Objective:To construct recombinant plasmids and express chimeric proteins of neutralizing epitopes of Human Enterovirus 71 and Norovirus P domain. Methods:Nucleotide sequences of the neutralizing epitopes of EV71 were designed and optimized according to their reported sequences of amino acids and the codon bias usage of E. coli for expression. These nucleotide fragments were cloned into the plasmid vector containing norovirus P domain and GST tag via the loop2 of P domain, which was confirmed by sequencing. The constructs were transformed into the bacteria BL21(DE3) and induced with IPTG for protein expression. The GST-fused proteins were purified via GST affinity beads. Immunoblot analysis was used to detect the GST tag and evaluate the immunogenicity of chimeric proteins. Results:Seven recombinant plasmids were constructed successfully as confirmed by sequencing. The expressed 7 chimeric proteins were expressed and soluble in E. coli, which was determined by SDS-PAGE and Commassie blue staining. Immunoblot analysis via the anti-GST antibody revealed the expression of GST-fused proteins. Further analysis of immunogenicity showed that all 7 chimeric proteins could react with anti-NoV P domain antibody. Five out of 7 chimeric proteins could recognize anti-EV71 polyclonal antibody except two epitopes of SP55 and SP28 chimeric proteins. Conclusion:The chimeric proteins of NoV P domain and EV71 neutralizing epitopes were successfully expressed and antigenic, which lays a solid foundation for developing the bivalent subunit vaccine and the detection kit for NoV and EV71 viruses.
Aim:To investigate the effect of folate chitosan Prdx6 shRNA nanoparticles on the growth of gastric cancer cells. Methods:Prepared the targeting folate conjugated chitosan Prdx6 shRNA nanoparticles and observed nanoparticles morphology by atomic force microscope and particle size of nanoparticles by laser particle size analyzer;observed the folic acid chitosan Prdx6 shRNA nanoparticles transfection efficiency by nverted fluorescence microscope;detected gastric cancer cell prdx6 protein expression changes using Western blotting; detected gastric cancer cells survival rate by the methods of CCK-8. Results:Preparation of folate chitosan shRNA Prdx6 nanoparticles. Efficiency of folate-chitosan Prdx6 shRNA nanoparticles transfecting gastric cancer cell was significantly higher than that of non targeting nanoparticles by fluorescence microscope; Prdx6 protein expression of gastric cancer cells transfected by folate-chitosan Prdx6 shRNA nanoparticles was significantly lower than that of non targeting nanoparticles. Compared with the control group, folic acid chitosan shRNA Prdx6 nanoparticles could significantly inhibit the proliferation of gastric cancer cells (P<0.01). Conclusion:Folate chitosan shRNA Prdx6 nanoparticles can be efficiently transfected into gastric cancer cells. The growth of gastric cancer cells was significantly inhibited after transfection with folate chitosan shRNA Prdx6 nanoparticles.
RC28 is a novel antiviral protein extracted from the mushroom Rozites caperata. In our previous studies, RC28 cDNA were obtained from Rozites caperata total RNA by 3'-RACE and inserted into pMD18-T plasmid by TA cloning. RC28 cDNA fragments were sub-cloned into E. coli expression vector pET28a(+) and Pichia yeast expression vector pPIC9K separately to form recombinant RC28 expression vectors with N'-terminal histidine tags. The two kinds of RC28 expression vectors were transformed into their respective host strains and positive clones were selected to construct stable expression systems. SDA-PAGE and Western blot were performed to analyze RC28 expression after induction. RC28 protein were purified and collected by using Ni-NTA columns and detection of the antiviral activity of the recombinant protein achieved by MTT assay. In our experiment, soluble recombined RC28 proteins were prepared both in E. coli and Pichica yeast and the yields of purified protein from these two expression systems were about 3.5mg/L and 0.2mg/L, respectively. However, the recombined RC28 protein expressed from E. coli system showed poor antiviral activity while the recombined RC28 protein from Pichica yeast system showed much higher antiviral activity which was close to that of the natural protein.
