Objective: To study the ability of Sus Mx1 and Bos Mx1 to inhibit the replication of PRV. Methods: Sus scrofa Mx1 gene and Bos taurus Mx1 gene were cloned from IBRS-2 and MDBK cells to pcDNA3.1/myc-His (-) B vector respectively, and transfected into PK-15 cells with Lipofectamine TM2000 to detect whether they can express successfully in cells by RT-PCR and Western blot respectively. And then the cytotoxicity to cells WST-1 and the ability of Sus Mx1 and Bos Mx1 to inhibit the replication of PRV were checked. The PK-15 cells were infected with 20TCID50, 100TCID50PRV after transfection 24h. Subsequently, samples were collected after infection 24h, 48h, 72h, and total viral copies were tested by extracting RNAs of frozen and thawed samples repeatedly. Meantime, the cytopathic effect after infecting 100TCID50PRV in 72h was observed. Results: both recombinant eukaryotic plasmids were constructed and expressed successfully. They were able to reduce the amount of RNA of PRV significantly by real time PCR (P <0.01). Conclusion: Sus scrofa Mx1 and Bos taurus Mx1 can inhibit the replication of PRV in PK-15.
Objective:The aim is to probe the histone epigenetic information in encoded region of SAF gene after inflammatory cells model from human THP-1 monocytic leukemia cell line are constructed, which is expected to provide basis for the further research about the mechanism of transcriptional regulation of SAF gene. Methods:The LPS-stimulated or unstimulated THP-1 cells were colletcted and fixed by 1% formaldehyde for 10 minutes at room temperature. After that, nuclei were isolated. Chromatin was sheared by sonication, and samples were precleared for 1 hour at 4℃ with 50% protein A/G. Chromatin containing target proteins were precipitated overnight at 4℃ with 5μg antibodies or isotype-matched control IgG. Input and immunoprecipitated chromatin were incubated at 65℃ overnight to reverse crosslinks. After proteinase K digestion, DNA was extracted and purified by phenol-chloroform. The DNA was used as templates for PCR and qPCR to get the histone epigenetic signals. Antibodies included rabbit polyclonal to Histone H3, rabbit polyclonal to Histone H3 (tri methyl K36), rabbit polyclonal to Histone H3 (mono methyl K9) and rabbit polyclonal to RNA polymerase II CTD repeat YSPTSPS (phospho S5). Results:During inflammation, the total histone H3 signals in the 4A and 4B exon of SAF gene were significantly reduced. However, histone H3K36me3 didn't obviously diminish. The level of ser5 phosphorylation of the polymerase C-terminal domain (CTD) heptamer repeat was reduced in the 4A exon of SAF gene in inflammatory stimulus conditions. Expression of H3K9me1 decreased in the 4A exon of SAF gene. During inflammation, the level of H3K9me1 decreased and the enrichment of H3K36me3 increased in the single nucleosome in 4A exon of SAF gene. At the same time, the level of ser5 phosphorylation of the polymerase C-terminal domain (CTD) heptamer repeat decreased. Conclusion:The low level of histone H3 enrichment in SAF coding regions support the idea that histone H3 in SAF coding region could be evicted by LPS induction so as to pave the way for RNA polymerase II elongation. All of these effects might increase the elongation rate of the RNA polymerase II.
Objective: Using a constructed enhancer sequence detecting vector, screening sequences that could enhanced heterologous protein expression in Escherichia coli, and preliminary identified its functional domain by deletion mutants. Methods: Chloramphenicol acetyltransferase (CAT) gene and truncated human papillomavirus (HPV) major capsid protein gene L1 (named L11) were fusion expressed as a report gene; to screen enhancer-like sequence from the collected samples, and detected the enhanced activity by protein expression. The functional regions were identified by construction of deletion mutants. Results: One enhancer-like sequence was successfully screened, which enhanced 11-folds of chloramphenicol resistance; fusion protein expression level increased 2.26-folds; the functional areas are located in the 1~265bp. Conclusion: An enhancer-like sequence were successfully screened, which can improve the expression of heterologous gene in Escherichia coli.
