Objectives:To establish a tumor model in mouse with expressing Luc and human PSCA for the development of new anti-tumor drugs or vaccines. Methods:The Luc and human PSCA genes were cloned and pcDNA-luc and pcDNA-PSCA plasmids were constructed. RM-1 cells were stably transfected with both pcDNA-luc and pcDNA-PSCA plasmids. The Luc-expressed cells were examined with luminometer and the PSCA-expressed cells were examined with RT-PCR and flow cytometry analysis. The male C57BL/6 mice were inoculated with RM-PSCA/Luc cells, then the growth and the survival time of mice were measured, respectively. In vivo bioluminescence imaging was used to detect Luc expression and immunohistochemistry analysis was used to detect PSCA expression. Results:RM-PSCA/Luc cells stably expressing Luc and PSCA were obtained and can establish tumor growth in 100% of inoculated mice. Conclusion:The Luc-expressed and PSCA-expressed tumor model in mice was successfully established and could be used for evaluating the activities of drugs or vaccines.
Peptide tyrosine tyrosine (PYY) is a 36 amino-acid peptide secreted from L-cells predominantly within the distal gastrointestinal tract. Two main endogenous forms of PYY have been identified, PYY1-36 and PYY3-36. The latter has an effect of anorexia and can inhibit food intake in rodent animals. Peripheral administration of PYY3-36 can reduce food intake and body weight gain. All PYY3-36 reported were chemically synthesized. Gene cloning of PYY3-36 will be an innovation. PYY3-36 gene and its derivative that were obtained by site-directed mutagenesis and artificial synthesis respectively were cloned into pET32a(+) vector. Recombinant plasmids were expressed in Escherichia coli. Fusion proteins were purified by Ni ion affinity chromatography. Purified fusion proteins were then digested by enterokinase and purified by another affinity chromatography to get the target peptides. The biological activities of PYY3-36 and PYY3-36-Gly37 were studied in vivo in Kunming mice. The result showed that when administrated at a dose of 800μg/kg intraperitoneally, both PYY3-36 and PYY3-36-Gly37 can reduce food intake within 9 hours. Interestingly, the inhibition ratio of PYY3-36-Gly37 can reach to 50%, which is obviously higher than that of PYY3-36.
The internal ribosomal entry site (IRES) from encephalomyocarditis virus (EMCV) was a popular element widely used in the plasmid to express proteins in eukaryotic cells or cell-free extracts, such as the pIRES2 plasmid developed by clontech. Although it had already been widely used, researchers often encountered in headache problems when manipulating it. Normally, the translation efficiency driving by the modified EMCV IRES was difficult to control. It’s necessary to study EMCV IRES mediated translation in detail.The relationship between the end sequence of EMCV IRES and its activity was determined. The red and green fluorescent protein genes were used as reporter genes, and "GCTAGA" bases were inserted at the end of EMCV IRES by molecular cloning. The expression efficiency was analyzed with fluorescence microscopy, fluorescence spectrophotometer, Western blot and other methods of molecular biology. As a result, it was found that additional bases at the end of EMCV IRES severely affected the IRES activity of EMCV, and the frequently used EGFP reporter gene may contain IRES element itself. These results indicate that investigators should pay more attention to the integrity of IRES sequences and their connection forms with the downstream sequence.
Chitooligosaccharide (COS) or oligochitosan has been found to induce plant defense response. In order to study the mechanism of COS induced tobacco defense response, nitric oxide (NO) generation was indentified in tobacco suspension cells induced by COS by using NO specific fluorescence diaminofluorescein diacetate (DAF-2DA) and Microplate Reader GEMINI EM. The results showed that tobacco cells treated with COS resulted in a rapid NO generation, whose fluorescence intensity was dependent of COS concentration. The NO generation was sensitive to NO scavenger Carboxy-PTIO potassium salt (cPTIO), NO synthase (NOS) inhibitor Nω-nitro-L-arginine methyl Ester (L-NAME), lanthanum chloride and ruthenium red. Nitrate reductase (NR) inhibitors sodium azide and sodium tungstate had no effect on NO generation induced by COS. L-NAME, cPTIO, EGTA, lanthanum chloride and ruthenium red reduced the expression of defense-related gene induced by COS. Above results showed that NOS mediated NO generation and Ca2+ participated in the process of COS-induced NO generation.
