LYC5 is a kind of c type human lysozyme protein. To optimized and designed the LYC5 gene based on the coding sequence of mRNA on GenBank and the preference of Pichia yeast codon, then cloned the encoding gene into the expression plasmid pPIC9K,and the resulting recombinant plasmids were named as pPIC9K- LYC5. Then they were linearized and electro-transformed into the Pichia pastorios GS115 express bacterium,the multiple copies of transformants were screened by Minimal Dextrose medium (MD),Minimal Methanol medium (MM),the antibiotic of G418 and PCR identification. The transformants were screened through culturing and induction under methanol in baffled flask,the results of SDS-PAGE indicated that the LYC5 target proteins were secreted into the supernatant, about 15 kDa in sizes. These proteins were identified as LYC5 lysozymes by LTQ orbitrap elite MS analysis,with expression levels being about 20 mg/L. The expression products had obvious antibacterial activity on Micrococcus lysodeikticus,with the activity was about 40 000 U/mg,the optimum temperature and pH for reaction of LYC5 lysozyme were 45℃ and 5.0. The successfully expressed LYC5 lysozyme protein with antibacterial activities,which laid a foundation for deeping discuss the antimicrobial spectrum and application of human lysozyme family.
The effects of glutamine and asparagine on CHO cell growth, metabolism and antibody expression in batch culture with cells being centrifuged were investigated. It was found that glutamine can't be replaced by asparagine simply, CHO-dhfr cells can't grow normally in adding separately experiments. When glutamine and asparagine concentration reached 4mmol/L meanwhile can support normal growth of CHO cells. As the total amount of glutamine and asparagine increased (0 to 24mmol/L), ammonia concentration increased linearly, however, ammonia production had no relationship with proportion of glutamine and asparagine. Furthermore, improving the asparagine and glutamine proportion moderately not only can increase the antibody production but also decrease the lactate production. In brief, the foundation of supplement of glutamine and asparagine in development and optimization of mediums and efficient fed-batch process for antibody production was established.
Objective: To express the soluble antiviral peptide, the cysteine auxotrophic expression system was used in Escherichia coli (E. coli). And the biological activities of the purified antiviral peptide were initially identified. Method: The gene sequence of antiviral peptide Se-GBVA10 was chemically synthesized according to the optimized codons of E.coli, and the gene was cloned into GST fusion expression vector pGEX-2T. Then, the plasmid pGEX-2T-GBVA10 was converted to cysteine auxotrophic expression strains (E. coli BL21 cysE51). Expressed proteins were purified by glutathione Sepharose 4B affinity column. Finally, the content of selenium, the antiviral activity and the antioxidant activity (the vitality of glutathione peroxidase) of the antiviral peptides were detected. Results: The fusion protein, sjGST-Se-GBVA10 and sjGST-GBVA10, were successfully expressed by means of cysteine auxotrophic expression system. After cutting with thrombin, the purified antiviral peptides, Se-GBVA10 and GBVA10 were obtained. The content of selenium of the Se-GBVA10 was 0.974 mol/mol peptide, the glutathione peroxidase vitality of Se-GBVA10 was 47.52 U/μmol, the concentration for 50% of maximal effect (EC50) of Se-GBVA10 was 21.73μmol/L, and the median cytotoxic concentration (CC50) of Se-GBVA10 was 849.41μmol/L. Conclusion: The antiviral peptide Se-GBVA10 has the same antiviral activity as the GBVA10, but also has some antioxidant activity.
Objective: To construct prokaryotic expression vector of the full length human thyroid hormone receptor interacting protein 15 (hTRIP15), to establish the strategy to express, purify and crystallize hTRIP15; To determinate its expression in human umbilical vein endothelial cells line (HUV-EC-C), platelet, mitral valve, gall bladder and glioma. Methods: Full length coding sequence of hTRIP15 was amplified from human hepatocyte cDNA library by PCR and cloned into pGEX-6P-1 vector after digested with BamH I and Xho I. GST-hTRIP15 was expressed in E.coli BL21 (DE3), and then protein was purified by affinity chromatography and gel filtration chromatography. Hanging drop vapor diffusion method was employed to screen and optimize the crystallizing condition. Crystal structure determination was tried by X-ray crystal diffraction technique. Further, the expression of hTRIP15 in HUE-EC-C, platelet, mitral valve, gall bladder and glioma was evaluated by RT-PCR and Western blot. Results: Recombinant hTRIP15 was successfully expressed in E.coli and purified. The preliminary crystallization condition was determined using the hanging-drop vapor-diffusion method. TRIP15 mRNA was determined by RT-PCR in human HUV-EC-C, platelet, mitral valve, gall bladder and glioma, and its expression in HUV-EC-C was further confirmed by Western blot. Conclusion: The strategy of cloning, expressing and purifying the hTRIP15 was established. The preliminary crystallization condition was obtained. It provides experimental data for final tertiary structure determination. The expression of hTRIP15 was preliminarily determined in HUV-EC-C, platelet, mitral valve, gall bladder and glioma. It provides experiment data for further function study.
