Beta 2-glycoprotein Ⅰ (β2GPⅠ)is the main antigen of antiphospholipid antibody (aPL) in serum of antiphospholipid syndrome (APS). β2GPⅠ binding to oxLDL via its the fifth domian and then subsequently recognized by aPL is a key event in the development of APS arterial thrombosis. In this study, a prokaryotic expression vector encoding β2GPⅠ fifth domain (β2GPⅠ-DⅤ), β2GPⅠ-DⅤ mutant and the Phe280-Ala320 fragment of β2GPⅠ-DⅤ were constructed, their induced expression and purification were performed, and the molecular mechanism of the binding of β2GPⅠ-DⅤ to negative phospholipids was analyzed. Results showed that the spatial configuration of the Cys281-Cys288 and Ser311-Lys317 segments in β2GPⅠ-DⅤ, which are maintained by the two disulfide bonds in C245-C296 and C288-C326, is a necessary precursor condition for the binding to CL. On this basis, the binding activity of rDⅤ to oxLDL and oxLDL in the serum of APS were further examined, indicating that rDⅤ has biological activity consistent with that of natural β2GPⅠ. The obtainment of rβ2GPⅠ-DⅤ and the establishment of the method in rβ2GPⅠ-DⅤ binding to oxLDL in this study lay a foundation for the early laboratory diagnosis of APS.
Objective: To study porcine interleukin-17(IL-17) eukaryotic expression and their biologic activity. Methods: The IL-17 gene was amplified by PCR and cloned into eukaryotic expression vector pVAX1,named PV17. PV17 was transfected into IPEC-J2 cells,HaCaT cells and L02 cells,thereafter the cells were collected on hours 24,48 and 72,the supernatants were collected on hours 48. Related genes expression levels in cells were analysed by qRT-PCR,bioactivity of antimicrobial peptides in supernatants were analyzed by bacteriostatic test in vitro. Results: The eukaryotic expression plasmid of the porcine IL-17 gene was constructed and transfected into the three eukaryotic cells,and could expressed in target cells. The expression levels of antimicrobial peptide genes (RegⅢ,S100A8 and BD2),JAK-STAT signaling pathway genes (JAK1,STAT1 and STAT3) and cytokine genes (IL-6,IL-12 and TNF-α) were significantly up-regulated. Furthermore,the supernatants have marked bacteriostatic effect on E. coli and S. aureus. Conclusion: The recombinant plasmid of the porcine IL-17 gene was constructed and expressed in target cells,and the expressed products elicited the significant increases of cytokines and antibacterial peptides,which manifested obvious antibacterial activities against E. coli and S. aureus drug-resistant bacteria,and facilitated the further development of porcine IL-17 as the antibacterial reagent.
R-2-haloacid dehalogenase can selectively hydrolyze R-2-haloacid and have important applications in the synthesis of chiral compounds.The analysis of the crystal structure provides a direct structural guide to improve the selectivity and activity of the enzyme,which is the frontier in the field of enzymatic structure research.The crystal structure of R-2-chlorpropionic acid dehalogenase (DehDIV-R) from Pseudomonas ZJU26 was studied.DehDIV-R was expressed in Escherichia coli BL21(DE3) using ppSUMO as vectors,and purified by Ni-NTA affinity chromatography,ULPI digestion,second Ni-NTA affinity chromatography and gel filtration chromatography.High-quality crystals were obtained in optimal conditions (0.1 mol/L HEPES pH 7,12% PEG 6000,0.2 mol/L MgCl2,8 mmol/L CHAPS).The diffraction data of crystals were collected at BL18U1 beamline of Shanghai Synchrotron Radiation Facility(SSRF).The crystal structure of DehDIV-R with a resolution of 2.35? was successfully resolved by Molecular Replacement(MR).The Ramachandran plot shows that 98.02% of the amino acids are in the optimum region,indicating the rationality of the structure.The purification,crystallization and structural analysis of the DehDIV-R have laid a foundation for further understanding the relationship between structure and function.