The neverland from the Drosophila melanogaster play essential roles in cholesterol dehydrogenation. To clone the neverland from the Drosophila melanogaster cDNA library and construct the eukaryote expression plasmid pIEx-6-nvd and pXY212-nvd to expressing the recombinant NVD in S2 cell and S. cerevisiae W303-1A for further analyzing its bioactivity. Results of Western blot indicates the recombinant protein expressed by S2 cell and S. cerevisiae W303-1A can specifically combine with 6×His antibody. Results of HPLC shows the cholestrol can be converted to 7-dehydrocholesterol by the recombinant S2 cell, however, the purified NVD and the recombinant S. cerevisiae W303-1A can not conversion cholesterol to 7-dehydrocholesterol, even the mixed recombinant S. cerevisiae W303-1A cell lysate and S2 cell lysate, neither. As the above result shows, the NVD need some chaperonins to complete the electron-transfer reactions when mediates the cholesterol dehydrogenation reaction.
The effect of different levels of elevated dissolved CO2 availability(CO2 stress) was investigated in batch and chemostat cultures of Aspergillus niger by gassing with CO2-enriched air. Under batch cultures, higher elevated dissolved CO2 availability in culture medium resulted in a more significant decrease in specific growth rate, whereas it appeared to have a positive effect on glucoamylase synthesis. Under chemostat cultures with low dilution rate (D1=0.05/h), high levels of dissolved CO2 led to no reduction of specific growth rate, but an increase of glucoamylase production. Whereas at high dilution rate (D2=0.08/h), both substrate uptake and cell grow were more severely inhibited by more elevated dissolved CO2. The effect of elevated dissolved CO2 availability on the fermentation process depends on CO2 levels as well as dilution rate. Therefore, the influence of carbon dioxide on cell growth and glucoamylase production dependent on CO2 levels, cultivation mode and specific metabolic activity, which would be helpful for the design of specific growth rate in scale-up of glucoamylase fermentation by Aspergillus niger.
Pichia pastoris is one of the most widely used eukaryotic expression systems. P. pastoris can express heterologous proteins with methanol as the sole carbon source. However, the expression can be repressed by glycerol. As reported recently, the glycerol transporter played a part not only in transporting glycerol,but also in the regulation between glycerol and methanol metabolism. Objective:A mutant P. pastoris X-33 ΔGT2 (PAS_chr3_1076) was constructed, and the glycerol de-repression effects was found. Methods:The X-EGFP and ΔGT2-EGFP cells were constructed respectively based X-33 wild-type strain(WT)and ΔGT2 cells, in which the EGFP was driven by PAOX1. The biomass and expression levels of AOX1 and EGFP were tested in different carbon resources (glycerol, methanol, glycerol plus methanol) mediums. The extracellular glycerol contents were tested. Results:The results showed that, for each OD strain, the enzyme activity of AOX1 of ΔGT2-EGFP was 35% higher than that of X-EGFP, and the fluorescence of ΔGT2-EGFP was 70% higher than that of x-EGFP. The x-EGFP harvested more biomass than ΔGT2-EGFP when glycerol as the sole carbon source resulting in less glycerol contents in the culture supernatant. Conclusion:GT2 involved in uptaking and metabolism of the glycerol, and the absence of GT2 could release the repression of glycerol on AOX1, which indicated that the glycerol transporter could be related to the transcription of PAOX1. The more efficient expression system of yeast is expected to be constructed based on these results.
Agarase was fermented with Vibrio sp. ZC-1, and separated through hollow fiber concentration, ammonium sulfate precipitation and DEAE-anion exchange chromatography. A pure AgaZC-1 detected by SDS-PAGE was obtained. Its relative molecular mass and specific activity was about 45kDa and 114.613U/mg, respectively. Furthermore, the enzymatic properties of AgaZC-1 were studied. The results showed that the optimum pH was 7. It was stable in the range of 5.0 to 9.0 and could maintain more than 80% of its relative enzyme activity after incubation for 1 hour. The optimal reaction temperature was 50℃ and 60% of enzyme activity was remained after incubation for 1 hour under 45℃. At high concentrations (5mmol/L), Fe3+, Cu2+, Sn2+ and Zn2+ could completely inhibit the enzyme activity. While at low concentrations (1mmol/L), Cu2+, Ba2+, Na+, Zn2+, Ag+, Sr3+ and K+ had obvious inhibition on enzyme activity. The Km and Vmax of AgaZC-1 was 0.538mg/ml and 6.33μmol/(L·min), respectively. It was highly specific for agar and the hydrolysates were neoagarotetraose and neoagarohexaose analyzed by TLC.