Objective: Through efficient shRNA interference to knockdown simultaneously IRS1 and IRS2 in porcine liver cells, and verify its effects on genes expression related to glucolipid metabolism, which may lay a solid foundation for producing the type2 diabetes mellitus model pigs. Method: First of all, cloning porcine IRS1 gene and the partial 3-untranslated region sequence of IRS2 gene by Overlap PCR and 3'RACE respectively. Then screening the most effective interference shRNA fragments of IRS1 and IRS2 by Real-time PCR. Finally, the glucolipid metabolism related genes experssion were detected in the porcine liver cells of both knocking down IRS1 and IRS2. Result: The knockdown of both IRS1 and IRS2 resulted in significant upregulation of gluconeogenic enzymes PEPCK and F-1,6-BP, as well as a decrease in Gck expression, at the same time, also led to the upregulation of lipogenic enzymes SREBP-1, Abcg8 and CYP7a1. Conclusion: The knockdown of both IRS1 and IRS2 may lead to an increasing level of blood glucose and also can cause a disorder of cholesterol metabolism. Therefore, IRS1 and IRS2 have important roles in the regulation of hepatic glucolipid metabolism.
The glutamyl endopeptidase gene from Staphylococcus aureus was successfully expressed by cloning into the expression vector pGEX-4T-2 and then transformed into E.coil BL21(DE3). Then the recombinant GluV8 was purified by glutathione-Sepharose affinity column. For deamidating activity assay Z-Phe-Leu-Glu-pNA (L-2135) was used as substrate. The reaction result showed that recombinant GluV8 can effectively hydrolyze the alpha-carboxyl of glutamic acid residue, releasing p-nitroanilide with an activity of 1568U/mg. The optimal temperature and the pH is 42℃ and 8.0 respectively. And it remained most of activity at 50℃ and pH9.0. The specificity of recombinant GluV8 by HPLC and LC-MS/MS was identified. These results suggested that the recombinant protein is a relatively specific proteinase that could be effectively utilized for protein identification.
Equol is an active and stable phytoestrogen. It can be produced by microbial conversion of a known compound daidzein. A recombinant Saccharomyces cerevisiae strain was constructed, which harbored a plasmid enabling expression of genes orf-1, orf-2 and orf-3 for the conversion of daidzein into equol. It was found that the recombinant strain was able to convert dihydrodaidzein, the immediate downstream product of the reaction sequence, into equol. The results provided valuable information for the construction of yeast cell factory for de novo biosynthesis of equol.
Acetate/butyrate metabolic pathways play an important role in ABE fermentation and their changes will influence entire carbon fluxes distribution. Several fermentations with intermittent feeding of acetate/butyrate were conducted in a 7 L fermentor and the results indicated that exogenous acetate/butyrate enhanced solvents productivities by 47.1% and 39.2% respectively, and butanol/acetone ratios were improved by 21.7% for butyrate added batch and decreased by 16.2% for acetate added batch. A nonlinear constraint was utilized for acids rates calculation and the results revealed that acetate/butyrate formation pathways were almost blocked by corresponding acids feeding. Then a metabolic model constructed by graph theory was utilized to dispose those cases with acetate/butyrate added and to predict solvents production, in which some improvements were adopted based on the calculation results and related references. The model predicted solvents production, butanol/acetone ratios accurately and constructed the directed signal flow diagrams of ABE network under different conditions correctly.