CBL is a member of newly identified calcium signal transmitting proteins. CBL-CIPK pathway played an essential role in responding to the biotic and abiotic stimuli in plant. CBL1 and its specified CIPKs are the main executors dealing with various environmental stressed such as low potassium, osmotic, drought, wound, and cold. Site-directed mutation of positive charged amino acid residue and methylation of Lys on the surface demonstrated that the non-specific aggregation of CBL1 from Ammopiptanthus mongolicus(AmCBL1) was result from the weak charges interaction between different molecules, and the trimer state may probably be the function unit for the AmCBL1 at the present of Ca2+. The homogeneous state of methylated AmCBL1 provided a solid foundation for its crystallization.
Streptococcus pyogenes is a gram-positive pathogen that causes a variety of human diseases. The zinc binding proteins of S. pyogenes were enriched through Immobilized-metal affinity chromatography (IMAC). The proteins were further digested in-solution and analyzed by the advanced LC-MS/MS. In total, forty-eight zinc binding proteins involved in protein translation, glucose metabolism, metal transport, oxidation and other physiological processes were identified. The data presented here are helpful in understanding of the pathological mechanisms of S. pyogenes and facilitate the discovery of potential antibacterial drug targets and vaccine candidates.
The Serpin gene has been cloned from the genomic DNA of Bifidobacterium bifidum WBBI02. A recombinant Serpin expression system in prokaryotic cells, namely, pBX2-WBBI02 was constructed, and the Serpin protein was successfully expressed and purified. An in vitro inhibition test of Serpin against intestinal proteinase and the effect of Serpin on the adherence of Bifidobacterium longum were approached. The results showed that the serpin gene of Bifidobacterium bifidum WBBI02 is 768 bp, whose similarity is 99.9% with that of Bifidobacterium longum NCC2705. The purified Serpin was efficient to inhibit the activities of eukaryotic α-chymotrypsin and pancreatic elastase by maximum 90% and 97%, respectively, microscopical observation proved that Serpin enhanced the adherence of Bifidobacteria to HT-29 cells.
Recombinant bacterium GS115-pPIC 9k-ChiA 4.0 fermented chitinase, which was purified by ammonium sulfate precipitation, dialysis , DEAE Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography, obtained electrophoretically pure chitinase. Rate of recovery was 19.64%, and purification factor was 17.74. Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) chitinase toxicological studies carried out by contact toxicity and stomach toxicity, chitinase liquefied Plutella xylostella larvae cell and caused death. The effect and the concentration of chitinase is proportional to time, and the effect caused by stomach toxicity was more intense than by action of contact toxicity. The result of antibacterial experiments which were performed on Helminthosporium sativum, Tomato botrytis cinerea, Tobacco brown spot, and Colletotrichum gloeosporioides, indicated that chitinase can inhibit fungal by enzymatic hydrolysis of chitin in fungal cell wall.
The partial mutants library of Methylobacterium sp. MB200 was constructed by using plasmid pTnMod-RKm’ and 11554 mutants were obtained which could survive in MMII containing two antibiotics (Nm and Km). Rescreening results showed that 333 strains could not use methanol as the sole carbon source,these strains were preliminarily considered that their one carbon metabolism way had been destroyed. This laid the foundation for cloning genes related to one carbon metabolism. The location analysis was carried out by TAIL-PCR, based on the characters of pTnMod-RKm’ and the full sequence of mtdA and mtdB genes were cloned .
Anti-microbial peptides sequence from two kinds of aquatic animals have been cloned into the prokaryotic expression vector pGEX-4T-1, constructing two fusion protein expression vector pGEX-Y18 and pGEX-CEC1,which were transformed into Escherichia coli Rosetta to express,and the existent form of expression fusion protein mainly in inclusion body. Fusion protein was renatured,and cleaved to produce antimicrobial peptides Y18 and CEC1. Antimicrobial tests showed that not only fusion protein GST-Y18 and GST-CEC1 but also antimicrobial peptide Y18 and CEC1 can effectively restrain the growth of E. coli DH5α、S. aureus、B. subtilis. and S. cerevisiae.
To improve the soluble expression level of extracellular domain of E2 envelope protein of Sindbis Virus (SINV) in prokaryotic expression system, the expression conditions were optimized. The extracellular domain of E2 gene was cloned from cDNA of SINV and sub cloned into expression vector pGEX-6P-1. The recombinant plasmid pGEX-6p-1-E2 was constructed and transformed into four host bacteria respectively. The optimum host bacteria and induction temperature were selected by SDS-PAGE assay. The molecular chaperone co-expression system was constructed and the optimum molecular chaperone vector was screened. The results were described as follows: the E2 gene was amplified and cloned into vector pGEX-6p-1 successfully; the quantity of E2 expression was similar in four host bacteria; the soluble expression level of E2 was highest when induced at 16℃; in the molecular chaperone co-expression system, the five vectors containing different molecular chaperones (pG-KJE8、 pGro7、pKJE7、 pG-Tf2 and pTf16) improved the soluble expression level of E2 in varying degrees when co-expression with recombinant plasmid pGEX-6p-1-E2, and pG-Tf2 was confirmed to contain the optimum molecular chaperones combination and made the soluble expression level of E2 improved 15.7%. These results can provide a potentia1 value for further structural and functional studies of E2.