The effect of adding intermediate metabolites, thiamine, magnesium on cell growth and L-phenylalanine production were investigated. The yield of L-phenylalanine (L-phe) improved when 1g/L sodium citrate, 1g/L α-ketoglutarate, 150mg/L thiamine or 3g/L magnesium was added. According to the metabolic network of E.coli YP1617, the reason was acquired by metabolic flux analysis. The addition of them can adjust the metabolic flux distribution of G6Pand PEPnode, which provide erythrose-4-phosphate (E4P), phosphoenolpyruvate (PEP) and NADPH for L-Phe production. In the optimal fed-batch fermentation, glucose consumption, cell and L-Phe concentrations, the yield of L-Phe was 24.49%, 23.50%, 62.87% and 30.88% higher than the control, respectively. Moreover, acetate production decreased 56.51%.
Objectives: To prepare photosensitizers-loaded Liquid Crystalline Nanoparticles, and study its cytotoxicity and photodynamic therapy (PDT) effects on tumor cells. Methods: Photosan Cubosomes were prepared with Glyceryl Monooleate (GMO), Poloxamer 407 (P407) and Photosan. Then the cubosomes were observed on the dynamic light scattering and scanning microscope. The photodymanic therapy effects and cytotoxicity of Photosan Cubosomes to L02 cells and HepG2 cells were studied by MTS assay. Results: Photosan Cubosomes were prepared successfully. The results showed that the blank cubosomes, Photosan and Photosan Cubosomes did not display any cellular toxicity against L-02 cells and HepG2 cells without laser irradiating; however, Liquid Crystalline Photosan mediated PDT by 5J/cm2 laser produced obvious inhibitory effects on HepG2 cells [inhibited (77.9±2.06)%] while showed slight inhibitory effects on L-02 cells [inhibited (32.9±1.19)%]. Moreover, Liquid Crystalline PhotosanPDT showed less damage on L-02 cells but stronger inhibitory effects on HepG2 than Photosan. Conclusions: New photosensitizer loaded Photosan Cubic Liquid Crystalline had higher security and stronger PDT effects on cancer cells and showed better characteristics than Photosan. It provided a novel method for cancer treatment by PDT.
Effects of different dissolved oxygen (DO) on the production rate of γ-PGA, at the 8, 32 and 56 hours were studied, using the 5L fermentor to synthesis γ-PGA with Bacillus licheniformis. Results indicated that neither high nor low DO value could efficiently accumulate γ-PGA based on the analysis of relevant data. The research also concerned about five kinds of enzyme activity which were key enzymes of metabolic flux of biosynthesis for γ-PGA. The enzymes includes hexokinase, glucose-6-phosphate dehydrogenase(G-6-Dp), pyruvate dehydrogenase(PDH), isocitrate dehydrogenase(ICDH)and glutamate dehydrogenase(GDH). And G-6-D Pcould depress the synthesis of γ-PGA. High activity of Hexokinase and GDH would promote the production of γ-PGA. PDH and ICDH might advance the production rate of cell. The higher activity of PDH and ICDH might be not conductive to the fermentation. Moreover, the extracellular metabolites profiles of fermentation under three different DO values were acquired and the metabolic flux redistribution of pathways related to γ-PGA biosynthesis was calculated based on the collected data. As a result, the metabolic flux favored to distribute toward glycolytic pathway at DO 30%, in which the ingestion rate of extracellular glutamic acid was higher and the subsequent γ-PGA biosynthesis was enhanced.
Corynebacterium crenatum SYPA5-5 is an L-arginine high-producing industrial strain of mutation breeding. The role of citrate synthase in L-arginine biosynthesis was investigated by overexpressing the citrate synthase (prpC2) gene in C. crenatum SYPA5-5. The resultant 5.37-fold increase in intracellular citrate synthase activity was achieved in the prpC2-overexpressing strain C. crenatum SYPA5-5/pDXW-10-prpC2 . The recombinant strain enhanced the L-arginine yield to 44.7g/L by about 23.1% in 5L fermenter, as compared to the control, with affecting glucose depletion rate slightly. While the L-arginine yield increased in the prpC2-overexpressing strain, the L-lysine yield, the most primary by-product formation during L-arginine fermentation, decreased to 1.21g/L from the original concentration 5.96g/L, correlating with an increase in the tricarboxylic acid cycle (TCA) intermediates (citrate and isocitrate) and an increase in the activity of citrate synthase.