α-hemolysin (αHL) is a channel-forming toxin released by Staphylococcus aureus, which can form a transmembrane heptamers nanopore on eukaryotic cell membranes and other lipid bilayers. The αHL nanopore has been popularly used as a single molecule biosensor. In the present study, αHL was expressed in the host strain BL21(DE3)pLysS and purified by using molecular weight cut-off of ultrafiltration membranes. αHL heptameric nanopore was formed on rabbit red blood cell memberanes and characterized by using patch clamp technique and single channel current recording. αHL heptameric nanopore showed a stable structure and good ion permeability on lipid bilayer. It can be used as a stable single-molecule detection device. These results make a useful work for the preparation of α-hemolysin heptameric nanopore and the further exploring in the filed of single-molecule detection.
Gene targeting technology mediated by homologous recombination is a powerful tool for gene function analyze, because of its high accuracy and the mutation we introduced can be predicted.While in eukaryotes, the ratio of homologous recombination is too low to do further screening and identification. For this reason, establishing a high efficiency and widely used screening strategy becomes important. A new screening strategy which can dramatically improve the homologous recombination efficiency based on visual "double fluorescence" screening was established. The green fluorescent protein and neomycin gene were used as positive selection markers, and red fluorescent protein gene for negative selection marker. After screening by G418, the cell clones which just expressing the green fluorescent protein gene were selected and detected by transboundary PCR. The new strategy was used to detecting the gene targeting of pig MSTN gene in pig cells combined with CRISPR/Cas9 technology. In the two target sites T1 and T2, the targeting efficiency screening by "double establishing" reached up to 80.5% and 86.7%, which the was dramatically improved. This visual "double fluorescent" screening strategy established will become a useful tool for animal gene editing in the future.
A process of one-pot enzymatic synthesis of L-2-aminobutyric acid was studied. The ilva gene encoding L-threonine deaminase (L-TD) from E. coli, leudh gene encoding leucine dehydrogenase (LeuDH) from Geobacillus stearothermophilus and kred gene encoding ketoreductase (KRED) from Novosphingobium aromaticivorans were cloned into pET-21a(+), then introduced into E. coli BL21(DE3) respectively. All of the three enzymes were overexpressed in soluble forms. L-2-aminobutyric acid was efficiently synthesized with low-cost L-threonine as substrate by the recombinant L-TD and LeuDH coupling with a NADH regeneration system based on KRED. Effects of pH and the concentration of L-threonine and isopropanol on the yield of L-2-aminobutyric acid were investigated. After 20h at the optimal pH 7.5~8.0, the yield of L-2-aminobutyric acid could achieve 99%, with a concentration of 43g/L, when using 50g/L L-threonine, 5% isopropanol, 0.5g/L NAD+, 0.6g/L L-TD, 2g/L LeuDH and 2g/L KRED. A new thought for the production of L-2-aminobutyric acid was provided.
Trehalose is a ubiquitous non-reducing disaccharide in nature, and is an excellent natural drying agent and preservative. Trehalose synthase capable of catalyzing the maltose directly into trehalose, and is the preferred production of trehalose. To obtain trehalose synthase having good catalytic surface, which is displayed in a highly efficient and stable surface of Bacillus subtilis, at the same experiments were selected enhanced green fluorescent protein (EGFP) and trehalose synthase (Tres) as a model protein, to come from Bacillus subtilis spore coat protein CotC as Bacillus subtilis anchored proteins displayed on the surface. Flow cytometry analysis of the situation in the spore surface display EGFP, the results showed that the capsid protein of Bacillus CotC can EGFP fixed spore surface. Then replace fluorescent protein gene egfp and trehalose synthase gene tres. The recombinant strains was hang up using pH 7.5 buffer suspension and the concentration of substrate for 30% of the maltose in 50℃ water bath roling 2h. Reaction products were analyzed by HPLC and the enzymatic activity can be detected, the enzyme activity of trehalose by calculating reached 252U/ml. This suggests that CotC is associated with the outer part of the coat. CotC can therefore be used as a molecular vehicle for spore surface display of exogenous proteins.
Ceramic Hydroxyapatite (CHT) has been widely used in the manufacturing of a wide variety of monoclonal antibodies (mAbs), vaccines, and other protein therapeutics. It is an effective tool for the removal of residual host cell proteins, nucleic acids, viruses, endotoxins, and product-related contaminants such as aggregates and degradation fragments. However, damage of the CHT media and increased column back pressure are often observed at production scale-up. A transient pH excursion during elution with increased salt concentration leads to Ca2+ depletion from the CHT column. In the present study,the effect of surface neutralization system (SNS) on the life cycles of CHT media is demonstrated. Following post-loading wash, a buffer with slightly elevated pH is applied onto the column so as to prevent significant pH drop during target protein elution. The level of Ca2+ depletion in response to pH and salt concentration of the SNS buffer have been investigated, and the optimal buffer composition for maintaining the pH of column effluent near neutral been identified. This newly developed protocol has been validated for the polish purification of mAb WLB304. The dynamic binding capacity of WLB304, impurity clearance efficiency, leached Ca2+ level, pH excursion and column back pressure during purification process were closely monitored. Data indicate that the implementation of SNS can significantly reduce Ca2+ depletion and effectively extend the life time of a CHT column, without compromising its chromatography performance.
