Objective: To identify the functional domain of α-Synuclein in affecting mitochondrial function and how the function to be impaired, especially, the mitochondrial membrane potential and the release of Cytochrome c. Methods: Harvest of α-Syn-N and α-Syn-△N by PCR, then subcloned into the pCMV-Myc mammalian expression vector. The recombinant plasmids were transfected into HEK293T cells by Lipofectamine 2000. After detecting the protein expression by western blot, the functional domain was detected by co-immunoprecipitation. The mitochondrial membrane potential through flow cytometry and immunofluorescence, at the same time, the release of Cytochrome c through flow cytometry to detect. Results: The recombinant plasmids were constructed successfully. CO-IP has proved that N-terminal may be the functional domain of α-Synuclein in affecting mitochondria. Over-expression of N-terminal could depolarize the mitochondrial membrane potential and induce the Cytochrome c releasing in MN9D cells. Conclusion: N-terminal may be the functional domain of a-synuclein and over-expression of N-terminal could decrease mitochondrial activity.
Objective:To evaluate the feasibility of repairing laryngic catilage deffect by mesenchymal stem cells and PLGA porous foam scaffolds under the effect of cartilage-derived morphogenetic protein 1 and transforming growth factor-β1 by tissue engineering techniques. Methods:In vitro ,under the effect of CDMP1 and TGF-β1 ,BMSCs and PLGA porous foam scaffolds were cultured to investigace the expression of cartilaginous phenotype The histological staining for glycosaminoglycan using alcian blue dye-binding method and immunohistochemical staining of cartilage-specific protein collagen Ⅱ were used to identify chondrogenic differentiation of BMSCs. Results:BMSCs cultured in the medium containing CDMP1 and TGF-β1 expressed collagen Ⅱ and glycolsaminoglycan. Transplanted into the body ,BMSCs - Biology stent can repair laryngic cartilage defects. Conclusion:under the dffect of CDMP1 and TGF-β1,BMSCs-Biology stent can differentiate into the chondrogenic phenotype and can effectively repair laryngic cartilage defects.
Trypanosoma is a human parasite severely affecting poor tropical areas. However, current frontline drugs for Trypanosoma treatment have severe side-effects with decreased effectiveness. Based on the fact that aminoacyl-tRNA synthetase is a bona fide drug target for several microorganisms, including bacteria and fungi, it is plausible that it may also be effective target of Trypanosoma. In this report, the Trypanosoma brucei Leucyl-tRNA synthetase (tbLeuRS) was cloned , expressed and purified to develop an in vitro enzymatic assay system. The assay conditions were further optimized for the effective screening of tbLeuRS inhibitors thus establishing an anti-Trypanosoma drug screening system targeting tbLeuRS. The results indicated that this system can be employed for the effective screening of anti-Trypanosoma drugs with satisfactory specificity. In addition, this system can also be used for compound optimization, as well as IC50 testing. Using this system a series of compounds are identified that are effective Trypanosoma inhibitors without toxicity to human cells. Therefore, targeting tbLeuRS may represent a new venue for the development of anti-Trypanosoma drugs.
Objective: To express and purify two truncated mutants of mouse RHOX5 protein and explore the ability of the intact RHOX5 and its two truncated mutants to bind MDFIC protein. Methods: The cDNA sequences encoding two truncated mutants of mouse RHOX5 protein RHOX5 N and RHOX5 C were amplified and then subcloned into the pGEX4T3 vector to construct the prokaryotic expression plasmids. The recombinant plasmids were transformed into E.coli RosettaTM2(DE3)strain respectively in order to overcome the codon usage bias problem, and the exogenous protein expression was induced by IPTG. After purification using GlutathioneSepharose 4B affinity chromatography, the GST fusion proteins were separated by SDS-PAGE.GST pull-down assay was used to determine the ability of the intact RHOX5 and its two truncated mutants to bind MDFIC protein. Results: GST-RHOX5, GST-RHOX5N and GST-RHOX5C fusion protein were expressed effectively in E.coli RosettaTM2(DE3)strain and purified using GlutathioneSepharose 4B beads. Furthermore, GST pull-down assay indicated that both RHOX5 and RHOX5 C could bind MDFIC in vitro, while the RHOX5 N truncated mutant lost the ability of interacting with MDFIC. Conclusion: GST-fused RHOX5 and its truncated mutants were expressed and purified successfully. In addition,it suggested that the homeodomain of RHOX5 protein is critical for its interaction with MDFIC protein.