In the present study, a recombinant strain that is able to express 4-hydroxyphenylacetate-3-hydroxylase (HHA). Hydroxytyrosol can be produced in this recombinant strain using tyrosol as the substrate. HHA gene was amplified using E. coli BL21 (DE3) as the template and then connected to the expression vector pET-28a. Obtained recombinant expression vector pET28a-HHA was transformed into competent cell BL21 (DE3). The suitable amount of tyrosol was used as raw materials to prepare hydroxytyrosol in the recombinant bacteria. Thin layer chromatography and gas chromatography were used respectively to detect the production level of hydroxytyrosol in supernatant. After the IPTG induced, two protein bands (58.8kDa and 18.5kDa) were detected by SDS-PAGE analysis. Hydroxyltyrosol was also detected by thin layer chromatography and gas chromatography. The results showed that the expression recombinant strain HHA was successfully conducted and this strain can effectively convert tyrosol to hydroxytyrosol.
PACAP 27-derived peptide RP2 which can promote promote the epithelial cell proliferation is efficiently producted by genetic engineering and be used to study its effects on corneal epithelial cells,which can provide an experimental basis for the treatment for corneal injury. Recombinant peptide RP2 was expressed by genetic recombinant technology after the prokaryotic expression, purification by Chitin-Beads column, and producted,analyzed、identification by HPLC, SDS-PAGE and mass spectrometry, then its effects on mouse corneal proliferation were studied. Experimental results showed that the molecular weight of RP2 is 3.3 kDa. and the purity is 96%. Mouse corneal epithelial cells were respectively treated with recombinant peptide RP2、 PACAP 27 and PBS for 24 h and 48 h,The proliferation rate of mouse corneal epithelial cells treated with recombinant peptide RP2,is (49.6±3.1)% and(93.0±1.7)%, the PACAP 27 group is (29.0±2.4)% and (78.8±2.6)%,while the PBS group is (20.2±1.1)% and (40.9±3.3)%. Therefore, the producted recombinant peptide RP2 can significantly stimulate corneal epithelial cell proliferation, so it will likely to become a new candidate drug for corneal injury.
Objective: Construction and screening the recombinant cell line that is highly expressed of original fully-human anti-human IgE monoclonal antibodies. Methods: The screened originally fully-human anti-human IgE single-chain antibody (scFv) by the ribosome display technology is re-constructed to full length IgG1κ antibody. Construct the recombinant eukaryotic expression vectors and electric-transfect CHO-S cells. Select multiple highly expressed clones for the 40ml flask batch culture by the Dot-blot method. Then according to the growth characteristics of the cells and the antibody expression quantity select highly expressed clones for the 40ml flask and 3L flask fed-batch culture study. The antibody biological activity before and after the reconstruction of the candidate cell lines are compared. Results: Four kinds of eukaryotic expression plasmids-pMH3-H, pMH3-L, pCApuro-H, pCApuro-L are successfully constructed and co-transfected CHO-S cells. Complete four times of electric transfection and eight rounds cell clones screens and obtain two highly expressed candidate clones-Mab1# and Mab2# whose antibody expression quantity reaches 470mg/L and 499mg/L in 3L flask fed-batch culture. The affinity result by the Bio-Layer Interferometry (BLI) technology shows that the affinity of two monoclonal antibody-Mab1# and Mab2# reaches nM (10-9) level and are comparable with the only existing anti-human IgE monoclonal antibody drug Omalizumab on the market. Compare the activity of Mab1# strain full-length antibody with its parent single-chain antibody by the surface plasmon resonance (SPR). The result shows the level of EC50 which inhibits the binding of hIgE and FcεRI in Mab1# is 3nM, with affinity increases 4.3 times, the neutral activity of EC50 increases 23.7 times and the neutral activity of EC90 increases 41.3 times. Conclusion: The single-chain antibody (about 25kDa) of originally expressed fully-human anti-human IgE is successfully transformed to full length antibody (about 150kDa) whose affinity and neutral activity level are significantly increased and obtain two candidate cell lines.