Objective: To generate mice with a liver-specific knockout of CD36 gene using the Cre-Loxp system,which will lay a foundation for the study of the biological function of CD36. Methods: CD36 targeting vector was constructed and electroporated into embryonic stem cells.Positive clones with correct homologous recombination were screened by long-chain PCR.Positive embryonic stem cell clones were amplified and injected into the blastocysts of C57BL/6J mice to obtain chimeric mice.And then mated with Flp mice to obtain Flox heterozygous mice.The Flox mice were mated with the introduced Alb-Cre mice,and CD36 fl/fl:Alb-Cre + mice were obtained in the F3 generation,which are liver-specific CD36 knockout mice.The genotypes of the mice were identified by PCR.PCR,real-time fluorescence quantitative PCR and Western blot were used to verify the knockout effects of CD36 gene in the liver.The expression of CD36 in kidney,adipose tissue and myocardial tissue was detected by Western blot.Morphological changes of the liver were observed by HE staining. Results: Flox heterozygous mice with CD36 gene were established.After mating with Alb-Cre mice,CD36 fl/fl:Alb-Cre - and CD36 fl/fl:Alb-Cre + genotypes were screened in F3 generation.DNA levels confirmed that CD36 fl/fl:Alb-Cre + genotype mouse liver CD36 gene was knocked out by the Cre/Loxp recombinase system.Compared with mice with CD36 fl/fl:Alb-Cre - mice,CD36 fl/fl:Alb-Cre + mice had significantly reduced expression of CD36 mRNA and protein in the liver,and there was no difference in the expression of CD36 protein in kidney,adipose tissue and myocardial tissue.There was no significant difference in the morphological characteristics of liver. Conclusion: Liver-specific CD36 knockout mice were successfully generated by the Cre/Loxp recombinase system,providing an animal model for the study of CD36’s function in hepatic metabolism and diseases.
Objective: To screen high expression monoclonal cell lines and optimize the production and quality of Anti-CD20rh MAb. Method: Screen high expression monoclonal cell lines by finite dilution method, and evaluated the yield of monoclonal cell lines by double-sandwich ELISA. In order to screen the best cell culture program, two to three cells were picked based on the cells’ growth state, yield, viability, and so on. The result were evaluated by the sugar type, the isoelectric point, the purity and the distribution of the acid and alkali base peak. Results: The production of CHOS cells tripled after a series of optimization, increased from nearly 500mg/L to 2 290mg/L. After the optimization of culture program, the purity of the target protein reached up to 97.48%, and the distribution of the acid and alkali base peak looks more close to the ideal state. Conclusion: The production and quality of the target antibody were optimized by cloning and culture optimization, these results are of great significance to the later experimental research and industrial production.
Lactose is one of the important carbon sources for infants to obtain energy, but lactose needs to be hydrolyzed by enzyme to galactose and glucose before being absorbed. Infants and young children lacking lactose hydrolase can fall in lactose intolerance when consuming lactose-containing food. Lactose intolerance is a symptom of high incidence in China. Therefore, lactose hydrolysis with high efficiency in vitro is of great importance to alleviate the symptoms. The lactose hydrolase (also known as β-galactosidase) was displayed on the cell surface of the food-satefy-grade yeast Yarrowia lipolytica, and the engineered yeast cells are used directly to hydrolyze lactose to produce galactose and glucose. The engineered yeast cell (HCY10) can completely hydrolyze 50g/L lactose in 24 hours to produce galactose and glucose. The method has the advantages of high efficiency and simplicity, and it can provide an alternative solution for the efficient lactose hydrolysis in vitro.
Baculovirus expression vector system (BEVS) is the ideal platform for Virus-Like Particles (VLPs) production. The key points of the Process Analytical Technology (PAT) in animal cell culture include obtaining cell physiological characteristic parameters, monitoring process sensitive parameters, and controlling critical time points. These parameters in turn can promote process optimization and process control. A close correlation between cell metabolic status and on-line parameter critical frequency (fc) was found in Sf 9 cell batch culture. Using the fc as on-line indictor of cell metabolic status and feeding the culture, a 1.7 time increase of Peak Viable Cell Density (PVCD) was obtained. Besides, the metabolic status was also improved. A close correlation was found between the proportion of S-phase cells and specific capacitance growth rate (με). It turned out that με could indicate the optimized infection time point. Additionally, a high correlation was found between ε and the maximum VLPs productivity. The parameter of ε can act as an on-line indicator. Overall, an effective product mode was established by using fc as on-line feeding indicator, με as on-line infecting indicator, ε as on-line harvesting indicator. Compared with traditional process, the volume productivity was increased by 76%, and the culture duration was reduced by 24h. A new VLPs production mode has been established successfully.
