Objective: To establish the cell lines which can stably express the RPB5-mediating protein (URI) and study the effects of URI gene on the biological function of human hepatoma SMMC-7721 cells. Methods: Get PFLAG-CMV-4-URI from the E.coli which was transfected the plasmid. Then to identify the recombinant eukaryotic expression by enzyme analysis, RT-PCR Transient transfected the plasmid PFLAG-CMV-4-URI into SMMC-7721 cells by Lipofectamine reagent. G418 selected the cell clone which stably expressed URI. The URI expression were detected by RT-PCR and cell proliferation was measured by MTT assay. Plate colony forming assay was used to observe the clone formation ability of cell lines. Results: The positive clones which stably expressed URI were selected. Result of RT-PCR and enzyme analysis showed that URI gene could stably expression in SMMC-7721 cells. MTT assay showed that URI promoted the growth of SMMC-7721 cells. And the URI gene could also improve the formation rate of cell clones. Conclusion: URI gene can improve the proliferation of SMMC-7721 cells probably by helping the cell through the G2/M checkpoint.
Objective: To amplify human PHP14 gene, and construct the N-terminal and C-terminal GFP fused vectors, establish the overexpress PHP14 NIH-3T3 cell line to investigate its effect on NIH-3T3 cell proliferation and anchor independent growth. Methods: The cDNA sequence of PHP14 gene was amplified and sub-cloned into pEGFP-N and pEGFP-C3 vectors. Transfecting of those vectors into NIH-3T3 cells and investigating the cell proliferation and anchor independent growth in PHP14 overexpressed NIH-3T3 cells using MTT and Soft agar colony assay. Results: The pEGFP-N2-PHP14 and pEGFP-C3-PHP14 prokaryotic expression vectors were constructed successfully and the expression of those vectors were detected in NIH-3T3 cells. Overexpression of PHP14 in NIH-3T3 cells did not affect the cell proliferation of NIH-3T3 cells, but gave the NIH-3T3 cells the ability of anchor independent growth. Conclusion: PHP14 prokaryotic expression vectors were constructed successfully and overexpression of PHP14 in NIH-3T3 cells did not affect the cell proliferation of NIH-3T3 cells, but gave the NIH-3T3 cells the ability of anchor independent growth.
Objective: To study the reaction conditions for the chemical modification of recombinant lysostaphin (Lysostaphin) with activated polyethlene glycol (PEG) and the methods for the purification of PEG-Lysostaphin. Methods: Disrupt the cell wall of bacteria by ultrasonic cell disruptor and purify the lysostaphin by Cation exchange chromatography (SP) and Hydrophobic chromatography (HIC); Lysostaphin was chemical modified with activated mono-methoxy polyethlene glycol succinimide propionate (mPEG- SPA) under various conditions, the ration of mono-mPEG-Lysostaphin was determined by SDS-PAGE and EMALDI-TOF-MS; The reaction mixture was purified by Sephacryl S-200 molecular size exclusion chromatography. Results: The optimal reaction was pH 8.0,temperature 4℃, 1:5 for the mass ratio of lysostaphin and mPEG, two hours for reaction time was selected. The reaction mixture was preliminarily separated by one step Sephacryl S-200 molecular size exclusion chromatography. Conclusion: It’s preliminarily determined that the optimal reaction conditions for the chemical modification of lysostaphin with activated polyethlene glycol and the methods for the purification of PEG-Lysostaphin.
Objective: Observe the growth of osteoblast-like cells induced and differentiated by human adipose stem cells (hADSCs) in the spongelike porcine small intestinal matrix (SIS). Explore ability of three-dimensional SIS promote osteoblast-like cells proliferation and differentiation. Methods: acellular SIS is prepared by combination of physical and chemical methods with porcine proximal jejunum. Particles was made with film-like SIS by milling-in liquid nitrogen cryogenic grinding. And the particles has been reshaped spongelike after crosslinked by freeze-drying technology. hADSCs is isolated and cultured by enzymatic digestion. And surface antigen has been identificated by flow cytometry. hADSCs has been induced and differentiated into osteoblast-like cells、chondroblast-like cells and adipocyte-like cells. The osteoblast-like cells is cultured in cavernous SIS. Cell morpholog is observed by scanning electron microscope. Osteoblast-like cells is cultured in material extracts of SIS. The cell viability is evaluated with MTT. Osteogenic differentiation is detected by ALP activity. Results: Spongelike SIS is three-dimensional stereo-scaffold with a great of regular three-dimensional pore. Stem cell-associated antigen is expressed by primary hADSCs. hADSCs can differentiated into osteoblast-like cells stained by alizarin red. Osteoblast-like cells can proliferate and express ALP obviously in cavernous SIS. Conclusion: spongelike SIS with three-dimensional pores presents superior cytocompatibility, and it can promote osteoblast-like cell differentiated by hADSCs proliferate and increase osteogenic activity significantly. It can became a novel three-dimensional natural biological materials for bone tissue engineering.
