/L and 58.82±3.51 mmol/min·mg. Tansgenic yeast stain transformed with LmAPX gene showed higher stress resistance than the control in the medium containing 8 mmol/L H2O2 or 100 mmol/L NaCl under the inducement of β-galactose. Conclusion: LmAPX enhanced the tolerance to oxidative stresses.
Object: To construct, express, purify anti-VEGF antibody Fab fragment in E.coli, and to assay its activity.Through controlling the fermentation conditions, Fab fragment can be achieved actively and largely by secreting to the periplasmic space or extracellular medium.Methods: The co-expression plasmid pET30a-LC-HC which has the OmpA signal peptide, SD sequence and the T7 promoter before Fd chain and L chain was successfully constructed.Then the plasmid was transformed into Escherichia coil BL21(DE3).The inducing condition including medium, temperature and IPTG concentration was also optimized. Results: The best shaking fermentation condition on Escherichia coil periplasmic secretory expression: in 1L XY1 medium that including 1.5% Tryptone, 1% Yeast Extract, 0.5% Glucose, 0.15% NaCl, 0.1% NH4Cl, 0.08% MgCl2~6H2O, the strains were cultured with 10% inoculum at 37℃, and then added 0.1mM IPTG until the late logarithmic growth phase(OD600 was about 2) to induced overnight(about 16h) at 16℃. Except periplasmic expression, there is also a considerable part of Fab that secrete into the medium. Fab fragment which secrected into the periplasm was extracted by breaking periplasm of bacteria.At the same time, the culture medium were concentrated with the hollow fiber column, and further purified Fab by ProteinG affinity chromatography.At last, we got the purified Fab antibody fragments with more than 90% purity by SDS-PAGE analysis.The protein concentration is measured with Brandford detection method.The amount of purified Fab from periplasm and medium is up to 0.4mg/L. Taking the VEGF165 as the binding antigen, the half-maximal effective concentration (EC50) of the purified Fab is 30ng/ml being analyzed indirectly by ELISA, which show a high antigen-binding activity. Then 3.7L fermentor with 2L volume of XY1 culture mediun is used to do small pilot scale fermentation.The final cell yield is 30g/L in the 3.7L fermenter with 2L culture medium, and the concentration of Fab antibody purified by affinity column is 1.94mg/L. Conclusion: Fab fragment can be successful and high-efficiently secrected into the periplasmic space and extracellular medium through constructing the co-expression vector and optimizing the fermentation condition, and has highly activity against VEGF. It provides research basis for large-scale preparation of Fab antibody fragment.
Enrichment and purification of spermatogonial stem cells (SSCs) is a prerequisite for genetic modification methods by SSCs. Stem cells antibody CD90.2 was used to enrich and purify mice SSCs through magnetic activated cell sorting(MACS) method, flow cytometry analysis(FACS) and Real-Time Polymerase Chain Reaction (RT-PCR) were used to detect the sorting efficiency. The sorting result using FACS showed that the purity of SSCs was 50.11% after sorting. However, the data by RT-PCR suggested that the expression of GATA4, a special-expression gene of sertoli cells, was downregulated about 6 times, and the expression of GFRα-1 and OCT4, two special-expression genes of germ stem cells, was upregulated 6.5 and 5.9 times respectively after-sorting. The relative expression of those three genes indicated that the enrichment efficiency using MACS was 6 times. The deviation between FACS and RT-PCR may come from undissociated magnetic bead and genetically modified fluorescence of SSCs.
Objective: To produce mitochondria deficiency transgenic mice, providing a useful animal model for the investigation of mitochondria related diseases. Methods: Transgenic mice were produced by injection of mitochondrial transcription factor A (TFAM) RNAi lentivirus into zygotes. Frozen sections of tissues wre prepared to observe GFP expression. Variations of TFAM mRNA and protein were separately detected by quantitative PCR (qPCR) and western blot. Changes of mtDNA copy number were measured by qPCR. Results: We constructed lentiviral vectors expressing shRNA targeting TFAM, which markedly repressed the transcription and translation of TFAM in transduced MEF cells. TFAM knockdown mice were successfully produced by infection of the purified virus into the perivitelline space of single-cell embryo. Green fluorescence was observed in heart, liver, spleen, lung and kidney of transgenic mice. Using qPCR and western blot analysis, we found that the transcription of TFAM in the tissues of transgenic mice was significantly decreased. Moreover, reduced expression of TFAM in myocardium led to a decline of mitochondrial DNA (mtDNA) copy number, as well as an impaired respiratory chain function. Conclusion: A TFAM knockdown transgenic mice line was successfully constructed.