A safe efficient microbial transglutaminase purification method applied for industrial manufacture was introduced in order to improve the purity of transglutaminase and extend its applications for pharmaceuticals. Cultures was centrifuged at 10 000 r/min 4℃ to remove the cells before adjusting conductivity to 4.1 mS/cm and pH to 5.5 for purification. The sample was purified by cation-exchange chromatography at a linear velocity of 60 cm/h in the first step. In this step SP Sepharose FF as a packing provided high selectivity and high loading for the target protein. After primary purification, the sample was further processed/isolated with hydrophobic chromatography packed with phenyl Sepharose 6 FF (high sub). SDS-PAGE pattern manifested a purity of over 95% and HPLC analysis indicated a purity of above 99% were achieved after this two purification step method. LPS of the purified sample was determined at 0.013 EU/ml with tachypleus amebocyte lysate, met the blood products requirement (<0.15 EU/ml) provided in Chinese Pharmacopoeia. Thus the method described/established is efficient and practical in microbial transglutaminase purification.
Objective: Different kinds of HSA/Exendin-4 fusion protein are rapidly constructed using BglBrick method of synthetic biology. It lays a foundation for screening and comparison of the bioactivity of fusion protein in each HSA/Exendin-4. Method: Two basic Brick expression vectors, pPICZαA-Exendin-4 and pPICZαA-HSA, are constructed based on the yeast expression vector, pPICZαA plasmid. Rapid assemblies of different amounts of Exendin-4 molecules at the terminals of HSA are achieved via BglBrick method. Ten constructed HSA/Exendin-4 fusion proteins are integrated into the chromosome of Pichia pastoris KM71H, and the corresponding target proteins are expressed after methanol induction. Result:The constructed HSA/Exendin-4 fusion proteins via BglBrick method successfully express the corresponding target protein in Pichia pastoris after methanol induction. Conclusion: The use of BglBrick method can help fulfill the rapid assembly of long-term HAS fusion proteins.
In order to detect antibodies against infectious bronchitis virus(IBV) in the serum, the N gene of IBV was cloned, expressed and then utilized in the enzyme linked immunosorbent assay(ELISA). N gene(1230bp) of IBV was amplified by RT-PCR from a strain of IBV H52,and confirmed by sequencing and blast analysis. The N gene was then subcloned into prokaryotic expression vector pGEX-KG in the form of vector pGEX-NP. The fusion protein N-GST which was expressed in E. coli BL21(DE3) was characterized by SDS-PAGE and Western blotting analysis as 80kDa with immunity and dissolubility, and was purified with GST affinity column, then to establish an indirect ELISA for the detection of antibodies against IBV. The results indicate the assay has excellent reduplication, high sensitivity and specificity. It could be applied to detect the antibodies against IBV fast, to provide technical support for sero-epidemiologic survey of IBV infection and to understand how IBV infect animals.
Aim: The purpose is expressing EGF in oil-body of transgenic safflower. Method: The Ddoil-EGF fusion gene was got with polymerase chain reaction (PCR) amplification, and the Ddoil-EGF fusion gene was cloned into plant expression vector pCAMBIA1390R to gain p1390Do-EGF. The recombinant plasmid p1390Do-EGF was transferred into Agrobacterium tumefaciens LBA4404. By using co-cultivated method,the fusion gene Ddoil-EGF was transferred into safflower cells. Transferred shoots were selected on solid medium containing mannose.EGF was detected by PCR and real-time RT-PCR in safflower. The expression of EGF in safflower was detected by SDS-PAGE and Western blot. Effect of EGF on the proliferation of Bab/c 3T3 cells assessed by MTT. Result: PCR result showed that EGF gene was transformed into safflower. Relative quantitative real-time RT-PCR showed EGF mRNA was transcripted in safflower. The transgenic safflower seeds expressing Ddoil-EGF fusion protein through SDS-PAGE were confirmed. Western blot result showed that recombinant protein expressed by transgenic safflower could eliciting immunoresponse to EGF antibody. MTT result showed that EGF can stimulate the proliferation of Bab/c 3T3 cells. Conclusion:The Ddoil-EGF fusion gene was transformed into safflower and was expressed in safflower seeds.It is a new ways to express EGF in plant.
The study on immobilization of lipase by sol-gel has become research hotspot in recent years.The aim is to find the optimum conditions for the immobilization of lipase from Aspergillus niger and also to select the most suitable support maximizing the immobilized enzyme yield under these optimum conditions.TMOS,MTMS,ETMS,PTMS were used as the precursors in the preparation of encapsulated lipase from Aspergillus niger. The 80.2% of immobilized degree and the 136.3% of relative activity were obtained under optimum conditions, ETMS/TOMS=5:1 and molar ratio of water/silane = 8.With emulsified olive oil as substrate,the kinetics properties for immobilized enzyme were assessed as follows. The Km of immobilized lipase and free lipase in pH 4.0 and 50℃ conditions were detected to be 1.899×10-4 mol/L and 2.789×10-4mol/L by double reciprocal plot,Lineweaver-Burk plot, respectively. The optimum pH of immobilized lipase and free lipase were analyzed to be 4.0 in optimum temperature. The relative activity of immobilized lipase was found above 95% in the range of pH4.0 to pH5.5. The optimum temperature of immobilized lipase was observed at 60℃ which was 10℃ higher than free lipase. The pH stability and thermostability of immobilized lipase were obviously improved in comparison with free enzyme. When immobilized lipase was cyclically used for twelve of reactions, it still retained 71.7% of original activity. When the immobilized lipase was storaged in the dark at room temperature for 180 days, it maintained 79.2% of the initial activity.