The ftsZ genes are a set of key genes in cell division regulation, and their proteins form a ring structure at the division site to control the process of cell division. In order to study the relationship between cassava starch formation and amyloplast division, three cassava ftsZ family genes were isolated and named as ftsZ1-3. To identify their functions, the ftsZ1-3 genes respectively fused with GFP were expressed in E. coli BL21(DE3). The result showed that the expression of cassava ftsZ genes actively affect the E. coli division although the genetic relationship between cassava and E. coli is far, and the homology of their ftsZ genes is low. This result will be a foundation for further study the function of cassava ftsZ family genes.
The results of experiment showed that the optimum condition for preparation of protoplast to Penicillium griseofulvum HL as the starting strain was incubation time of mycelium of 48h, osmotic stabilizer of 0.7 mol/L sodium chloride, enzyme combination of 0.5% snailase +0.5% cellulase, hydrolysis temperature of 30℃ and enzymolysis time of 3h with pH value of 6.0. Under this condition the yield of protoplast could reach 3.14×107/ml.The optmium condition for regeneration of protoplast of Penicillium griseofulvum HL was improved examine’s medium as the regeneration medium with osmotic stabilizer of 0.7 mol/L sucrose and regeneration method of double-layer solid culture. Under this condition the regeneration rate of the protoplast was 24.93%. The protoplasts of Penicillium griseofulvum HL were induced by ultraviolet ray, diethyl sulfate, ultraviolet ray and diethyl sulfate, ultraviolet ray and LiCl,a high-yield strain of erythorbic acid ZD4 was obtained by first screening of flat plate and second screening of shake flask.The yield of erythorbic acid was increased from the initial 1.23mg/ml to 5.28mg/ml. And it can descend stably after six generation.
In order to improve microbial production of L-glutamate oxidase, the L-glutamate oxidase gene (LGOX) from Streptomyces sp.X-119-6 was successfully expressed by connected to the expression vector pET28a and then transformed into E.coli BL21(DE3). The HisTrapTMFF affinity chromatography column was used for the purification of LGOX. Enzymatic properties of the recombinant LGOX were also investigated. Results showed that the recombinant LGOX activity could reach 1.1 U/mg at the IPTG concentration of 0.4 mmol/L at 30℃ for induction 6 h. The optimum reaction temperature and pH were 37℃ and 5.0, respectively. The Km was 2.12 mmol/L and Vmax was 1.06μmol/min·mg. The LGOX had high substrate specificity of L-glutamic acid with application prospect.
Objective: To realize high level prokaryotic expression of the tachi2 gene from Trichoderma asperellum and characterize the recombinant enzyme. Methods: The tachi2 gene was amplified by PCR, and recombinant plasmid pEHISTEV /tachi2 was constructed successfully by inserting the tachi2 gene into pEHISTEV. The recombinant plasmid pEHISTEV/tachi2 was transformed into Escherichia coli BL21, which was induced with isopropyl-β-D-thiogalactoside (IPTG). The recombinant protein Tachi2 was purified and renatured to analyze enzymatic properties. Results:The recombinant protein Tachi2 was expressed successfully in the form of inclusion body in the recombinant E.coli. The molecular weight of Tachi2 was about 44 kDa, Tachi2 was purified and renatured by a serial of treatments, the renatured protein had high chitinase activity. The optimum temperature and pH for the enzyme activity were 40℃ and 7.0 respectively. The enzyme activity was stable under 40℃ and in the pH range of 6~9, its activity was significantly reduced by 0.05 mol/L of Cu2+ and Zn2+. Conclusion:The recombinant strain Escherichia coli BL21 showed high level prokaryotic expression of the tachi2 gene. And the enzymatic properties were further defined. It will provide theoretical basis for application and further research in this chitinase.