Mammary gland bioreactor is considered as a promising approach for generating pharmaceutical proteins. Yet, the low-level expression of the transgene is one of the bottlenecks in this technique. Here, a strong and constitutively expressing promoter instead of a mammary gland specific but weak promoter was used to improve transgene expression; meanwhile, a Cre/loxP system driven by a β-lactoglobulin (BLG) gene promoter was used to restrict the strong expression in mammary gland. Methods: Construct the mammary gland-specific expression unit (Polyadenylation signal-pBLG-cre), this unit was then flanked by a pCAG strong promoter and a lacZ reporter. Thus, a mammary gland-restricted expression vector named pCBCZ (pCAG-loxP-PolyA-pBLG-cre-loxP-lacZ) was constructed. The in vitro experiment results: Cre-loxP recombination was detected in the mouse epithelial (HC11) cells transfected with pCBCZ vector. X-Gal staining experiment showed that the vector can strongly drive β-galactosidase expression in HC11 cells, but weak in NIH 3T3 cells. These data indicate that the pCBCZ vector can effectively drive gene expression at a mammary gland-restricted manner, which provide a new strategy for the generation of an effective mammary gland bioreactor.
Trichoderma reesei can be considered a candidate for consolidated bioprocessing (CBP) microorganism. However, its ethanol yield needs to be improved significantly. Here we improved the ethanol tolerance of a wild-type strain CICC40360 by genome shuffling while simultaneously enhancing the ethanol productivity. The starting population was generated by nitrosoguanidine treatment of the spores, and then subjected for the recursive genome shuffling. The positive colonies from the mutant library, created by genome shuffling were screened for growth on regeneration of protoplasts medium plates containing different concentrations of ethanol. Characterization of all mutants and wild-type strain in the shake-flask indicated the compatibility of ethanol yields enhancement. After two rounds of genome shuffling, the best performing strain, S2-254, was obtained. It was found capable of completely utilizing 50g/l glucose, producing 6.2g/l ethanol, and tolerating 3.5% (v/v) ethanol stress. The result demonstrates that this method was effective and easy to operate for the construction a recombinant strain with desired phenotypes in a short time.
Genomic imprinting is a kind of epigenetic mechanism, which affects monoallelic parent-of-origin-specific expression in mammal's development. Noncoding RNA (ncRNA) is a polynucleotides that does not code protein, it can regulate gene transcription. At least one non-coding RNA transcript is existed in most of the imprinted loci and genomic imprinting is mainly regulated by lang-non-coding RNA which the length is longer than 200nt, through cis-transcriptional interference. Aberrant expression of imprinting gene and related lncRNA are the cause of some congenital diseases. So far, dozens of genetic imprinting is found to be related with human genetic diseases, in which genomic imprinting regulated by lncRNA plays an important role in the occurrence and treatment. The regulation disscussed mechanism of the genomic imprinting by lncRNA and its related diseases.
Fusion enzyme is one of the strategies to modify the characteristic of enzymes. Multiple-functional enzymes may be constructed by enzyme fusion. To our knowledge, fusion enzymes were applied in the production of oligosaccharide, biofuel, biomaterial, amino acid and biosensor. Fusion enzyme can be formed by rational design and non-rational design. Each stratagy has the advantage that the other don't. According to the application of these two strategies be used in rescent years, we illustrated the application of fusion enzyme in industry.