As the good targeting and anticancer activity, antibody-drug conjugates (ADCs) have become the new hot spot and important trends for anticancer antibody drug development, and get more and more attention. ADCs consist of a monoclonal antibody (MAb), a linker, and a cytotoxic drug. ADCs combine the targeting of MAb and the anticancer effects of cytotoxic drugs, reduce the adverse effects of cytotoxic drugs, improve the selectivity of cancer therapeutics, and better cope with the drug resistance problem of MAbs. Compared with traditional monoclonal antibodies, because of the complexity of ADCs, the establishment of analysis methods for ADC quality attributes has greater difficulty and specificity. The current development situation, analytical methods and challenges associated with characterization of ADCs were summarized. It will provide reference for researching and quality control of ADCs.
Cloning of interest DNA is necessary for functional analysis of genome sequences and research on metabolically engineered pathways of modern producer strains. Methods to precisely clone large DNA fragments include library constructing and screening, PCR amplification and various different DNA assembly methods in vitro. In addition, direct cloning by homologous recombination in vivo has more advantages in the cloning and engineering of long DNA sequences. The main methods of direct cloning of large DNA fragments in vivo via Red/ET recombination system and its applications were introduced.
Microenvironment effects proliferation, migration and functions of cells. The factors of microenvironment that effect fate of cells include interactions between cells and extracellular matrix, soluble signal molecules, anoxia and nutrition. To prepare engineering tissue scaffold need to simulate the cells microenvironment furthest following the bionics theory so that the scaffold can be used in studies of cell behaviour and clinical therapy. Comprehensive understanding the effects factors of microenvironment on cells played an important role on tissue engineering scaffold preparation, and the studies about tissue engineering scaffold also promoted the knowledge about microenvironment effect on cells. Researches on tissue engineering scaffolds have broad prospects in the field of tissue engineering. And new preparation techniques also play a huge role in promoting the research on tissue engineering scaffold.
Flavonoids, plant-specific polyphenolic compounds, have significant physiological activities, including antioxidant, anti-inflammatory effects, and improvement of blood circulation. They have promising markets for health products, cosmetics and medicines. At present, flavonoids are mainly extracted from plants, but their growth are restricted by the seasons, location and varieties, their separation and purification processes are complex and low efficiency. With the development of synthetic biology and metabolic engineering, the flavonoids metabolic circuits have been constructed, optimization and combination of gene sequences from different sources and chassis modification have been achieved. Now engineered yeast, Escherichia coli and other microorganisms have been able to produce 2s-flavanones, flavones, isoflavonoids, flavonols, anthocyanins and flavanones which can be modified to creat new products with noval biological activities by methylase or glycosylase. The research achievement on flavonoids production in engineered microorganisms was reviewed and the future trend of development was explored.
Ubiquitin/26S proteasome pathway (UPP) is the most effective, highly selective proteolytic pathway. It mediates 80% to 85% of the protein degradation in eukaryotic cell, participates in various cellular life courses, and plays an important role in maintaining the normal physiological function of the cells. The results show that many aspects of plant growth and development as well as some processes such as drought stress response are all affected by this pathway. It summarizes ubiquitin/26S proteasome pathway, which roles in the processes of plant growth and development, and emphatically elaborates the progress of the plant drought stress response and its mechanism mediated by the ubiquitin-protein ligase E3.
L-Arginine, a semi-essential amino acid, has lately attracted considerable attention because the amino acid has been shown to be a precursor to nitric oxide (NO), a key component of endothelium-derived relaxing factor. Currently, L-arginine is primarily produced by microbial fermentation, and thus rapid and efficient breeding of arginine high-yield strains has become the focus in industry. The progress of breeding methods for arginine-producing strains was reviewed, and the current problems and perspectives on molecular breeding methods were discussed.
