Because of the safety concerns of blood sources and whole blood products, genetically engineered recombinant factor Ⅷ have begun to replace blood-derived factor Ⅷ for the treatment of hemophilia and related diseases. However, the relatively low expression level of recombinant factor Ⅷ remains a major technology bottleneck to reduce the cost of recombinant factor Ⅷ production. To explore the possible means to increase recombinant factor Ⅷ expression at molecular level, site-directed mutagenesis techniques were used to generate potential glycosylation sites around A and C domains of a B-domain deleted recombinant F Ⅷ and tested the activity, expression and secretion of various glycosylation constructs. The results showed that generating glycosylation sites at the outer edges of domain A increased secretion of factor Ⅷ while those glycosylation sites generated at the Bip-binding core not only greatly reduced the secretion but also abolished the activity of Factor Ⅷ. Different from A-domain, the glycosylation sites generated in C-domain abolished most factor Ⅷ activity without increasing its secretion regardless whether they were located at the core protein-C binding site or at the edge of C-domain. Taken together, the results suggest that the only glycosylation sites with the potential of increasing factor Ⅷ secretion are located around the edge of A-domain.
ImmunoglobulinG-degrading enzyme of Streptococcus pyogenes (IdeS) is a kind of typical cysteine hydrolase, which cleaves IgG at a specific site in the hinge area and leads to the formation of F(ab)2 and Fc fractions. Because of its unique specificity and hign activity, IdeS could be used in the preparation of subunits, structural analysis and charaterization of IgG. The recombinant GST-IdeS-His6 was efficiently expressed in Escherichia coli by using a two-tag system. And an enteropeptidase cleavage site was inserted at the N-terminal of IdeS for removing the GST-tag easily. The enterokinase site was also added between amino terminal and GST, in order to remove the GST-tag easily. Purified by affinity chromatography, IdeS was identified and analysised by SDS-PAGE, HPLC-SEC and LC-MS.The results suggested that this system was efficient for the production of IdeS. There would be about 25mg protein, with the purity more than 90%, obtained from one liter bacteria liquid. The protease and antibody IgG could be mixed with 1:100 (m/m) ratio, reacted at 37 ℃ for 30 min, then digested completely. The recombinant IdeS could meet the quality requirement of protein structural analysis and could be further utilized effectively in the characterization analysis of antibody drugs. And this enzyme could be also applied in the research of biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.
Objective: To construct the eukaryotic expression recombinant plasmid of human ANKRD49, study its function, and identify the RNA interference targets of ANKRD49. Methods: Total RNA was extracted from A549 cells and the cDNA was synthesized. The open reading frame of human ANKRD49 gene was amplified from the cDNA by RT-PCR, and cloned into p3×Flag-CMV-14 to construct recombinant plasmid p3×Flag-CMV-14/ANKRD49. After confirming by colony-PCR, double restrict enzyme digestion and DNA sequencing, the eukaryotic expression recombinant p3×Flag-CMV-14/ANKRD49 was transfected into HEK 293T cells. The targeted protein expressed in host cells was detected by Immunoblotting and immunofluorescence staining. The distribution of ANKRD49 in host cells was detected by immunofluorescence staining. The cell proliferation of ANKRD49-expressed HEK 293T cells were measured through MTT assay. The RNA interference targets of human ANKRD49 were identified via Immunoblotting assay followed by co-transfecting both p3×Flag-CMV-14/ANKRD49 and siRNA into HEK293T cells. Results: The product of RT-PCR was 720 bp. The recombinant plasmid p3×Flag-CMV-14/ANKRD49 was confirmed successfully by colony-PCR, double restrict enzyme digestion and DNA sequencing. Immunofluorescence staining and Immunoblotting showed that human ANKRD49 was expressed successfully in the p3×Flag-CMV-14/ANKRD49 transfected-293T cells than mock transfected cells with molecular weight was approximately 27 kDa. Immunofluorescence staining also revealed that ANKRD49 was distributed in nucleus. MTT assay showed that ANKRD49 had no effect on cell proliferation. Co-transfection and Immunoblotting assays showed that the expression of human ANKRD49 was efficiently knockdown by number 1 and 4 siRNA. Conclusion: The eukaryotic expression recombinant plasmid p3×Flag-CMV-14/ANKRD49 was constructed and expressed in nucleus of HEK 293T cells. This protein had no effect on cell proliferation. Two ANKRD49 interference targets were identified.