M2e gene of three copies for H5N1 subtype AIV was synthesized and fused with human mycobacterium tuberculosis hsp70 gene.The fused gene was cloned into the prokaryotic expression vector to get pET-3M2e and pET-3M2e-hsp70.Recombinant protein r3M2e and r3M2e-hsp70 were successfully expressed induced with IPTG and purified with Ni2+-NTA collumn.Following that,the immunity of the recombinant protein was analysized with Western blot. 20-day-old AIV non-immunized chickens were vaccined with r3M2e and r3M2e hsp70,at the same time,Trx and KLH-M2e inoculated chickens were served as vector and positive controls.Two weeks after the primary vaccination,every group was boosted with the same vaccine as in the primary vaccination.The humoral immunity of the vaccined chickens was evaluated with antibody detection against M2e,cytopathic suppression test,and indirect fluorescence assay.The cellular immunity was estimated according to lymphocyte subtype analysis with flow cytometry and M2e specific cytokine detection.Four weeks after the boost vaccine,all groups were challenged with 100EID50 AIV of H9N2-subtype,and the virus from swabs was detected with Real-time PCR.Results indicated that r3M2e hsp70 vaccined chicken developed the better humoral and cellular immune response,also,made a better performance compared with r3M2e vaccined group in virus challenge.
To research a new transient oviduct bioreactor expressing recombinant human tissue kallikrein,hKLK1 cDNA-expression cassette was subcloned into avian adeno-associated virus transfer vector pAITR,and transfected into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper using calcium phosphate precipitation method.The recombinant AAAV-KLK1 were injected into laying hens via wing vein at 1×1010 virus particles per hen.Oviduct-specific expression of hKLK1 was demonstrated by reverse transcription-PCR; the enzymatic activity results showed that the kallikrein expression could be detected at the second day after injection,reaching the highest level of 107.3U/ml at the third week,and lasted for 6 weeks after injection; Oral administration of hKLK-contained egg white had significant hypotensive effect on spontaneous hypertension rats，which lasted for 5 days. These data suggest that the transient oviduct reactor mediated by recombinant avian adeno-associated virus can be used for high level and long-lasting hKLK1 expression,and the production of other recombinant proteins.
The purpose is to obtain optimal cultural conditions for Ganoderma lucidum in the solid medium mainly containing the residual of Pteridium aquilinum,and to provide theoretical foundation for the further use of this material from Pteridium aquilinum.Response surface methodology was applied to optimize the main culture conditions including proportion of the residual compound in medium,water content in medium and culture temperature.The results showed that proportion of residual of Pteridium aquilinum in medium,water content in medium and cultural condition had noticeably significant effects on daily average growth rate of mycelium of Ganoderma lucidum(p<0.01).Obviously correlation was found to be existed between proportion of residual of Pteridium aquilinum and water content in medium,or water content in medium and the cultural temperature.The optimal proportion of residual of Pteridium aquilinum in medium was 85.0%.The optimal water content in medium was 62.4%,and the optimal cultural temperature was 27℃.Under these optimized conditions,daily average growth rate of mycelium of Ganoderma lucidum was 3.48mm/d.Multivariate regression analysis showed that the model for regressing proportion of residual of Pteridium aquilinum in medium,water content in medium,cultural temperature and daily average growth rate of mycelium of Ganoderma lucidum might be available for the culture of the fungi.Practical growth rate of mycelium of Ganoderma lucidum could be forecasted by applying the model. The application of residual of Pteridium aquilinum as a main additive in the culture of Ganoderma lucidum was firstly reported.It has laid a pave for the further investigation of residual of Pteridium aquilinum in medium.
Fungal diseases cause a considerable damage to many economic crops,while chemical control of diseases has a huge threat to human health and the environment.Therefore,it is becoming extremely urgent to develop biological pesticide with high activity,low toxicity and low residues.Burkholderia cepacia CF-66,isolated from the compost,shows strong antifungal activity due to various secondary metabolites,such as pyrrolnitrin,phenazine,diketopiperazines,siderophores and so on.One of diketoperazines,namely,cyclo(Phe-Pro)(cFP)was purified through the following steps.Firstly the crude extract was obtained from the fermentation broth of B.cepacia CF-66 by centrifugation,reduced pressure distillation and ethyl acetate extraction.Then cFP was isolated and purified by silica gel column chromatography and RP-HPLC.Three peaks were exculsively detected.The third one(cFP)was collected and analyzed by HPLC and GC-MS,showing single peak with a concentration of 15mg/ml.Finally,the antifungal activity was carried out by detecting minimal inhibitory concerntration(MIC),which demonstrated that cFP significantly inhibited the growth of Sclerotinia sclerotiorum,Rhizoctonia solani kühn O-28,Curvularia lunata and so forth.The result of microscopic observation showed that cFP could cause twisted hypha,fractured hypha and swollen tips during hyphal development.In conclusion,cFP has great potential for developing biological pesticide.
pPIC9 was used as a template and α-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/α-factor（pYCα）was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man（pKLman）was cut by restriction enzymes and linked with pYCα.This recombinant vector pYCα-man was used to determine the secretory ability and stability of pYCα.The excellent secretory ability of pYCα was proved by two experiments.One showed that INVSc1/pYCα-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYCα clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYCα were not detected,while INVSc1/pYCα-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYCα was also very good proved by continuous cultivation for about 150h.