The lipopeptide, a high performance biosurfactant, has a wide range of applications in areas such as medicine, food, cosmetics and microbial enhanced oil recovery. HPLC and ESI-MS were used to identify the composition of lipopeptide produced by B. subtilis CICC 23659 and the product of B. subtilis CICC 23659 was identified as surfactin which included lipopeptide homologues C13, C14 and C15. The index of biomass, lipopeptide yield, lipopeptide composition, emulsion index and critical micelle concentration were used to evaluate the influence of Fe2+ on fermentation of lipopeptide for B. subtilis CICC 23659. The addition of Fe2+ could not only improve the biomass of B. subtilis CICC 23659 but also substantially increase the yield of lipopeptide. When the addition of Fe2+ was 5 mmol/L, the maximum yield of biomass and lipopeptide was 3.69 g/L (6 times increased) and 234.08 mg/L (9.5 times increased), respectively. The addition of Fe2+ changed the relative content of lipopeptide homologues C13, C14 and C15 in the lipopeptide product, the relative content of lipopeptide homologues C13 and C15 decreased and lipopeptide homologues C14 increased. The relative content of lipopeptide homologues C13 was higher, the CMC value of crude surfactin was bigger and the lipopeptide homologues C14、C15 had the opposite influence.
In order to improve the safety of lentiviral vector and improve the preparation of novel hepatitis C virus (HCV) particle vaccine,the traditional three plasmid-base lentiviral system was modificated: the packaging plasmid pHR’CMV Δ R8.2 was transformed into the integration-deficient packaging plasmid pCMVΔR8.2D64E, and confirmed the integration-defective properties in vitro. The reporter gene (GFP) in transfer plasmid was replaced by the HCV non-structural gene NS3. HCV envelope plasmid with different subtypes (from three representative subtypes 1a or 1b or 2a) combined with the above two modified lentiviral plasmids were cotransfected 293FT cells. HCV integration-defective pseudotyped lentiviral particle (IDLVpp) vaccines carrying HCV NS3 were packaged. The HCV IDLVpp were identified by Western blot, and the particle structure was observed by electron microscope. The expression of transgenic HCV NS3 in Huh7 cells infected by HCV IDLVpp was also observed. So a scientific and experimental basis for novel effective HCV vaccines development and applications of HCV IDLVpp vaccines was provided.
Objective: To construct the prokaryotic expression vector of human gAd gene and obtain the no-tagged recombinant human gAd protein. Methods: Total RNA was extracted from human adipose tissue. The gAd cDNA was obtained with RT-PCR technique and subcloned into a prokaryotic exprssion vector pET-22b(+) to generate pET-22b(+)-gAd. The expression of no-tagged recombinant human gAd protein was induced by IPTG in E.coli BL21(DE3). The gAd protein produced as inclusion bodies at an elevated level. The inclusion bodies were solubilized by alkaline shock, and then refolded and purified after acetone precipitation. SDS-PAGE and Western blot were used for identification of the purified gAd. The abilities of gAd to induce the phosphorylation of AMPK in HUVECs and to protect hearts after myocardial ischemia/reperfusion in mice were used for its biological activity assay. Results: The human gAd coding sequence was correctly cloned into pET-22b(+) vector. After expression and purification, the recombinant human gAd significantly induced the phosphorylation of AMPK in HUVECs and protected hearts from myocardial ischemia/reperfusion injury. Conclusion: The no-tagged recombinant human gAd was successfully expressed and purified in prokaryotic expression system with high biological activity.
Saccharomyces cerevisiae was used as host to express recombinant human parathyroid hormone (PTH)(1-34)ab-human serum albumin (HSA) fusion protein steadily and completely. A genomic DNA fragment, encoding a fusion protein hPTH(1-34)ab-HSA, was fused to α-factor signal peptide gene by PCR, and a recombinant plasmid pYX-α-hPTH(1-34)ab-HSA was constructed. The recombinant plasmid was introduced in S. cerevisiae strain W303 to produce fusion protein. The results of Western blot and N-terminal amino acid sequencing indicated that the fusion protein hPTH(1-34)ab-HSA was expressed steadily and integrally in S.cerevisiae W303-1A. To solve the incompleteness and instability of hPTH(1-34)ab-HSA fusion protein expressed in Pichia pastoris.