Streptomyces albulus M-Z18 with 1.60g/L yield of ε-Poly-L-lysine was used as initial strain to screen high-yield mutant,and the physiological and biochemical properties of high-yield mutant were compared with M-Z18.The ribosomal engineering technology was used to breed the high-yield mutant with double antibiotic resistance. A genetically stable strain named S-7, was successfully screened by streptomycin mutagenesis. The ε-Poly-L-lysine yield of S-7 was 2.03g/L. S-7 was used as initial strain to screen mutant with paromomycin,and obtained SP-14, with 2.37g/L yield of ε-Poly-L-lysine. The use of streptomycin and paromomycin to breed high-yield mutant with high antibiotic resistance is an effective method.
The development of biologics based on mammalian cell culture technologies has increasingly rapid advances for the pharmaceutical markets in the recent years. Economic concerns and time constraint as the critical factors and the driving force have accelerated bioprocess development of delivery of new biopharmaceutical drugs to market. Dramatically, advancement of semi-high-throughput micro-bioreactors in bioprocess development has shown a significant alternative for the conventional approaches due to automation, increased capability of throughput, and excellent parallel level compared to costly and laborious bench-top bioreactors. There are several commercially available micro scale bioreactors, such as Simcell TM, Ambr 15 TM and Ambr 250 TM, being applied in different stages of cell culture development to enhance throughput. This research reviewed and summarized the strengths and challenges of high-throughput bioreactors for the mammalian cells culture, showing the potential as scale-down models for process development and further improvement in the future.
Mesenchymal stem cells (MSCs) have capacity of self-amplification and multi-direction differentiation, which is a hot research topic as popular seeding cell in recent years. It is well known that micro-environment modulates MSCs’ development and differentiation. Mechanical stimulation is one of the factors effecting on MSCs’ differentiation. The effect of extracellular matrix stiffness and mechanical stress, like shearing stress, hydrostatic pressure, stretch stress, and microgravity on MSCs remains a hot point. In the present article, the effects of external matrix stiffness, mechanical stress and the mechanical force on three-dimensional scaffolds on the MSCs’ differentiation were mainly reviewed.
Betalains is a kind of botanical water-soluble natural nitrogen-containing pigments, which used as food additives and cosmetics. Betalains and anthocyanin pigments do not coexist in a same plant, and their metabolic pathway is an important phytochemical classification index. Betalains have potential pharmacological effects of anti-tumor, anti-oxidation, anti-malaria and liver protective, which have promoted the further study of them. The key enzymes of betalains synthesis pathway and research progress of synthetic biology strategy for betalains production at home and abroad are reviewed. It provides a reference for establishing synthetic biological method of betalains production.
Cell wall can maintain the shape and integrity of cell and resist internal expansion pressure during bacterial growth.The synthesis, division, regeneration, and recycling of cell wall are closely related to bacterial growth and the response to environmental stress. At present, the mechanism of cell wall growth, how to regulate cell wall growth and how to coordinate with other cellular processes remain largely unknown.The regulation mechanism of cell wall is very important for understanding the function of bacterial cell wall, determining the action of new drugs and developing the new generation of treatment methods. In this review, the bacterial regulatory mechanism of cell wall growth is summerized and the mechanisms of the scaffolding proteins, transcriptional regulators as well as small non-coding RNA and the protein-protein interaction to control the synthesis of cell wall, cell division and stress response are highlighted. In addition, the application of cell wall regulatory mechanism in the development of antibacterial drugs is summed up, and the future research direction is proposed.
Patent is an effective carrier of technology. Through the patent situation analysis, the context and trends of technological development can be effectively grasped. In this paper, all the data is extracted from the patent database. Through the combination of quantitative data analysis, qualitative research, and expert wisdom, the patent situation analysis can be obtained in the field of agricultural biotechnology from various dimensions, including application trends, protection of markets, distribution of fields, and important patentees. Through the patent situation analysis, the overview of global patent protection of agricultural biotechnology can be grasped and it point out the key directions of China's agricultural biotechnology research and development. This article discusses the status quo and existing problems of China's agricultural biotechnology patent protection, and promotes the innovative development in the field of agricultural biotechnology. In this paper, the current situation and existing problems of patent protection of agricultural biotechnology in China is also discussed, in order to promote the innovation and development of agricultural biotechnology.