Xdh encoding xylitol dehydrogenase(XDH)gene was amplified from the genome DNA of G.oxydans NH-10.The recombinant plasmid pSE-xdh was constructed by inserting xdh genes into expression vector pSE380 and transformed into E.coli JM109.The recombined XDH was purified through two steps including HisTrap HP affinity chromatography and SephacrylS-300 gel filtration chromatography,and then the enzymatic properties were investigated.The optimum pH and temperature for reduction conditions of XDH were 5.0 and 35℃,while that for the oxidation conditions were pH 11.0 and 30℃.XDH was a kind of NADH-dependent dehydrogenase,and the Km value for NADH was 57.8mmol/L and the Vmax was 1209.1mmol/(ml·min).The XDH activity of recombinant strain was 13.9 U/mg.16.7 g/L xylitol was obtained from 28.0 g/L D-xylulose in 16 h by mixed fermentation of resting cells which was composed of original strain and recombinant strain,whereas control strain produced 8.3 g/L xylitol.These results demonstrated that increasing XDH activity could improve xylitol productivity.
Alliinase is immobilized on pre-activated chitosan microspheres by glutaraldehyde.The optimum immobilization conditions are as follows: glutaraldehyde concentration is 4%,alliinase concentration 20.2U,immobilization time 2h.For immobilized alliinase,the highest activity was allowed at pH 7.0 and temperature 35℃,and the Michaelis constant(Km)was disclosed to be 7.9mmol/L by Lineweaver-Burk plot,and after 10 times reuses the immobilized alliinase lost no more than 10% activity.
The antioxidant activity of lycopene is highest among the current carotenoids. Recently the researches on lycopene are focused on ingredient of functional foods in the world. In this study, the fermentation production of lycopene by Streptomyces rimosus was reported first time in China. The determination methods, such as spectrophotometric method and HPLC were constructed. Using a stain Fc of Streptomyces rimosus as a original strain, a high-yield lycopene producing mutant strain Fc’ was selected after UV mutation. The lycopene yield of the strain is 2.5 times higher than the original strain Fc. The optimal fermentation conditions were determined by flask experiments, and the lycopene yield of strain Fc’ reached 230mg/L in flask under the optimal fermentation condition，meanwhile the Streptomyces rimosus strain could produce purer lycopene without adding any blocking agents in its fermentation process. Our results lay a good foundation for lycopene commercial production by fermentation using Streptomyces rimosus .
Fermentation for EPA production by diatom Nitzschia laevis under different mediums was investigated.As a result, the basic medium was selected,which is 8LDM that is enhanced the concentration of nutritions 7 times. By applying 8LDM medium, the EPA yield reached 93.22mg/L. This result was 4.24 times and 4.36 times to f/2 and LDM, respectively.
Npu DnaE intein was used to produce some large proteins, which were difficult to obtain through conventional expression systems. A T7 expression system was described, by which the gene of T7 RNA polymerase is split into two pieces, and each piece fuses with Npu DnaE N and C terminal sequences respectively. Functional T7 RNA polymerase is created by mixing the two kinds of fusion constructs in vitro.The approach of split inteinmediated production of large proteins, in theory, readily generalizable to the purification of other large, cytotoxic or membrane proteins.
Objective: To establish a multiplex PCR-microarray method for detecting important enteropahogens.Methods: Uniplex and multiplex PCR were performed to obtain the best primer sets for identifying the target bacteria at species and multi-species level. Fluorescent dyes were mixed into PCR reaction to determine whether it can affect the efficiency of amplification. To improve the efficiency of microarray, a 35 pairs primer-labeling system was optimized based on the hybridization results to find the best combination to avoid false negative results. Results: Specific PCR products were all obtained using species-specific primer sets. More preferential amplification may happen when more primer pairs were added to the reaction. The hybridization results showed a positive association between the efficiency of multiplex-PCR and signal intensity. Conventional PCR yielded more products than fluorescent dyes labeled PCR. Thirty-five primers were divided into three different combinations to label target respectively, hybridization results showed a high specificity. Conclusion: Mixing fluorescent dyes into PCR may reduce the efficiency of amplification and hybridization，but may have no effect on the analysis of hybridization results. The hybridization efficiency of microarray depends on the amplification efficiency of multiplex PCR. For microarray target labeling, three primer sets could be used to avoid negative hybridization led by preferential amplification of multiplex-PCR.It indicates that the multiplex PCR-microarray method is an attractive diagnosis tool for the high-throughput identification of enteropathogenic organisms especially for multiple causative agents and epidemiological investigations.
