It was carried out on a Bacillus megaterium NK13 strain which to a certain extent could asymmetricly hydrolyzes the rac-ketoprofen chloroethyl ester to (S)-ketoprofen. By constructing Bacillus megaterium NK13 gene library, one positive clone containing the plasmid pUC18-NK- HYD3 was obtained. The analysis of sequence detection showed that the positive clone included one Open Read Frame of 741 bp nucleotide sequence which contains esterases’ conserved motif —— GXSXG. The results of blast in NCBI database showed that it was a novel esterase gene. Then the esterase gene was cloned to pET-21b(+) vector and transformed into E.coli BL21(DE3). After being induced by IPTG, SDS-PAGE analysis showed that the relative molecular mass of the esterase was about 28 kDa. The results of TLC and HPLC indicated that the esterase is a (R)-ketoprofen enantioselective enzyme. In the recombinant strain suspension system, The highest enantiomeric yield of (R)-ketoprofen was 62.74% when conversion rate was 15%. In PBS system, which contained the recombinant strain wet cells, when the conversion rate increased from 10% to 50%, the e.e.% of (R)-ketoprofen were maintained at 73%~76%.
Aim:to detect L1-ORF1, an ubiquitous protein in tumor tissue, its polyclonal antibody with rabbits were prepared. Methods: L1-ORF1 gene was prepared by PCR amplification and cloned into pGEX-6P-1 with GST fusion. After expressing in E.coli, GST-L1-ORF1 fusion protein was purified by Glutathione Sepharose 4B and subjected to immunity with rabbits. The titer of anti-serum was measured by ELISA and the specificity was examined by Western blot with the cell lysates derived from HepG2 tumor cells. Result: The anti-L1-ORF1p antibody exhibits a high sensitivity and specificity, which was evaluated by L1-ORF1p transfected normal cells and tumor cells. Conclusion:Anti L1-ORF1p polyclonal antibody with high sensitivity and specificity were successfully acquired.
Examination DNA vaccine-binding proteins in the immune cells. Cytoplasmic proteins were extracted from splenocytes of mice and were incubated with DNA vaccine, pVAX-OVA as the experimental group, and empty vector pVAX, as the control group. The bound proteins were isolated by an agarose gel and SDS-PAGE and examined by MALDI-TOF mass spectrometry to analyze their sequences. In splenocytes, the potential proteins interacted with pVAX are Foxl2,SUV420H2 and gamma actin, and the potential proteins interacted with pVAX-OVA are IQ motif containing F4. Foxl2 may improve nuclear import of DNA vaccine, which includes nuclear localization signals (NLS), conserved DNA-binding forkhead domain and interaction with transporter proteins. The interactions between DNA and those proteins may or may not be beneficial for DNA vaccine entering nuclear before its expression.
IgE plays a key role in type I hypersensitivity reactions. The blocks of IgE binding to its high affinity receptor FcεRI can efficiently eliminate allergic reaction. The sFcεRIα/mIg(IgG2)was constructed by gene fusion of the extracellular portion of α subunit of FcεRI receptor with human IgG2 hing region, CH2 and CH3 region. The chimera could be a markedly longer plasma half-life than the soluble extracellular domain. This chimeric protein was produced by transfecting Chinese Hamster Ovary cells (CHO). Following affinity purification, the apparent molecular weight of sFcεRIα/mIg(IgG2), which showed high affinity with human and mouse IgE, was above 170 kDa. A good foundation for industrialization of this new engineered soluble protein for type I hypersensitivity diseases was provided.
Heat shock transcription factors (HSFs) are critical regulators of heat shock response of plants and participate in various plant stress responses. Rice HSFs are encoded by a multigene family but their specific functions are not well-understood. Study on expression profiles of rice HSF genes is important for illuminating the molecular mechanism by which rice respond to environmental stresses.The identification of rice HSFs genes were conducted through sequence comparing and their expression profiles in various tissues under hormone treatments and abiotic environmental stresses were also analyzed. In a genome-wide scan, twenty-five rice HSF genes were identified and called OsHsf1~ OsHsf25, respectively. The phylogenetice tree of rice and Arabidopsis HSFs was constructed through amino acid sequence alignment, twenty-five rice HSFs were classified as three groups, class A, B and C,and each group comprised one or several subgroups. Class A, B and C included thirteen, eight and four genes, respectively. The gene structure analysis showed that most rice HSF genes contain a small number of exons and introns, and are spliced into a single gene model, alternative splicing was observed only in eight members (OsHsf2, OsHsf7, OsHsf9, OsHsf11, OsHsf12, OsHsf21, OsHsf24 and OsHsf25). The expression profiles of 24 rice HSF genes were derived through quantitative PCR. Most rice HSF genes presented the tissue-specific and development-specific transcription patterns. OsHsf4, OsHsf12 and OsHsf13 showed high expression levels in all tissues analyzed, whereas OsHsf17 expressed with low transcription levels. Ten HSF genes were significantly regulated by gibberellin 3 (GA3), kinetin (KT), and abscisic acid (ABA) treatments at transcription level, among them, OsHsf1, OsHsf3, OsHsf21and OsHsf24 were down-regulated by GA3 and KT, and OsHsf1and OsHsf24 were also up-regulated by ABA. Thirteen HSF genes were observed to have a significant response to drought, salt, and heat stresses. OsHsf1, OsHsf3, OsHsf5, OsHsf11, OsHsf12, OsHsf14, OsHsf21, OsHsf23, OsHsf24, and OsHsf25 were strongly up-regulated by all three stresses. OsHsf2 and OsHsf16 were significantly induced by drought and salt stresses. OsHsf15 was up-regulated only by heat stress. Rice HSFs expression profiles, especially in response to drought, salt, and heat stresses, can provide a foundation for dissecting the functions of rice HSFs in response to abiotic environmental stresses. The HSF genes responding to abiotic stresses identified may be used to improve rice tolerance to abiotic stresses through genetic engineering technology.
