Modern biotechnology is moving into large-scale industrialization phase, which makes global bio-economy developing rapidly. As a key pillar of bio-economy, industrial biotechnology is supporting the development of bio-manufacturing, bio-energy, bio-agriculture, bio-medicine, bio-environmental protection and biological services. The progresses and achievements of fundamental research, applied research, technology transfer and industrial development made in the field of industrial biotechnology in China recently were analyzed and demonstrated. The current situation and development trend of industrial biotechnology in China, as well the challenges and opportunities for future development were discussed.
Object:To study the effect of glycosylation on the conformation, stability and bioactivity of recombinant TNFR-Fc fusion protein. Method:Using PNGase F to remove the oligosaccharide chains in the Fc region of TNFR-Fc fusion protein, and the differences between glycosylated and deglycosylated recombinant proteins were tested by size-exclusion chromatography(SEC), fourier transform infrared spectroscopy(FTIR)and fluorescent spectroscopy, followed by study of the thermal stability of the proteins. In addition, the difference of the bioacitivity by cell killing experiment is tested. Results:Although the molecular weight of TNFR-Fc became smaller after deglycosyltion, but there were no detected change of charge property, bioactivity, secondary or tertiary structural. However, deglycosylated TNFR-Fc exhibited less thermal stability. Conclusion:Deglycosylation didn't affect the conformation, bioactivity or charge property of TNFR-Fc, but induced a decrease in protein stability.
Objective:To investigate the effect of Bone morphogenetic proteins 9 on induced osteogenic differentiation of human umbilical cord mesenchymal stem cells in vitro and in vivo.Methods:BMP9 gene-recombinant adenovirus was infected into hUC-MSCs and paralleled with the Ad-GFP as negative control group. Cells in two groups were detected in 3d, 5d, 7d of alkaline phosphatase activity;Immunohistochemical staining was used to calculated the expression of osteocalcin and osteopontin after 14d;Alizarin red staining was used to observe the formation of mineralized nodules after 21d;Then collect both groups of infected hUC-MSCs for the establishment of ectopic osteogenic model through nude mice subcutaneous injection, removed the ectopic induced bone tissue after 4 weeks and present to Micro-CT scan and analysis, H&E staining, Masson Trichrome staining, Alcain Blue staining.Results:It is shown that ALP activity and mineralized nodule formation of BMP9 group was significantly higher than that in control group, Immunohistochemical staining showed positive expression of OCN, OPG in BMP9 group was significantly higher than that of control group; the results of ectopic osteogenic model showed that the control group had no visible subcutaneous mass, Only Ad-BMP9 infected hUC-MSCs can observed the formation of ectopic bone obviously, average value of bone mineral density is 396.05+0.60; H&E staining results showed that the generation of mature bone matrix and bone trabecula in the BMP9 induced ectopic bone tissue, Masson Trichrome staining results showed that BMP9 induced of matrix mineralization, Alcain Blue staining results showed that BMP9 induced endochondral osteogenic effect. Conclusion:The BMP9 significantly induces osteogenic differentiation of hUC-MSCs in vitro and in vivo, to provide the feasibility for clinical cell therapy in bone tissue engineering.
Objective:To study the biocompatibility and osteoinductivity of typeⅠcollagen/poly (caprolactone)/attapulgite composite scaffolds in vitro. Methods:The ColⅠ/PCL/ATP composite scaffolds materials of three different content (0%, 10%, and 30%wt) ATP were fabricated by the solution perfusion-salt-leaching process. D1 cells were co-cultured with the materials for 7d or 1~6d, the biocompatibility of scaffold was evaluated by scanning electron microscopy, Phalloidin and H&E staining and CCK-8, respectively. D1 cells were co-cultured with the materials for 7d, 14d, and 21d; the relative expression of bone-related genes including Runx-2, Osterix, ALP, Col I, OPN, and OC were measured by RT-qPCR, and evaluated osteogenic effect comparison of three scaffolds. Results:Three scaffolds had suitable cell adhesion and appreciation properties, especially 30%wt ATP materials. The results of RT-qPCR show that Runx-2 expression of 30%wt ATP was significant increased in 7d, but decreased in 14d, 21d; and ALP expression was significant increased in 14d, but significant decreased in 21d; and there were significant increased in the relative mRNA levels of Osterix, Col I, OPN, OC, compared with the 0% and 10%wt ATP materials (P<0.05), which had time and dose dependent manner. Conclusion:ColⅠ/PCL/ATP composite scaffolds may be used as an ideal scaffold for bone tissue engineering.