Rhamnolipids are excellent biosurfactants which are widely used in biomedicine, oil recovery and environmental protection. Response surface methodology (RSM) was employed to optimize the medium for rhamnolipids production by Pseudomonas aeruginosa O-2-2. The result of the Plackett-Burman design showed that phosphate, nitrate and trace elements had significant effects on rhamnolipids production. The Box-Behnke design suggested that the optimum values of phosphate, nitrate and trace elements were 3.2g/L, 13.76g/L and 5.17ml, respectively. Rhamnolipids production reached 8.85g/L, which was in agreement with the predicted production of 8.48 g/L. Compared with the production of original level (6.24g/L), 30.8% increment had been obtained. By scaling up the fermentation to 200L fermentor with the optimal medium, the concentration of rhamonlipids reached to 70g/L, and the fermentation period was kept in 110h. When the secondary-distillation was used as a new separation process, the rhamnolipid purity was 86.6%. In general, relative content of each ingredient in this product was: mono-rhamno-mono-lipid 1.79%, mono-rhamno-di-lipid 36.83%, di-rhamno-mono-lipid 39.64%, di-rhamno-di-lipid 20.70%.
Under very high gravity (VHG) ethanol fermentation, aeration is an essential operation parameter for yeast cell to improve the performance of ethanol production. Flocculating yeast was used to convert 300 g glucose/L medium under 5 aeration schemes, including non-aeration, controlled-aeration regulated by redox potential (ORP) at -150 mV and -100 mV, constant aeration by pumping air at the rate of 0.05 vvm and 0.2 vvm. ORP was monitored under all conditions and taken as a criterion to distinct anaerobic, microaerobic and aeraobic conditions. The results showed that anaerobic fermentation produced the least ethanol (119±1.5 g/L) and left the highest glucose in 72 h. Microaerobic fermentation achieved the accurate air supply depending on the real-time cell oxygen demand, which lead to higher ethanol concentration (131±1.8和125±1.7 g/L). Aerobic fermentation brought about a quick biomass formation, and corresponding fast substrate utilization. However, too large aeration rate like 0.2 vvm caused the low yield (decreased by 12.2%) due to the huge formation of biomass and by-product such as glycerol. On the other hand, the lost of ethanol by air flow was highest under this condition. Moreover, it was observed that the flocculation quantified was promoted by increasing the air supply. In order to undertake a comprehensive evaluation for ethanol production and flocculation characteristic under different aeration schemes, a series of radar plots were illustrated based on data normalization. Constant aeration at 0.05 vvm was the preferable aeration condition thanks to its performance balance at all investigated aspects.
In recent years, increasing attention has been paid to synthetic antitumor peptides. Despite many technical hurdles, such as the half-life period of most peptides is short, peptides are advantageous because they are small, specific to tumor cells, possess low immunogenicity and are relatively easy to modify and design. Several antitumor peptides and their modified derivatives have advanced to clinical trials. Modern chemotherapy based on synthetic oligopeptides has the potential to provide an effective anti-tumor treatment for cancer patients while minimizing severe side-effects. Here, a review of the potential of novel natural anticancer peptides such as necrotic peptides, apoptotic peptides and function-blocking peptides in the context of their ability to induce tumor regression is presented. And the focus is on the therapeutic prospects of antitumor peptides and their possible application in tumor therapy.
The precise insertion of a foreign DNA molecule at genome through homologous recombination remains low efficiency in plants. Genome editing is an important tool to precisely integrate DNA molecules at a defined genomic location. Extensive efforts have been made to understand the mechanisms governing gene targeting and to establish efficient systems to achieve precise and efficient targeting. A set of genome editing techniques, engineered meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, have recently emerged that enable targeted editing of genomes in plants. The recent development of genome editing technique based on the CRISPR/Cas system demonstrate that it is efficient and specific for wide application. The rapid progress in the field of genome editing was summarized, and then the potential perspective of the genome editing technology to be used in agriculture and plant engineering was discussed.
Bioethanol as a renewable clean energy is causing widespread concern. Saccharomyces cerevisiae is the most commonly strains in the production of ethanol, but ethanol tolerance often become the most important factors that limit the Saccharomyces cerevisiae ethanol production.Improvement of ethanol tolerance of yeast cells is beneficial for ethanol production. However, traditional breeding methods have many shortcomings, such as long breeding cycle, variable mutation direction and so on. Recent research results about S. cerevisiae molecular mechanisms of tolerance to ethanol were reviewed, and the genetic engineering, metabolic engineering to improve S. cerevisiae ethanol tolerance was summarized.