To develop a reverse dot blot assay for rapid detection of HBV genotypes. Specific oligonucleotides probes were desighed and immobilized on nylon membranes. The DNA sample to be tested was PCR-amplified with DIG labeling primers and then hybridized with the immobilized probes. This procedure for detecting HBV genotypes was simple,rapid and specificity. 30 specimens in Chongqing area were collected and detected by this method, and results were evaluated using direct sequencing. Results showed that: This new method was applicable to precise detection HBV genotypes for specimen with copies up to 103, and the HBV genotyping results showed that genotype B was the predominant genotype in Chongqing area．
The MegaBACETM 1000 DNA Sequencing System was made by Amersham Pharmacia, which is a high throughput fluorescence-based DNA sequencer that can be used for DNA sequencing and genetic analysis etc. But the markers（ET550-R Size Standards）produced by this corporation which are used for genetic analysis in this instrument are very expensive. In order to cut the cost,a set of markers were developed and after authentication,that can be used in the MegaBACE 1000 DNA sequencer. Using these markers and our own recognition software,an improved, high throughput AFLP work protocol based on MegaBACETM 1000 were constructed.
To construct knockout vectors containing ampicillin resistant gene and partial sequence of hyaluronidase gene(Hyl)so that Hyl can be knock out by transforming the plasmid into Streptococcus zoopidemics mutans.First,partial sequence of Hyl（Hyl-1）was cloned into the vector of pMD19-T by using DNA of Streptococcus zoopidemics as template,and then a knockout vector pMD19T-SA was constructed, in which Hyl-1 gene was disrupted by inserting ampicillin resistant gene(Amp)from reverse PCR. As expected, the vector was proved to be consisted of Hyl-1-Amp-Hyl-1-pMD19-T.Thereafter,DNA fragment of Hyl-1-Amp-Hyl-1 was subcloned into pBR322 vector,the resulting construct was then checked by PCR and restriction analysis for the proper configuration of the knockout vector pBR322-SA.Both of the knockout vectors were used to transform Streptococcus zoopidemics and one recombinant was obtained in result. From results of PCR and Hyl activity assay, it was indicated that in the recombinant the Hyl gene was disrupted completely.
ρ-Hydroxyphenylpyruvate dioxygenase(HPPD)catalyzes the formation of homogentisate from ρ-hydroxyphenylpyruvate and O2. In plant, HPPD is one of the key enzymes in the biosynthesis of prenylquinones plastoquinones and tocopherol. Since plastoquinone is the final electron acceptor in carotenoid biosynthesis, a lack of this component in thylakoids impairs carotenoid biosynthesis, which results in bleaching symptoms in leaves as in phytoene desaturase inhibition. Currently, three bleaching herbicide families which target for HPPD, isoxazoles, triketones, and pyrazoles, have been reported. Some kinds of this herbicides family are used control of a wide range of important broadleaf and grass weeds in maize and rice filed. Advances in this new bleaching-herbicide target enzyme and herbicide-resistant transgenic plants were overviewed.
The arbuscular mycorrhizal(AM)symbiosis is a mutualistic endosymbiosis formed by plant roots and AM fungi. The AM symbiosis is manifested in bidirectional nutrient exchange: AM fungi obtain carbon from their plant host while assisting the plant with the acquisition of mineral nutritions(in particular phosphate)from the soil. AM symbiosis facilitate phosphate uptake is the central reason of plant growth and development. Phosphate transport into the root is mediated by phosphate transporters, and phosphate transporters play a role in the acquisition of phosphate released by the fungus in the AM symbiosis.The molecular biology of arbuscular mycorrhizal symbiotic phosphate transporters in plants was summarized. AM phosphate transporter belong to Pht1 family, is not only essential for the acquisition of phosphate, also critical to AM symbiosis. Study of phosphate transport roles and their gene regulation will further our knowledge of the interaction between the two symbiotic partners, and so as to provide innovative approach to improving phosphate efficiency and agricultural yield.
With development of wide-host-range vector systems, Tn5 transposon and its derivative vectors have been widely applied to genetic research of gram-negative bacteria.The applications of Tn5 transposon mutagenesis technology to genetic researches of bacteria were briefly discussed, including researches on biological control mechanisms of biocontrol bacteria, identification of bacterial essential genes, discovering virulence genes of bacterial pathogens, characterization of metabolism regulatory genes and genetic improvements of bacteria.
Saccharomyces cerevisiae strains may have some defects in beer production. Purposeful alteration of metabolic pathway with molecular biology techniques after analysis the metabolic fluxes has been a main way of S.cerevisiae breeding. The researchers have done a lot of work on some aspects of S.cerevisiae, such as the substrate utilization, maneuverability, reducing the no use by-products, improve the beer flavor, and got many delightful results,all of this are summarized here.