Exendin-4 is the first-approved drug of the incretin mimetic family, which acts like the naturally occurring hormone GLP-1 in vivo. Exendin-4 decreases fasting and postprandial plasma glucose levels. Exendin-4 may play a role in enhancing glucose-dependent stimulation of insulin secretion, and maintaining of β-cell mass by promotion of β-cell proliferation and neogenesis, and inhibiting of β-cell apoptosis. This experiment studied the standard Fmoc strategy solid phase synthesis of Exendin-4. Using NMP as the coupling solvent, HOBt/DIC as the coupling reagent, AIM as the containing reagent. Synthesized peptides were clearaged by TFA/Phenol/TIS, and identified by high performance liquid chromatography and quadrupole time of flight LC/MS. The yield of pure Exendin-4 was 21% with a purity of 99.4%. Using the SRB method to measure the bioassay of Exendin-4 cell regeneration experiment. It is an influential research into the therapy of type 2 diabetes mellitus.
The sequence of polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14) was analyzed using bioinformatics. A peptide sequence was selected according to hydrophilicity, antigenicity, flexibility and accessibility. The cDNA of interest was subcloned into a eukaryotic expression vector pET-DsbA to construct pET-DsbA-GALNT14. The expression of recombinant fusion protein was induced by IPTG,then purified by Ni-IDA Resin. Polyclonal antibody against GALNT14 was prepared from immuned New Zealand white rabbit. The titer and specificity of the prepared antibody were analyzed by immunodiffusion and Western blot. The result showed that the fusion protein was expressed successfully, and the interest protein with high purity was obtained. The generated antibody was specific. Both of MCF-7 and 786-O cell expressed GALNT14.A good foundation for further study on the function of GALNT14 and its role of tumorigenesis and development was laid.
A rapid and convenient multiplexed, microsphere-based immunoassay was simultaneously developed for Cephalexin(Cex) and Ractopamine(Rac) in raw milk. Synthetic antigen of Cephalexin and Ractopamine were covalently coupled to fluorescent-coded polystyrene microspheres as capture antigen, which were used to build a competitive detection system with standard sample of antibiotics and monoclonal antibody against Cephalexin and Ractopamine. Phycoerythrin-labeled goat anti mice IgG as fluorescent probe labeled monoclonal antibody specifically react with capture antigen. Fluorescent intensity associating microsphere and probe were detected respectively by laser on suspension array system to complete final detection. No cross-reactivity of the antibody was observed in reaction system, indicating that the antibody is highly specific for Cephalexin and Ractopamine. Optimum concentration of antigen covered microsphere was set up (8.4μg and 16.64μg per 100μl microspheres). Finally, the performance of beads-based immunoassay for simultaneous determination of Cephalexin and Ractopamine were evaluated demonstrating that the beads-based immunoassay is more adjective method for Cephalexin and Ractopamine determination in raw milk (LOD Cex = 20.59 ng/ml;LOD Rac = 23.51 ng/ml).
Three kinds of defoamers—soybean oil, organic silicone and polyether were used for the antimicrobial lipopeptide fermentation by Bacillus amyloliquefaciens ES-2, and the results showed that soybean oil not only function as a good defoamers, but could also enhance lipopeptide production and got a high yield of 2497.67mg/L. After extraction, the purity of the antimicrobial lipopeptide is 55.68%, and the extraction rate reached 88.72%. The effect of adsorption and desorption of different macroporous resin were also studied, and the result indicated that X-5 macroporous resin was the most suitable for the purification of antibacterial lipopeptide. The optimized technological parameters of the purification were as following: loading concentration 14.4mg/ml, flow rate for adsorption 1ml/min,desorption flow rate 2 ml/min, and elution volume was 2.5 BV. With above conditions, the recovery ratio and purity of product reached 90.01%and 74.4% separately.
Objective: To eliminate the interference of precipitate caused by inorganic salt in liquid medium on bacterial growth determination by turbidimetry. Methods: Using Methylobacterium organophilium ME25 as a model system, the effects of chelator EDTA on optical density determination and precipitate removal with spectrophotometer are investigated. Results: Chelator EDTA of 1.25×10-2mol/L could eliminate completely the precipitate in medium within a minute in the pH from 4.0 to 11.0 at room temperature, and the stability of sample is sustained more than an hour and didn’t affect the bacterial density determination at 600 nm (OD660). The data of optical density measured with real samples and standard samples showed the relative error of 3.0%, recovery of 98.0%~100%, relative standard deviation (RSD) of 0.5%. Conclusion: Chelator EDTA was quickly able to remove inorganic salt precipitate in medium and eliminating the interference of precipitate on bacterial growth determination.A relatively reliable, accurate and precise method used to measure bacterial optical density in precipitate-containing liquid medium was proposed.