The metabolome refers to a set of small molecular metabolites. The main purposes of metabolism are the conversion of fuel to energy to run cellular processes. The metabolome can reflect the process of metabolism, As a result, a cell's metabolome can serve as an excellent probe of its function. Cell function abnormality leads to the abnormal changes of metabolom, so metabolome can be assessment standards of the function of cell. The process of extraction and identification of metabolites and the analysis of metabolic data were briefly reviewed, including the pre-processing and network constructed of metabolome datasets.
Terpenoids, a family of compounds with a wide range of diversity and structural complexity, play important roles in the fields of pharmaceutics and energy. The terpenoid compounds are mainly extracted from plants or chemically synthesized, which are both cost-effectiveness. On contrary, the economic and efficient way of microbial synthesis is more promising. However, due to the complexity of theterpenoidsynthesis pathway and the difficult manipulation of the host metabolic networks, most terpenoid compounds have notbiologically synthesized inan agreeable yield. Here,the strategies for improving the yield of microbial terpenoids production.
In recent years, the breaking-through progress has been made in the research of tumor immunotherapy, and the explosive growth has been obtained in the R&D of related technology and product. Great investment has been made by many Biotech Corp and pharmaceutical companies, the international competition is becoming increasingly fierce. The emerging question confronting China is how to seize the opportunities and stand out in the international competition. An overall understanding of the development situation of the field through a comprehensive analysis of the main products, technology and future research direction of the dendritic cell-based vaccine and adoptive cell transfer therapy (ACT) featured are gtiven by CAR-T and TCR-T and anti-tumor immune checkpoint inhibitors targeted to PD-1/PD-L1, and reference for R&D work through the analysis of the technical features and development trend of immunotherapy represented by the above mentioned three approaches are provided. And for the R&D situation and the existing problems in this field, suggestions such as strengthening the overall planning, focusing on basic research, emphasizing the patent protection, enhancing the scientific supervision, are put forward to provide a reference on how to make a better development of China's tumor immune therapy.
Vaccine is the most effective weapon for the prevention of the disease, which is the public medical product that everyone can't be separated from and has made great contributions to the health of all mankind. There are about 68 kinds of vaccines in the global market at present and can prevent 34 kinds of diseases. Global vaccine markets maintain steady growth. Vaccine sales increased year by year and the market sales were 29.6 billion dollars in 2015. The top 5 companies, Pfizer, Merck, Sanofi, GSK, Novartis, occupied more than 85% of the market share. A variety of vaccine products had good sales performance. China vaccine markets also grow consistently with a market value of 18.37 billion in 2015. The issued amount of vaccine was 704 million bottles in 2015. The types of vaccine productions are increased and the ability of vaccine research and development is gradually strengthened. The vaccine markets in emerging countries are gradually developing and India performs excellently. The internationalization of Chinese vaccine industry is not as good as India, but it also made a lot of progress. Chinese vaccine industry entering the international market has become an irreversible trend in the future.
21st century is the century of biotechnology, and speeding up the development of transgenic technology has become the world's strategies to enhance the core competitiveness of countries all over the world. The global status of the development, planting and application of genetically modified (GM) crops are sammarized. Agricultural biotechnology has entered into a new stage of global distribution, countries have planned the layout with a view to gain the upper hand in the technology and market. After 20 years of development, China has made great progress in the field of biotechnology research and development, large-scale planting of insect-resistant cotton results a good economic and social benefits. But the commercialization of other genetically modified crops is slow due to the influence of the negative public opinion environment and other factors. China is a populous country, the contradiction between grain supply and demand is outstanding, vigorous development of the biotechnology industry is the inevitable choice of China's future. On the one hand, China should insist on the road of independent innovation on the basis of ensuring safety, on the other hand, the government should establish a timely information disclosure mechanism, create a favorable public opinion environment, make a scientific decision from the people's livelihood to promoter the healthy and orderly development of biotechnology industry.