In order to improve the thermotolerance of Saccharomyces cerevisiae and decrease the energy consumption cost for controlling temperature in ethanol fermentation process, 5heat shock protein (HSP) devices aredesigned and constructed, then transformed into S. cerevisiae through mining heat shock protein genes in Thermus thermophiles HB8.All the HSP devices could transcript normally at 42℃. The cell growth of the engineered yeast with heat-resistant device FBA1p-groes-SLM5tis improved 29.2% than the control under the graduallyenhanced high temperatureincubation.And thecellgrowth of S. c-GroES cultured at graduallyenhanced high temperature is nearly identical to the controlincubated at 30℃. Therefore, the heat-resistant deviceFBA1p-groes-SLM5t which endows yeast with better thermotolerant property is screened. Then, the thermotolerance of S. c-GroES is further verified through constant high temperature incubation. The engineered strain S. c-GroES shows better cell growth than the control by measurement of OD660 and cell viability under 37℃ (heat shock temperature)and 42 ℃ (heat lethal temperature).For instance, the cell viability of S. c-GroESdisplays3 times higher than the control at 42 ℃,48h. Moreover, the cell morphology of S. c-GroESis normal after heat shocked which indicates that the metabolism of S. c-GroESis not damaged. The above results of high temperature incubationshow that the engineered S. cerevisiaewith heat-resistant device FBA1p-groes-SLM5tcould adapt to various high temperature fermentation type. Meanwhile, the S. cerevisiae with heat-resistant device FBA1p-groes-SLM5tisendowed with anti-oxidation. The ROS level of S. c-GroES is 36.7% lower than the control at 42 ℃. Additionally, after treated with H2O2 of final concentration of 2mM,the cell viability of S. c-GroES shows1.62 times higher than the control.These results indicate that heat-resistant device could not only improve the thermotolerance of S. cerevisiae but also help cell defense oxidative stress. Under the 40 ℃ethanol fermentation,the cell growth of S. c-GroES and the control isworse than the above graduallyenhanced high temperature incubation and constant high temperature incubation owing to the anaerobic and heat shock cultural condition. However, the cell growth of S. c-GroES is better than the control cultured at 40℃ and nearly the same to the control cultured at 30℃. Under the batch fermentation, the ethanol yield of the control cultured at 30℃ is lower than the control cultured at 40℃, 60 h in the YPD medium containing 40g/L glucose, which is the same to the previous research, while its OD660 shows excellent than other strains. That is because nutrition is used by the control cultured at 30 ℃ to cell growth and ethanol can be the carbon resource when lack of glucose. However, the ethanol yield of S. c-GroESisimproved by 25% and 13.8% than the control cultured at 30 ℃ and 40℃, respectively. Meanwhile,the ethanol yield of single cell of S. c-GroESwas improved than the control trough calculating owing to the protection of heat shocked cells by heat-resistant device FBA1p-groes-SLM5t. These results show that the thermotolerance and ethanol synthesis efficiency of S. cerevisiae could greatly be improved byintroducing heat shock protein from thermophilus. This method provides a platform to increase production efficiency and reduce energy consumption substantially. Moreover, properties of other strains can be also improved by this valuable method.
Refolding process of proteins is a hot spot in the area of recombinant protein drug. Dilution or dialysis requires a large amount of refolding buffer and results in a low yield. Liquid chromatographic refolding has successfully proven its capability to achieve a high yield at high protein concentrations and it is easy for productive scaling up. Partial purification of recombinant protein could also be achieved simultaneously. The recent development of liquid chromatographic refolding process is reviewed in this paper. The principles, advantages and disadvantages are discussed with respect to various chromatographic refolding processes, including size exclusion chromatography, ion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography. The refolding process evaluation procedures are summarized, including characterization of refolding proteins, recovery yield and industrial viability.
Transgenic pigs are animals that have been genetically altered by inserting a transgene (natural or artificial gene) into their genomes to express a new trait, and intend to be stable in the progenies. Therefore, it has most important application in the animal genetics &breeding, drug evaluation, disease animal model, animal bioreactor and xenograft donor, etc. In this paper, the research progress of transgenic pigs, technical methods and application status were briefly reviewed.
Amino acids, which play the irreplaceable role in maintaining the body's normal physiology as a kind of nutrient substances, are usually used as additives in food, pharmaceuticals, and cosmetics. Production of amino acids mainly relies on microbial fermentation. However, high yield amino acid strain by selection hinders the large-scale industrial production. Application of metabolic engineering has become a hot spot of research in microbial metabolism network and genetically modification for screening high yield amino acid strain with the development of metabolic engineering strategy and technology in molecular breeding. The characteristic of C. glutamicum metabolism network and metabolic engineering strategy in molecular breeding of C. glutamicum-producing amino acids are introduced.
One hundred and eight biomedical parks in China have been investigated and development statuses of them were obtained. After investigating, some biomedical parks possess rational layout, which can promote local economic development. Three biomedical industrial clusters have been established. Biomedical parks have been an important basis of biomedical industry. At last, five suggestions on the development of biomedical parks in china were given.