Objective:To observe the effect of combined-silencing MMP-9 and FAK on migration of malignant melanoma highly metastatic B16F10 cells in vivo. Methods:Construct recombinant plasmid vectors pGV102-MMP9-siRNA and pGV102-FAK-siRNA, then Lipofectamine TM2000 mediated the plasmid vectors were transfected into B16F10 cells. To observe the growth of the tumor, the C57BL/6 mouse model of subcutaneous transplantation was established, and the pathological characteristics of tumor tissue was observed by H&E staining. B16F10 cells were stably intravenously injected into C57BL/6 mice by 5×105. After 24 days of injection, the number of mouse lung metastasis nodules was counted to evaluate the migration ability of tumor cells in vivo. Results:RT-PCR results showed that the mRNA level of MMP-9 and FAK in the transfected cells was significantly reduced than that of normal cells(P<0.01). Compared with the normal cells, the tumor growth rate and the number of lung metastasis nodules of C57BL/6 mice in the transfected cells were significantly reduced(P<0.01). Conclusion:Interference MMP-9 and FAK can significantly inhibit malignant tumor growth and metastasis in vivo.
Objective:In order to investigate and improve activity of anti-aflatoxin B1(AFB1) single chain Fv fragment (scFv), anti-AFB1 scFv was expressed in Sf9 insect cells and then further studied according to previous research on prokaryotic expression of anti-AFB1 scFv. Methods:The recombinant plasmid pFastBac 1-scFv2E6VHVL was constructed, and then transformed into competent cell of Escherichia coli DH10Bac. Blue-white selection was performed for positive clone selection. Subsequently, the corresponding Bacmid-scFv from positive clones were extracted to infect Sf9 cells, and anti-AFB1 scFv was expressed, purified and determined by ELISA. Results:After blue-white selection, the positive clones were confirmed by colony-polymerase chain reaction (PCR) and sequencing, and Bacmid-scFv from positive clones were extracted to infect Sf9 insect cells. Successful expression was confirmed by Western blot. After purification, the scFv from Sf9 insect cells was analyzed by ELISA. The results suggested that the sensitivity of scFv obtained was 30μg/ml. Conclusion:Compared with anti-AFB1 scFv expressed in E. coli BL21(DE3), the sensitivity of scFv expressed in Sf9 insect cells was little better, but there was still room for improvement.
An Arabidopsis thaliana AGG3 homologous gene was cloned from Jatrophacurcas L., which was named JcAGG3. JcAGG3 had an open reading frame of 834bp, encoding a protein of 277 amino acids. Its theoretical isoelectricand molecular weight point were 30.514kDa and 8.61 respectively. The protein was predictedlylocalised in plasma membrane.Through Biologysoftwareprediction analysis, a lot of promoter-binding elements related to the development of endosperm, hormonal regulation, light response and abiotic stresses were found in the cis-acting elements of the gene. The expression of JcAGG3 in different tissues and different development stages were examined using real-time polymerase chain reaction(RT-PCR). The results showed that JcAGG3 was expressed in root, stem, leaf and seed, with the highest expression in developing seed but least in stem. Young lealves had a significantly higher expression level than older leaves. After 6h dark treatment, the level of JcAGG3 expression was significantly reduced. However, under abscisicacid(ABA) and drought treatment, the level of JcAGG3 expression were increased.
Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are a large class of biopolymers that have attracted extensive attention as renewable and biodegradable bio-plastics. They are naturally synthesized via fatty acid de novo biosynthesis pathway or β-oxidation pathway from Pseudomonads. The unconventional yeast Yarrowia lipolytica has excellent lipid/fatty acid catabolism and anabolism capacity depending of the mode of culture. Nevertheless, it cannot naturally synthesize PHA, as it does not express an intrinsic PHA synthase. Here, a genetically modified strain of Y. lipolytica was constructed by heterologously expressing PhaC1 gene from P. aeruginosa PAO1 encoding PHA synthase with a peroxisomal signal (PTS1) at the C terminal. The gene was coden optimized and linked onto the vector of pINA1312 to construct pINA1312-oPHA. The expression cassette derived from pINA1312-oPHA was integrated onto the genome randomly through zeta element above the vector resulting strain PSOC. The recombinant yeast of PSOC barely produced mcl-PHA when grew in YPD, but can produce 0.67% DCW of PHA after 72 cultivation in YPD medium containing 0.5% oleic acid. Furthermore, when PSOC was grown in the medium containing triolein, PHA accumulated up to 1.51% DCW. It successfully demonstrated the potential use of Y. lipolytica as a promising microbial cell factory for mcl-PHA production, which laid the foundation for using food waste that contains lipids and other essential nutrients to produce mcl-PHA.
Objective:To clone the dioxygenase dio6 gene from Aeromonas sp. XJ-6, and explore the function of dioxygenase dio6 to provide genetic resources for biodegradation of aromatic compounds. Methods:Dioxygenase dio6 gene from Aeromonas sp.XJ-6 genome was amplified by PCR and induciblly expressed in Escherichia coli BL21(DE3). The expression products of dio6 was purified by Ni-NTA.TLC and HPLC to detect the degradation effect of dioxygenase on Tyr, and the degradation products were also detected by LC-MS so as to analyze the possible degradation pathway. Results:The size of Aeromonas sp. XJ-6 dioxygenase gene dio6 was 1194bp, the expression product of dio6 purified by MCAC was 44.9kDa. HPLC analysis showed that dio6 has strong degradation ability for Tyr under the optimal conditions that the amount of enzyme is 60μl per reaction mixture, temperature 30℃.Metal ion Mg2+ and Ca2+ slightly inhibit the enzymatic reaction, and Mn2+、Zn2+、Cu2+、Fe2+ and Ca2+ can improve the substrate degradation, of which Mn2+ has the most impact on dioxygenase dio6. LC-MS analysis results showed that Tyr was turned into fumaric acid with the help of dioxygenase dio6. Conclusion:Aeromonas sp. XJ-6 dioxygenase dio6 is a kind of benzene ring-opening enzyme, which may be provided as a genetic resource for aromatic compounds biodegradation.
Objective:Using insertional mutagenesis in Chlamydomonas to identify genes required for flagellar assembly, so as to researching the function of the gene related to flagellar assembly. Methods:By transforming the DNA fragment harboring paromomycinresistance gene into Chlamydomonas randomly, the mutant strainwith flagellar defect is obtained and identified the disrupted gene was CrPP2C(Chlamydomonas reinhardtii type 2C protein phosphatase).Then the CrPP2C gene function is analyzed by biochemistry methods. Results:Theflagella abnormal mutantsthat show short flagella or none flagella are obtained by electroporation transformation.DNA sequence analysis show that the CrPP2C gene is disrupted. Transformation of mutants with CrPP2C-HA could rescue the flagellar phenotype.The mutants could regenerateflagella to the original length and shorte normally. Electron microscopyimages show incomplete Y-link structure in the transition zone of flagella. Conclusion:The CrPP2C gene is associated with the transition zone structure and affects the assembly of flagella.
Objective:By using multiple gene expressing plasmids, fusion protein of single-chain variable fragment of alpha fetoprotein and allophycocyanin alpha subunit, phycobiliprotein lyase (cpcS), and enzymes (Ho1 and pebS) for conversion of heme to phycoerythrobilin were co-expressed in E. coli, with an aim to prepare fluorescent fusion protein which is covalently bound with phycoerythrobilin. Method:fragments of alpha fetoprotein and allophycocyanin alpha subunit were fused by fusion PCR. Then the fused gene, together with cpcS gene, were ligated into vector pCDFDuet-1. Ho1 and pebS genes were ligated into another vector pRSFDuet-1. The two vectors were then transformed into E. coli. Recombinant proteinwere expressed efficiently in E. coli by addition of IPTG and purified by affinity chromatography.The activities of the recombinant protein were evaluated by spectral analysis and competitive ELISA.Results:The recombinant protein, with a molecular weight approximately to 45.0kDa, has an absorbance maximum at 549.5nm and an emission maximum at 660nm. Competitive ELISA results showed that inhibition rate reached 48% when the concentration ratio of scFv and parent monoclonal antibodies was 16:1.Conclusion:A recombinant protein, which was fluorescent and immunologic active, was successfully expressed in E. coli.