Ectoine and its derivatives are de novo synthesized by halophilic or halotolerant bacteria as compatible solutes.The characteristic of ectoines in biochemical and physiological role of osmotic stress defence in microorganisms was introduced, the protective properties of ectoines for cells were descirbed and transferred to enzyme, nucleic acid, and human skin ete. Data were analyzed and summarized in the synthesis pathway of ectoines and transporters system in the halophilic bacteria on the cell and molecular level, and the production of ectoines by halophic bacteria was described, such as downshock and milking process. In addition,the applications of ectoines were also summarized, which would be useful in the field of biology industry, medicine treatment and cosmetic for prevent aging of skin etc.
L-lactic acid is widely used in food, pharmaceutical, cosmetic and industrial fields. Recent years, with the constant shortage of fossil resources, many synthetic polymer materials production has been limited. Therefore, L-lactic acid, which is maded based on biomass resources has been widely used for processing into poly L-lactic acid and other environment-friendly biodegradable materials. Due to the increasing demand for L-lactic acid, the efficient production of low-cost L-lactic acid has become particularly important.The breeding of L-lactic acid producing strain, the cheap resources development and utilization for L-lactic acid fermentation, L-lactic acid fermentation process and purification of L-lactic acid products are systematiclly focused on.For strains breeding, a number of excellent L-lactic acid producers have been obtained which can fermented cheap carbon source to lactic acid efficiently, and strains with low nutritional needs were also breeded. However, strains that have the integrated advantages needs to be further breeding; For fermentation substrates, application of a variety of cheap, abundant carbon sources for lactic acid efficiency fermentation have been already developed, but industrial-scale application of these substrates remains to be further studied; For fermentation process, environment-friendly and low labor intensity fermentation process has been developped, but the problem of high cost is still exists. For purification of products, the post-extraction process has been simplified through the production of breeding species with low nutritional requirements and the using of new fermentation process, but the actual applications still under the constraints of the high cost of the fermentation process. Finally, a short review on the chemical processing of poly L-lactic acid and biodegradation of poly L-lactic acid, and some suggestions were given.
After zearalenone (ZON) and its six derivatives are introduced,its biosynthesis pathway is described. Genetic organization of ZON biosynthetic gene cluster is not only depicted but expression style of the gene cluster is illuminated as well. It is discussed how inactivation and degradation of ZON occurs. Application of ZON hydrolase on genetic engineering is summarized and several proposals for ZON degradation with biotechnology in future are also put forward.
The spinosad, produced by Saccharopolyspora spinosa, is an new-style microbiological insecticide. Spinosad with the properties of both safety of biological pesticide and fast effect of agrochemical, has a weak toxicity towards mammalian, natural enemies of insects and environment. Thus, it has obtained permission and used in many countries. At present the research of spinosad in our country is still in the laboratory, and the lower yields of spinosad limited its industrial production.The structure of spinosad and its biosynthesis were summarized. The fermentation medium optimization, breeding of high spinosad producing strains, and its derivatives were reviewed.
Riboflavin (Vitamin B2) is a precursor to coenzymes such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). It can be commercially produced either by a semi-chemical synthesis or by fermentation with some microorganisms. Because of the advantages of biotechnical process, such as cost effectiveness, reduction in waste and energy and use of renewable resources, biotechnical route has become popular for riboflavin producer. In order to acquire riboflavin over-producing strains, research has been concentrated on metabolically modified strains by finalizing the relationships of genes and enzymes, dissecting the complex regulatory apparatus that governs expression of the rib genes and identifying transport gene. The engineered riboflavin overproducing Bacillus subtilis is one of the most successful examples. Metabolic engineering is a new developing subject in recent twenty years, which made celluar genetic modification to improve production or celluar peculiarity. To maximize the conversion of substrate carbon to desired end products, building blocks, energy equivalents, and redox cofactors must be guaranteed at an appropriate rate and stoichiometry by cellular catabolism. The applications and the trends of metabolic engineering in riboflavin production by Bacillus subtilis were summarized. The applications involved the improvement of the efficiency of carbon and energy utilization, enhancement of metabolic flux and deregulating of the process in riboflavin biosynthetic pathway.