The content of protein is usually 38%~50% in the cottonseed meal. However, some properties of the protein make cottonseed meal unsatisfiable for the animal feed industry. To solve the problem, four predominant strains, which can efficiently degrade the macromolecular protein of cottonseed meal, were isolated from natural samples. Based on biochemical characteristic and molecular biology identification, they could be classified as B. subtilis (2 strains, marked as H1 and H7), B. cereus (1 strain, marked as H9) and A. niger (1 strain, marked as P1). Either individual strain or combined strains fermentations were carried out. The results showed that under the optimal strain combination (B. subtilis H1 and A. niger P1) fermentation of cottonseed meal, the content of acid-soluble protein increased by 25.34%, the content of small peptides increased by 11% and the content of the total soluble amino acid increased by 24%, meanwhile, the digestibility of protein increased from 44.56% to 78.61%, which were significantly higher than other strains combination. The results showed that the fermentation by strains combination (B. subtilis H1 and A. niger P1) can improve the forage utilization ratio of the cottonseed-meal-protein effectively.
Succinate is an important bio-based platform molecule. In the present work, the growth and glucose metabolism of Escherichia coli mutant QQS101 with the deficiency of formate transporter A (focA), formate-pyruvate lyase (pflB) and lactate dehydrogenase (ldhA) under strict anaerobic condition were investigated. The degree of reduction per carbon of glucose and the products of E. coli mix-acids fermentation were compared, and then recognized that non-strict anaerobic condition favored QQS101 producing succinate from glucose. Furthermore, effects of the carbon sources for aerobic growth on fermentation were performed. Results showed that QQS101 could accumulate succinate with a concentration of 31.01 g/L with a yield of 1.258 mol Succinate/mol Glucose, when growing on xylose as the aerobic substrate. During the fermentation, addition of alanine could enhance the molar yield of succinate to glucose utilized.
Objective: To explore the effect of jet nebulizer on plasmid DNA (pDNA) integrity and the protective methods. Methods: In the experiment to determine the effect of nebulization time on pDNA integrity, 5ml pDNA solution was aerosolized. Aerosol samples were collected in the 1st min, 2nd min, 3rd min, 4th min, 5th min and the 10th min. In the experiment to determine the effect of aerosolized solution amount on pDNA integrity, 2 ml、4 ml、6 ml、8ml pDNA solution was aerosolized for 4 min respectively. Aerosol samples were collected in the last minute. In the experiment to determine the protective effectiveness of the two polyplex, 4 ml naked pDNA solution, 4ml PEI-DNA solution and 4 ml liposome-DNA solution was aerosolized for 10 min respectively. Aerosol samples were collected in the last minute. Degradation associated with the aerosolisation process was investigated with agarose gel electrophoresis. Results: As aerosolization time increased from 1min to 10min, the intact pDNA ratio decreased from (83.5±2.2)% to (37.1±2.8)%. As the pDNA solution amount added from 2 ml up to 8ml, the intact DNA ratio ascended from (32.1±3.5)% to (93.6±0.6)%. There was almost no damage to the PEI or cationic lipid protected pDNA solution. Conclusion: The damage to which jet nebulizer exerted on pDNA was related with time and dosage. PEI and cationic lipids provided plasmid DNA with excellent protection, which laid a good foundation for the application of jet nebulizer in future gene aerosol treatment.