CTxs are small peptides which specific target variety of voltage-gated ion channels and membrane receptor. Conotoxins are important tools for the study of receptors structure and function and their related diseases. MrIA is a T-superfamily conotoxin which was entering clinical research for analgesia. The traditional methods to acquire conotoxin are chemical synthesis, while these methods are cost-consuming but low yields. Tandem repeats of mria gene fragment were constructed and ligated with prokaryotic expression vector. The rMrIA was expressed successfully in E.coli. 95% purity rMrIA were obtained by CNBr cleavage and purification, 30mg rMrIA could be obtained from 1L media eventually. Mice hot plate test showed that rMrIA had good analgesic activity. An effective method to obtain large quantities of MrIA and a model for other small conotoxins expression were provided.
Active vitamin D3 has a wide range of physiological activity and phamaceutical value. Construction of key enzyme system for the selective hydroxylation of VD3 in Escherichia coli cell is an effective mean to realize the biological conformation of active vitamin D3 through molecular manipulation. The recombinant expression vector pET28b-FdR-Fdx and pET28b-Vdh were constructed, VD3 hydroxylase (Vdh, EC 1.14.13.159) originated from Pseudonocardia, ferredoxin and ferredoxin reductase originated from Acinetobacter sp. OC4 were actively expressed in Escherichia coli as the host cell and purified by nickel column. Reduced CO difference spectra assay was carried out to evaluate the activity of VD3 hydroxylase in vitro. 2, 6-dichlorobenzenone-indophenol sodium salt and cytochrome c, serving as an electron acceptor, were used to evaluate both the oxidation activity of the electron transfer chain for NADH/NADPH and the coupling effect with the hydroxylase Vdh. Finally, 25(OH)VD3 is selectively hydroxylated by Vdh and its electron transport chain using vitamin D3 as the substrate.
Construction of tolerant yeast to the inhibitory compounds from the pretreatment of lignocelluloses is quite required for efficient and economical cellulosic ethanol production. An industrial Saccharomyces cerevisiae strain YYJ003 was developed to be tolerant to mixed inhibitors, including 1.3g/L furfural, 5.3g/L acetic acid and 1.0g/L phenol. Compared with parent strain S, YYJ003 strain exhibited 7.8-fold ethanol productivity and a 6-fold conversion rate of furfural in the presence of inhibitor cocktail at pH 4.0. At pH 5.5 with inhibitor cocktails, the fermentation time for strain YYJ003 was shortened to 16 hours while 22 hours for S strain, respectively. Furthermore, YYJ003 achieved the ethanol yield of 0.50g/g glucose, and ethanol productivity of 4.16g/(L·h) in liquid hot water pretreated corn stover hydrolysate without detoxification at pH 4.0 while strain S failed to ferment.
Cancer is becoming one of the major diseases with serious hazards to human health. To find efficient treatment protocols has always been the important subject in cancer studies. While surgical management, radiotherapy, chemotherapy and immunotherapy have achieved certain effects, there are distinct limits in each method. With the development of data mining methods and molecular experimental technologies, more and more cancer related targets and signaling pathways were discovered from human genome. Based on such knowledge, gene therapy has been proved to be with significant efficacy and superiorities in both experimental and clinical trials. The principle of gene therapy and some recent technologies in cancer gene therapy were summarized. Then the prospects of cancer gene therapy in future clinical application were discussed.