The commercial therapeutic antibodies manufacturing began with construction of antibody-producing cell line, and the productivity of engineering cell line for antibody expression has reached 20~70pg/cell/day at present. Many industrial cases of various animal cell lines as host cell were summarized, accounting to their different attributes. Furthermore, the latest technology progression of cell line developments was introduced, as follows: Cell engineering technology ensured the host cell with robust growth and high productivity; The New vector element and site-directed integration technology contribute to overcoming the "position effection" by random integration. Lastly, the application of cell-sorting by flow cytometer and automatic screening device have remarkably improved the efficient and throughout of recombinant cell line screening, etc. Additionally, the phenotypic variation and instability of cell line and the technological trend in this field are also discussed.
After more than 30 years of development, monoclonal antibodies have become an important class of clinical drugs. Currently, these antibody drugs are used to treat many kinds of diseases, such as cancer, autoimmune diseases, infectious diseases and transplant rejection reactions. Ended in 2012, there are 29 kinds of therapeutic monoclonal antibody drugs through the FDA approval and on sale. And in 2011, there were more than 1000 kinds of antibody drugs undergoing clinical trials in the European Union or the United States, of which about 164 kinds of antibodies in clinical phase III trials. The safety and efficacy of therapeutic monoclonal antibody drug is largely decided by the target. Some monoclonal antibody drugs in research have the same targets with the antibodies on sale, and some have the unique targets. At the same time, more and more new targets are being found. There are four kinds of the most popular targets of research: tumor necrosis factor-α, cluster of differentiation 20, epidermal growth factor receptor and vascular endothelial growth factor. Detailed review which focuses on the different targets of the monoclonal antibody drugs, whether in research or on sale, is expected to provide valuable references for the monoclonal antibody drug development trend. The status quo of monoclonal antibody drug in our country was discussed, and the development strategies in the future were proposed.
For further investigating the enzymatic characterizations, evaluating the allergenicity and feed safety of the phytase generated from transgenic phytase corn, A more effective purification system using immuno-affinity chromatography was established. In order to increase the loading capacity and maintain the specificity, four monoclonal antibodies recognizing different epitopes of phytase were combined together. The crude extraction could be concentrated by 80% ammonium sulfate, and then subjected to dialysis and immune-affinity chromatography. The purification results showed that the obtained protein has an exclusive specific band in SDS-PAGE and 470.99 U/mg in specific activity. Furthermore, comparing to ion exchange chromatography, the immune-affinity based method was more stable and effective, the resultant product can meet the demands for various analysis.
IL-4 gene transformation mediated by pollen-tube pathway method of Chinese cabbage for molecular detection of descendants using PPT resistance screening, PCR amplification, PCR-Southern hybrid, ELISA, Western hybrid methods, combined with statistical analysis study IL-4 inheritance of genes in transgenic Chinese cabbage in future generations. Results indicated that: (1) PPT resistance in transgenic Chinese cabbage, PPT resistance positive rates of T4 generation was 33.43%, the positive rate of PCR amplification of IL-4 gene was 1.39%. IL-4 separation plant does not meet Mendel’s law of segregation. (2) ELISA test on 14 ones of PCR-positive strains of 5 strains in the T4 generation, 7 samples of which are positive, IL-4 expression was about 326.87~1233.13 ng/g FW, the ones of IL-4 expression within the same strains significance of difference. (3) Western confirmed IL-4 in transgenic Chinese cabbage hybrid offspring in the regular expression. It indicated that IL-4 gene has been incorporated into the Chinese cabbage in the genome with PCR-southern hybrid, and that gene still exists in transgenic plants’ future generations.
In recent years, the communication of genetically modified organism (GMO) has been impeded in China. While science community and government departments try to overcome the barrier by enhancing science popularization efforts, it cannot be neglected that behind the resistance to GMO, there are hidden cultural factors. It identifies three cultural factors that are involved: the holistic view of nature that pursues "harmony between man and nature", anti-intellectualism, and the psychology of the indulgence with traditional agriculture. All three factors are related to traditional Chinese thinking mode of intuitive extrapolation. After tracing historical concepts of heaven and man, it find that the communication of GMO faces the conflict between constructivism natural view and holistic natural view. In fact, the holistic view of nature has been misinterpreted amidst the pervading anti-intellectualism against high technologies to which GMO belongs. Because of the holistic view of nature and "anti-intellectualism", the indulgence with traditional agriculture is very popular in modern China. But it is completely impossible to back to traditional agriculture by questioning GM technologies. In the situation, if we want to overcome the barriers of GMO communication, we need to reconstruct a friendly intellectual environment. While we need to promote GMO by economic benefits, it is also necessary to integrate the technological development into the generally accepted view of nature.