Objective: in order to improve the processing of construction the mutant with double mutations at two different residue sites. Methods: According to the method for construction of the full-length DNA fragment by DNA shuffling, the primer pairs were designed at the mutation site, three small DNA fragments were PCR amplified using wild type gene as template, respectively, then separately mixed three DNA fragments and used as templates to carry out PCR without primer. The PCR product was subjected to the last-ste PPCR with primers to amplify the full-length gene AamanA with double-site mutations. Results: the DNA sequenced results indicates the mutant with double mutations at residues E151 and E231 was succeed to create. The Thin-layer chromatography and enzyme activity assay clearly shown the catalytic activity of the mutant with double mutations was lost. Conclusion: this is a simple, economic, rapid and effective method to construct two mutations in the DNA fragment. It has the potential application value in the field of molecular biology, such as characterization the reaction mechanism of enzyme and modification the structure of the proteins, and so on.
Quantitative real-time reverse transcription-PCR (RT-qPCR) is an efficient tool to measure absolute transcript abundance and provides valuable quantitative information on gene expression of biologic samples from different sources. Thousands of research laboratories worldwide have embraced RT-qPCR as a frequently used method for measuring genes expression in transcript levels because of its relatively low cost, high precision, and high sensitivity, as well as flexibility and simplicity. However, despite its popularity, more and more researchers begin to realize that the accuracy of RT-qPCR gene expression analysis depends largely on a proper normalization. However, the simplicity of the technology itself makes it vulnerable for abuse in experiments in which the operator does not perform the required quality control throughout the entire procedure. Here, the entire RT-qPCR workflow were reviewed and where and how critical issues can be resolved were indicated point by point, such as experiment design, sample and assay quality control, selection of proper reference genes for normalization, data analysis and reporting guidelines. Following the advice, any user should be able to do (more) successful gene expression profiling using the RT-qPCR technology.
Innate immune initiates at recognition of broad classes of conserved microbial molecular structures known as pathogen-associated molecular patterns (PAMPs) by a diverse set of germ line-encoded receptors, termed pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). TLRs are one of the largest and best studied families of PRR. TLRs activate innate immunity pathway, regulate the secretion of cytokines such as TNF-α, ILs and IFN-α, and ultimately modulate the function of immunity system. Over ten TLRs have been discovered in human genome that may be the therapy targets for cancer, viral and bacterial infection, inflammation, autoimmunity and radiation injury. Several compounds targeting TLRs are now undergoing preclinical or clinical evaluation, including anti-tumor drugs, anti-virus drugs, anti-infection drugs and anti-radiation drugs. The structural feature of TLRs, the characteristics of their signaling pathways, and the development status of related compounds for therapeutic manipulation were summarized.
The TAT protein transduction peptide rich in basic amino acids, encoded by human immunodeficiency virus type 1, was a member of the protein transduction domain family. It has been widely known that the full-length TAT peptide and the core domain (YGRKKRRQRRR) play important roles in transduction of heterologous biological macromolecules such as proteins, peptides and nucleotides to across all kind of biomembranes in vivo, although the mechanism is still unknown. Recently, the TAT core domain has another function for promoting heterologous protein expression in E. coli was demonstrated. Accordingly, the current progress of TAT peptide from the structural features, factors affecting protein transduction and the underlying mechanism were reviewed in order to promote the application of the TAT peptide.
Prion diseases are neurodegenerative diseases, harmful to animal and human health. Mechanisms of prion diseases are not fully understood. Systems of prion replications in vitro are used to studying prion pathogenesis, however, mock environment in vitro for studying conversion mechanisms from normal cellular prion proteins to disease-associated scrapie prion proteins is relatively difficult and very important. Cell-free conversion assay, cell-lysate conversion assay, Protein Misfolding Cyclic Amplification, Autocatalytic conversion assay, several methods of prion conversion in vitro were described, and discussed them in reflecting prion propagation in vivo, a lot of target samples for studying Prion disease were provided, in order to facilitate to further study prion pathogenesis in the future.
Maintenance of oxygen homeostasis is important to cellular and systemic function. Under hypoxia condition, a powerful defense of organism is triggered by the hypoxia signal to protect tissue and organs. In the process of defense, some genes are up-regulated, such as hypoxia inducible factor-1 (HIF-1)、vascular endothelial growth factor (VEGF)、erythropoietin (EPO) and nuclear factor-κB (NF-κB). HIF-1 is a anti-hypoxia stress-related genes and encodes a transcription factor, The regulation of HIF-1 activity is related to the oxygen concentration. VEGF is a target gene of HIF-1, which is one of the most critical antigenic factor identified to date. EPO can promote the differentiation and proliferation of red blood cells under hypoxia conditions. NF-κB can inhibit cell apoptosis which is induced by hypoxia. Obviously, these genes play important roles in protecting organism from hypoxia stress. The function, expression pattern and regulation mechanism of HIF-1, VEGF, NF-κB and EPO in hypoxia are summarized.