Numerous studies proved, recombinant human keratinocyte growth factor play the significant role in promoting hair growth, Successfully build rhKGF2-EGFP (enhanced green fluorescent protein) fusion protein expression vector, and gain pure rhKGF2-EGFP fusion protein. Rehydrated lyophilized protein pure liposomes prepared by rhKGF2-EGFP. The reuse of the characters of liposomes beneficial to the skin penetration and sustained release characteristics, and EGFP fluorescence characteristics, liposomes rhKGF2-EGFP hair-growth related skin permeability study.
The Culture and characterization of lysozyme transgenic goat mammary epithelial cell(TGMEC) were focused. The primary cell were isolated by using tissue culture method, and then purified by differential trypsin digestion. When cultured in the presence of insulin, hydrocortisone and prolactin, its growth curves comply with a typical S-type, and the chromosome number is normal when analyzed by Giemsa staining. The positive immunostaining to cytokeratin18 suggested that the resulting cell line exhibited the specific character of epithelial cells, and RT-PCR analysis showed the cell expressed endogenous milk protein (casein and lactoglobulin); Furthermore, the ELISA analysis demonstrated that this cell line also secreted recombinant lysozyme stably into the culture medium. The goat mammary epithelial cell line obtained provides an important cell model for mammary gland bioreactor studies.
TNF-α is one of the secretory products of adipocyte and is a multifunctional cytokine that plays an important role in regulating lipogenesis. miRNA is a kind of endogenous RNA with length of about 22nt, which regulates 60% of mammalian gene via its seed sequence. Author's previous work found miR-181a, which targeting porcine TNF-α, is highly expressed in a fat-rich pig breed and has an effects on adipocyte differentiation by regulation of TNF-α. In order to find out which miRNAs that targeting the porcine TNF-α, 3 end untranslated region of porcine TNF-α were amplified and the PCR product was digested with Xba I and Hpa I, then ligated to pGL3-control at the corresponding sites to construct the luciferase expression plasmid pGL3-TNF-α-UTR. Then five miRNA miR-19a,miR-124,miR-130a,miR-301, miR-506 were predicted to target TNF-α using bioinformatic analysis(softwares of TargetScan:http://www.targetscan.org/、miRanda:http://www.microrna.org/microrna/home.do and Pictar:http://pictar.mdc-berlin.de/). Mimics of these five miRNAs were synthesis and cotransfected with PGL3-TNF-α-UTR into CHO cells respectively, taking scrambled sequence as negative control. The results showed that TNF-α was the target of miR-19a, miR-124 and miR-130a by a dual luciferase assay (P<0.01). To verify whether TNF-α expression was inhibited by the seed sequences of these three miRNAs, binding sites of miR-19a, miR-124 and miR-130a on TNF-α3' UTR were mutated to design the rite-directed mutagenesis primers, after PCR amplifying, the products were digested with Dpn I to remove the originally un-mutated template. Then the mutated vectors PGL3-TNF-α-UTR-mutant1(miR-19/130a) and 2(miR-124)were constructed. The mutated plasmids were cotransfected with miR-19a, miR-124 and miR-130a respectively, and the result of dual luciferase assay showed that they all have no significant effect on depressing the expression of TNF-α(P>0.05).The result showed that porcine TNF-α is the target of miR-19a, miR-124 and miR-130a and they all inhibit the expression of TNF-α by their seed sequences, which provided the evidence of interrelationship between TNF-α and the three miRNAs.
As an intracellular kinase, protein kinase A (PKA) functions in many tissues morphogenesis, including the central nervous system, the eye and the limb, and regulates diverse cellular activities. However, its role in kidney development is still unclear. The expression pattern of zebrafish PKA was obtained by in site hybridization and frozen-section technology; Morpholino was used for gene knockdown through microinjection; Microstructure of the pronephron was checked by immuno-fluorescence. Results showed that prkaa1 and prkab1b, coding catalytic and regulatory subunit of zebrafish PKA respectively, were maternal genes and were specifically expressed in the renal tubule epithelial cells; Inhibition of prkaa1 or prkab1b resulted in high proportion of heart edema (54.4% and 77.3% respectively) and body curvature (48.5% and 72.6% respectively) and very low proportion of polycystic kidney disease(less than 5%); Inhibition of PKA prevented migration of the renal tubule epithelial cells, especially multi-ciliated cells, resulting in prominent dilation of pronephric duct and then embryonic death. This indicated that PKA may function in zebrafish kidney development by regulating the migration of renal tubule epithelial cells.
Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. A novel strategy to express a nattokinase from Bacillus natto in a live delivery vehicle E. coli BL21(DE3) was developed. The mature peptide-coding fragment of NK gene was amplified from B. natto total DNA by polymerase chain reaction (PCR). The sequence analysis indicated that the fragment of NK gene consisted of 825 bp, which encoded 275 amino acid residues. The sequence of NK gene has 99% homology with those of the reported ones. The expression plasmids NK-pep-pMAL-p2X and NK-pep-pMAL-c2X are constructed by inserting NK gene into vector pMAL-p2X and pMAL-c2X, and then fusionally expressed with maltose-binding protein (MBP) in Escherichia coli BL21(DE3). SDS-PAGE analysis revealed that the NK protein were secretion expressed after induction with IPTG for 4 hours under 16℃ or 37℃.The characteristic of this recombinant nattokinase is comparable to the native nattokinase. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.
AgrC is a membrane-embedded histidine kinase of Staphylococcus aureus that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm so as to regulate and control a series of related pathogenic gene expression. Restriction-Free cloning and Nested PCR were used to construct an expression vector of pET-28a-AgrC successfully. Then, expression vector pET-28a-AgrC was transformed into E.coli C43 (DE3), and then, IPTG was added to induce expression. The expressed AgrC protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). SDS-PAGE test showed that AgrC proteins were successfully separated and purified.
The inhibitory effect of Bacillus amyloliquefaciens KN-BL-1 and its fermented soybean meal were compared by oxford cup method. The crude peptides which separated by acid-precipitation were determined as the effective antibacterial ingredients. In order to study the inhibitory effect of each component of the crude peptides, DEAE-Sepharose ion exchange chromatography and Sephadex G-25 gel filtration chromatography methods were used to separate the crude peptides and the molecular weight of the antibacterial peptides were initially identified by 4000 Q TRAP HPLC-MS. The results showed that the KN-BL-1 and its fermented soybean meal had a significant inhibitory effect to S.aureus and other Gram-positive bacteria. After separated and purified through chromatography, three elution peaks showed antibacterial properties.one of the three had the strongest inhibitory effect, and its diameter of antibiotic circle was 19.8 mm.
Near-infrared fluorescent protein (iRFP) which effectively reduces light absorption and scattering in animal tissues is suitable for living animal deep tissue imaging. Eukaryotic expression vector, pAAV-iRFP713, near-infrared fluorescent protein 713 (iRFP) was constructed. Recombinant adeno-associated virus, rAAV-iRFP713, was packaged. In vitro infection of cancer cells with rAAV-iRFP713 resulted in the strong expression of near-infrared fluorescent protein after 48h. rAAV-iRFP713 was injected into skeletal muscle of mice. Strong in vivo fluorescence signal was detected with the infrared fluorescence imaging system 48h after injection of rAAV-iRFP713. The results suggest that rAAV could deliver near-infrared fluorescent reporter gene in vitro and in vivo and iRFPs might be favorable fluorescent molecular labeling agents for living dynamic animal imaging.
The amount of viable biomass is an important physiological parameter, which is correlated with the cell growth, metabolism and productivity during the rifamycins SV fermentation process. The measurement of viable biomass was studied employing on-line Biomass Monitor. It was able to utilize the dielectric properties of cells, and the capacitance measurement was correlated well with the viable biomass concentration excluding the interference of solid materials. At the same time, there was close connections among the capacitance measurement, OUR, CER and mycelial morphology. The diauxic growth phenomenon in the former fermentation was detected using capacitance detection, and the slow-release nitrogen source soyben powder instead of the original expensive fast-release nitrogen source of peptone was used for rifamycins SV fermentation, successfully eliminated the diauxic growth caused by nitrogen source using the conversion of stagnation, the physiological parameters of OUR and CER reached and maintained at 14.8 and 15.3 mmol/L/h at early growth phase, significantly higher than that under peptone conditions for only 8.6 and 11.3 mmol/L/h, which promote the continued higher rifamycin SV biosynthesis, the fermentation titer reached to 5969 + 19 U/ml, which was 18.7% higher than that of control (5030 + 17 U/ml).