Transcription activator-like effectors nucleases (TALENs) has been a novel landmark genetic engineering tool for targeted genome site-specific editing in recent years, which consists of sequence-specific DNA-binding domain TALE and non-specific DNA cleavage domain FokI endonuclease. TALEs recognize and bind to specific DNA sequences and FokI generate a double-strand break (DSB) by its nuclease activity, and then DNA damage repair system induced. TALENs are constructed to mediate high efficient multiple genetic manipulation, through forming DSB, including target genes specific sites of gene segment knock-in, knock-out or correction, etc. TALEN technology with its simple design, high specificity, low toxicity and target selection flexible becomes the most widely used genome site-specific editing technology. Here, we will review the recent progress, clinical application and prospects of TALEN technology.
Biosurfactants are a class of surface active metabolites produced by microorganisms. Compared with chemical surfactants, biosurfactants have the advantages of high biodegradability, low toxicity, and high efficiency. Summarizes the research progress in the impacts of promoting factors such as amino acids, yeast extracts, metal ions and organic acids on yield, structure and homologue composition of biosurfactants. In addition, the promoting mechanisms of these factors on biosurfactant production are also concluded. At the end of this review, the prospect on this field are introduced.
Glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH), a tripeptide composed of glutamate, cysteine and glycine, is the most abundant non-protein thiol compound widely distributed in living organisms. There are too many papers on biosynthesis of glutathione. Glutathione is mainly synthesized by the consecutive action of γ-glutamylcysteine synthetase (GSH I)and glutathione synthetase (GSH II), whose evolutionary history is more complex than anticipated. Many organisms without GSH I or GSH II were proved to have the other biosynthetic patheways used to produce glutathione, while they also have complex metabolic pathway. This review summarizes the advance of the biosynthetic pathways and metabolisms of glutathione and the strategies to improve the intracellular level of glutathione using genetic engineering.
Hydrogen is a clean and effective energy. Development of hydrogen production technologies is one of the research focuses currently. Application of new-type technologies and materials on biological hydrogen producing is a research emphasis to enhance hydrogen production and promote its engineering application. The latest progress in research on photo fermentative hydrogen production by immobilized photosynthetic bacteria is reviewed. The fundamental of immobilization, progress of its application and impact factors on hydrogen production are summarized. Three immobilization methods including entrapment, suspended carrier and fixed biofilm are discussed in detail, mainly from the aspect of their effects on photo fermentative hydrogen production. New materials which have been worldwide utilized on immobilization are introduced. An outlook on research direction and focuses are also proposed.
With the support of government, bioenergy industry has developed rapidly, but at the same time, along with the development of bio energy industry in the economic, social and environmental problems continue to surface. This paper tries to construct the system framework of bioenergy industrial ecosystem, analyze the basic connotation, organization form, composition as well as the boundary, and further summarizes the main features and functions.
Seed cell is another main research content of tissue engineering. To obtain enough quantity and quality of seed cell is necessary for research and application of tissue engineering. The seed cells for tissue engineering must have an ability of developing new tissue structure, which are mainly autologous, allogeneic and heterogenou and have different advantages and disadvantages when using. Some adult stem cells including hematopoietic stem cell, bone marrow stem cell, neural stem cell, adipose-derived stem cell and skin stem cell are important seed cells for no ethic disputes and relatively simple developing differentiation conditions, etc. Human embryonic stem cell and its tissue engineering still need a long way to go if they are applied in clinical medicine. Other cells can also be used as tissue engineering seed cells, including endothelial cells, epithelial cells, fibroblast cells, bone cells, osteoblast cells, horn cells, preadipose cells, adipose cells, tendon cells, etc. These cells are differentiated and have a limited ability of division but still applied in tissue engineering. Ideal seed cells have certain standards.
Accomplished with the development of the worlds' pharmaceutical Industry, a new pattern that most profit is brought by few enterprises or products is established. Compare to the developed countries which have advanced pharmaceutical Industry, ours are huge but weak. Few enterprises are growing gradually while the others are short of R&D investment seriously. According to the situation of R&D and our pharmaceutical industry, we proposed a new viewpoint that helps the production of blockbuster drugs independently in this paper. And detailed recommendations on seizing the opportunity, improving innovation ability, Participating in international competition and nurturing environment for innovation were also given.