Snake venom is a mixture of many proteins and enzymes with distinct biological activities, which may have significant values both in basic researches and clinical applications. However, limitation exists in the purification of sufficient active fractions for practical use. Pichia pastoris is the most commonly used eukaryotic expression system nowadays. The ability of eukaryotic processing, folding and post-translational modification endow the recombinant proteins expressed with similar biological activities of the corresponding natural proteins, is thus widely adopted to express snake venom proteins with glycosylation or rich in disulfide bonds. To date, about twenty-five snake venom proteins of 12 genus have been expressed successfully in Pichia pastoris, (including different snake venom family members such as serine proteinases, metalloproteinases/disintegrins, L-amino acid oxidases, C-type lectin like proteins, neurotoxins, vasoconstrictors, nerve growth factor,etc.),so far,snake venom cysteine-rich protein, Bradykinin-potentiating peptides etc. expressed in Pichia pastoris have not been reported. The reasons for the failure of expression of snake venom protein in Pichia pastoris maybe as follows: the codon bias, RNA secondary structure of the gene transcripted, heterogeneity of glycosylation and the difference between glycosylation types, the toxicity of expressed toxic protein on yeast cells etc., The measures to these problems are discussed also.
Transcription factors binding to cis-elements can regulate series of downstream gene expression. The over-expression of a disease-resistance transcription factor in plants which can improve comprehensive disease resistance by means of genetic engineering correspond to transgenic a number of resistance genes. They have become a research hotspot in recent years. Five families of transcription factors that are related to disease resistance (AP2/EREBP, MYB, WRKY, bZIP and homeodomain) were introduced, and their protein structure, functional properties, regulation mechanisms, their research progress on gene engineering of diseases resistances in plants, their applications were discussed.
With the completion of the human genome project, it has come into post-genomic era and now for the proteome. Proteomics aims at elucidating the expression, structure and function of all the proteins which are after all responsible for life’s activities. With the full sequencing of the grapevine genome, the grape proteomics research was significantly facilitated and be widely adopted. Recent development of grape proteomics were summarized. The contents include the protein sample extraction protocols for grape samples, results in berry development and quality formation, progress in berry skin, cell wall and plasma membrane proteomics, achievements in grapevine stress response and somatic embryo formation. In the end, the possible breakthrough in the near future were predicted.
The endospore-forming rhizobacterium Bacillus subtilis is able to produce a variety of polypeptides and peptide-derived antimicrobial compounds that are synthesized ribosomally or generated non-ribosomally. Non-ribosomal peptides contain lipopeptides and oligo-peptides, the molecular weight of which is less than 3 000 Da and that are catalyzed by employing multienzyme complexes (nonribosomal peptide synthetases, NRPSs) after growth has ceased, while the molecular weight of ribosomal peptides is much more than 30 kDa and they are synthesized during active growth. Both biosynthesis and regulation of NRPSs and ribosomal peptides are controlled by a series of gene, called gene clusters. Biosynthesis and gene regulation of non-ribosomal and ribosomal peptides were reviewed.
D-lactic acid (D-lactate) is an important chiral intermediate and one raw material for PLA production. Due to the scarcity of petroleum resources and the improvement of environmental awareness, polylactic acid industry is rapidly expanding, which increases the demand for production of D-lactate. However, low optically pure D-lactate has limitations in many of its end uses. Microbial fermentation can produce high optically pure D-lactate. In resent years, quite a lot of research works have been done in this field. Properties of D-lactate and its applications in industrial production, chemical processing and PLA production are simply introduced. The main purpose here is to summarize the current situation of D-lactate producing strains, including lactic acid bacteria, Escherichia coli, Corynebacterium glutamicum and yeast, etc. Studies on these microorganisms indicated that the approach of metabolic engineering has been widely used to make D-lactate producing strains which have higher production level, yield, productivity and optical purity, lower by-product levels, and improved ability in utilizing more simplified, cheaper and easier available materials. Metabolic pathway reconstruction by metabolic engineering and genetic manipulation is a development trend of screening D-lactate producing strains. At last, some prospects about D-lactate fermentation were made.
The formation of the concept of bioeconomy and the approach of the bioeconomy era are promoted by the rapid development of life sciences and biotechnology, which then are making changes fundamentally to the styles of produce and consume of agriculture, healthcare, environmental protection, industry. By analyses to impacts on future agriculture, healthcare, environmental protection, and industry respectively based on the bioeconomy through life sciences and biotechnology, the strategy of the bioeconomy development (GREW) have been put forward. As a strategic system, the GREW includes four sub-strategies as below: the strategy of green agriculture based on Green biotechnology, the strategy of healthcare based on Red biotechnology, the strategy of environmental protection based on grey or Environmental biotechnology, and the strategy of industry based on White or industrial biotechnology.