Soil pollution, as a result of industrial development, is becoming a serious problem in China and raising increasing concerns from the public. Soil remediation through plant genetic engineering is a widely used method to tackle this problem. The theory is to provide a new genetic resource for heavy metal phytoremediation through the study and manipulation of heavy metal responsive rate-limiting enzymes in plants. The gene LcGS, coding glutathion synthetase (GS) of Lycium chinense was cloned using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Expression of LcGS in L.chinense under CdCl2 stress over a 24h time period was monitored and analyzed using semi-quantitative RT-PCR. The expression of LcGS gene up-regulated initially following the increase of Cd stress time, then remained at a relatively high level from 9h, till the end of the monitoring period at 24h. The expression vector containing this gene was also constructed and named pCAMBIA2300-LcGS. Then the LcGS expression cassette was transformed into tobacco via Agrobacterium-mediated transformation. Integration of this foreign gene was confirmed using PCR. Under CdCl2 stress, transgenic plants carrying LcGS gene contain higher contents of glutathione (GSH), phytochelatins (PCs) and chlorophyll than wild-type plants, confirming higher tolerance to CdCl2. Therefore, over-expression of glutathion synthetase genes through genetic engineering can be a promising strategy for heavy metal phytoremediation.
Stress refers to the organism under the stimulus of constant exposure to the environment. Plants have the ability to set up the system of protection and adaptation. Adversity stress inhibits the growth, development and reproduction of the organism. It often determines the distribution of the species. What is important is that it provides a selective dynamic evolution for a specific population. Plants can cope with stress through three different strategies, and these three different strategy is to endure, resistance and avoid or final escape. DNA methylation, as an epigenetic phenomenon, can make the gene function change without changing the DNA sequence via the effect of methylase, in response to external environmental stimuli. This change can often be passed on to offspring and form the epigenetic memory, this provides a possibility for cultivating plants resistant varieties. The research progress of plants DNA methylation modification under adversity stresses weve reviewed, aimed at further understanding the effects of DNA methylation changes to plant resistance.
Plant gene engineering techniques are developing fast, offering technical support and paving the way towards the development of transgenic plants for oral vaccines. Tomato is one of the most useful transgenic acceptors. Transgenic tomatoes for oral vaccine which can be ate directly without heating are easy to administer. This largely reduce the level of damage to the foreign protein. With the demonstration of the immune protection mechanism of genetically modified plants and the completion of the international tomato genome project, new opportunities for transgenic tomatoes are emerging. However, there still are some challenges facing in the production of transgenic tomatoes for oral vaccines. These challenges include low expression rate of foreign protein, potentiality to be digested and the food safety of genetically modified plants. Status, problems and strategies of transgenic tomatoes for oral vaccines were reviewed, which may be helpful to inspire further studies.
The evolution process and the dynamic mechanism of the bioenergy industry has its own rules and characteristics, and the evolution process was analysed; the dynamic mechanism of the endogenous system, external system and symbiotic system of the bioenergy industry were discussed respectively. Insight into the evolution mechanism of the bioenergy industry has important significance not only for policy makers to construct the industry strategy, but also for manufactures to adapt to intense competition. A new perspective for the development of bioenergy industry research was provided.
On the basis of survey to the evolution of the bioeconomy concept in European Union (EU), the process of the bioeconomy policy in EU has been systematically surveyed from both at EU and member states level. Thus, several characteristics have been induced by comprehensive analyses of the bioeconomy policy in EU, which are concluded as below: improving the sustainable development of economic-society by combining the bioeconomy with the green transformation of industries; improving regional rural and agricultural development by combining the bioeconomy with the agriculture multifunctionality; paying great attention to the construction of synthetic policy and technology platform for bioeconomy development. Then the driving mechanism of the bioeconomy development, the underpinning function of agriculture for the bioeconomy, the platform values of the bioeconomy and the implications of the bioeconomy era, have been further expounded by relative discussions.
The involved clinical departments of tissue engineering include orthopaedics department, general surgery department, ophthalmology and otorhinolaryngology department, rehabilitation medicine department, urology department, oral dentofacial surgery department, neurosurgery department, plastic surgery department, chest surgery department, ophthalmology department, hepatobiliary surgery department and vascular surgery department, nervous tissue, liver tissue, cornea tissue, urinary bladder tissue, blood, ligament, ear, genital tract, hand, fat, breast, heart, kidney, pancreas and tube tissues that applied in constructing intestinal canal, esophagus, trachea, blood vessel, kidney and urethral canal, etc. The relatively successful fields of research and application include skin tissue, cartilage tissue and bone tissue. It is a long course that from the aboriginal initial research of engineered tissue or organ to the final authorized clinical application, which needs many researchers of different disciplines to work hard together. With the deep development of fundamental research and clinical application, modern tissue engineering is becoming an effective treatment and supplementary means for therapy of tissue or organ failure.