The innate immune system deploys pattern recognition receptor(PRR) to detect pathogen-associated molecular pattern(PAMP). Microbial DNA could be recognized by various host DNA sensors that are either membrane-bound or located in the cytosol, and stimulate the production of type I interferons and proinflammatory cytokines. DNA-stimulated innate immune activation plays an important role in antimicrobial response as well as certain autoimmune diseases. The recent progress in identification of novel DNA sensors in host cells, and the related signaling pathways that lead to the innate immune activation were summarized.
Recombinant protein expression in Escherichia coli (E. coli) is sound and effective with the protein expression taking up to 50 percent of the total protein. The soluble recombinant proteins usually have biochemical activities, these proteins include antibodies and enzymes. Here are many soluble recombinant proteins have been approved as drugs, so it is crucial to research how to promote soluble expression of recombinant proteins in E. coli. Here the researches about improving yield of soluble expression of recombinant protein by E. coli have been summarized thoroughly. Variables at stages of a protein expression such as promoter system, SD sequence, signal peptide, host strains, co-expression of other proteins and high cell density cultivation to optimize soluble expression in E.coli are discussed.
MicroRNAs (miRNAs) are a class of 20~24 nucleotides (nt) non-coding small RNAs with wide distribution in plant genomes. As one of the most important post-transcriptional regulators, miRNAs play a key role in gene expression and stress responses of plants through cleavage or translational repression by complete or partial complementarity to target mRNAs. MIRNA genes are generally known to be transcribed in the nucleus by RNA polymerase II (Pol II) into a primary miRNA with two following continuous cleavages by Dicer-like 1 to produce a precursor miRNA and a miRNA:miRNA star duplex. Promoters are indispensable components involved in gene expression and contain transcription start sites (TSS), TATA boxes and upstream cis-acting elements. It is known that the promoters of miRNAs are important in regulating the expression of miRNA in specific plant tissues, development stages, biotic and abiotic stress responses of plants. As a consequence, investigations on promoters of MIRNA genes are fundamentally important to reveal the molecule mechanism of spatial and temporal expression of miRNAs and will be helpful to reveal the function of miRNAs involved in the biological processes of plants. Consequently, it is highly indispensable to review and analyze the characteristics of miRNA promoters, identification methods of miRNA core promoters and cis-acting elements. Furthermore, the advances of researches on miRNA promoters of plants such as Arabidopsis and rice, the possible problems and prospects of miRNA promoters investigations were also discussed to provide insights into the evaluation of miRNA-mediated gene regulation mechanism.
Spider silk is a kind of macromolecular protein fiber with high strength, high elasticity, and many other excellent characteristics, so it has been applying widely in military, medicine, industry, construction, textile and other fields. However, spider's silk production quantity is small, and spider can not be high density cultivated for getting a lot of spider silk, it is difficult to meet the needs of practical application. So people can only focus on the biological engineering methods, and try to introduce spider silk protein genes into other organisms to produce spider silk protein. After years of research, a lot of important progress has been made. The research course of spider silk proteins expression in different biological carriers, such as microorganisms, plants, mammals and silkworm were focused, also the deficiency of the existing research and future research prospects were discussed, so as to provide reference for further exploration and development of large-scale production of spider silk.
Cell drug includes the agent, medicine or product that can be based on different cells to treat disease, which is an effective clinical therapeutical method after radiotherapy and chemotherapy and may be implemented individualized treatment. There are many types of cells drugs which are divided into the traditional somatic cells, immune cells and different types of stem cells, etc., according to their biological characteristics. The cells were operated outside the body including liver cells, islet cells, cartilage cells, dendritic cells, cytokine induced killer cells, lymphatic factor activated killer cells, processed bone marrow or blood stem cells in vitro, and treated tumor cells (tumor vaccines) in vitro, etc. Cell drug has already been used in some refractory diseases, including blood system diseases, cardiovascular system diseases, digestive system diseases, nervous system diseases, immune system diseases, and anti-aging. Cell therapy is involved in many cell types, and different cells or different treatment methods have different characteristics. Different cell drugs are used to repair the diseased cells for rebuilding the damaged function cells and tissues and restoring their biological function, which provides a new train of thought for the prevention and control of cell loss or traumatic disease.
