Mutant construction is a prerequisite work in bacterial gene function studies. In this report, we constructed a new suicide plasmid to construct Brucella mutant. A kanamycine gene was inserted at the multi-cloning site of pUC19, and on either side of the resistance gene, several enzyme sites were inserted, resulting in the new plasmid pUC19K. Using this plasmid, we constructed replacement mutant of Brucella omp25 gene. As the results showed, the mutant could be generated by only one round of selection with high efficiency. Therefore, the construction and application of pUC19K provides a new suicide plasmid for Brucella mutant construction and would accelerate gene function analysis of Brucella.
Mitochondrial dysfunction has been implicated in the aetiology of sporadic Parkinson’s disease but its role in the disease mechanism remains unclear. To investigate the effect of synuclein on mitochondrial dysfunction induced by rotenone. We used the human dopaminergic SH-SY5Y cells as a cell model. The cells over-expressed the wild-typeα-synuclein were treated with complex I inhibitor rotenone. The cell viability,complex I activity, Mitochondrial swelling and O2¯ content were tested at different time point-1W, 2W, 4W after rotenone treated. CCK-8 test results showed that the cell viability of overexpressed α-synuclein (SH-SY5Y-Syn) was much lower than the control group(SH-SY5Y-Ctr). After administrating with rotenone about 1or 2 weeks the cell viability of SH-SY5Y-Syn became higher than that of SH-SY5Y-Ctr. On the 4th week the results were contrary to the first 2 weeks. Similar results were got when test the mitochondrial function. In the first 2 weeks after rotenone administrating, the mitochondrial function of SH-SY5Y-Syn was better than that of SH-SY5Y-Ctr. This suggest that the α-synuclein could protect the mitochondrial against the injury induced by rotenone in the early stage-1W, 2W, while this effect disappeared in the final stage-4W.
Metallothioneins (MTs) are a family of low-molecularweight, cysteine-rich, metal-binding proteins, widely distributed in nature and have very important functions such as heavy metal detoxification and essential metal metabolism. It is very difficult to express recombinant MT directly because of toxicity to host cells, presumably owing to its thiol groups, and because of general difficulties encountered in expressing small proteins. A DNA coded fusion protein GST-SUMO-MT was constructed and cloned into vector pET-28a. The fusion protein was expressed in E.coli Origami (DE3) and the amount of expressed fusion protein in cultural media using described strategy was 70 mg/L. The fusion protein, GST-SUMO-MT was purified using the combination of Glutathione Sepharose chromatography and Sephardex G-25 and the purity was higher than 95%. GST-SUMO-MT could improve the endurance of host for the accumulation of Cd2+, Zn2+ and Cu2+and the endurance activity was 4.2, 4.0 and 1.6 times than that of control respectively. Moreover, every fusion protein, GST-SUMO-MT, could combine 2-3 Cd2+ detected by Atomic absorption spectrum.
The gene of mutaed TNF-αD4 gene was amplified by overlap PCR and cloned into the prokaryotic expressive vector pBV220. TNF-αD4 contains two changes: substitutions of pro8arg,ser9lys,asp10arg,ile157phe,leu 29ser,arg31val and a deletion of the N terminal four amino acids . The recombinant vector pBV220-tnf-αD4 was transformated into E.coli strain DH5α, and the high expression strain was obtained by screening monoclones. The level of expression was about 45% of total cell protein. After purification, the purity of fusion protein was above 90% by HPLC and relactive ability was 8 ×107 .TNF-αD4 was modificated by mPEG-ButyrALD。After purification， the purity of mPEG-TNF-αD4 was above 85% and relactive ability was 8.6 ×107. and the in vivo systemic toxicity of mPEG-TNF-αD4, which is indicated by LD50, is lower than that of rhTNF-a .These results strongly supported for the further study and exploitation of TNF-antitumor drug.
After Sumo molecular chaperone and Antifungal Peptide Drosomycin were synthesized by PCR, the recombinant plasmid pET-3c-SD was constructed successfully. After the recombinant plasmid was transformed into BL21(DE3), fusion proteins expressed in the E.coli 21 (DE3) by the induction of IPTG. Target proteins attained percent 80 in total proteins. Meantime, Soluble protein exceeds 80％ in total target proteins. The fusion protein was purified by Ni-NTA affinity chromatography，and its purification exceeds percent Ninety-five. The experiment shows the fusion protein own the antifungal activity, and this construction strategy help to facilitate small molecular peptide expression that had several two disulfide bonds
A novel gene, located between dnrX and drrB in the genome of daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482, was cloned and named as dauW. The full sequence of dauW was submitted to GenBank (Accession No.EF523565). Blast result indicated that it showed high homology with dnrW in GenBank. The exact function of dauW is as yet unknown despite the possibility that it might belong to a family of FAD-dependent oxidoreductases on the basis of conserved domain analysis. dauW was cloned into expression plasmids pET-28a(+) and pET-32a(+), respectively, and was successfully expressed in E.coli DE3 after induction with IPTG. The preliminary results of the expression of dauW suggested that it might be involved in the self resistance in Streptomyces coeruleorubidus due to the increased resistance to daunorubicin in the E.coli host.
To obtain a number of extracellular penicillinase, the gene (penp) encoding penicillinse of Bacillus cereus ATCC10987 was cloned into expression vector in Bacillus subtilis , transformed into Bacillus subtilis DB104 deficient in tow proteases。When recombinant bacteria was cultured in LB medium for 24 hours, the result of SDS-PAGE showed that the molecular weight of the protein was 28KDa and the penicillinase activity reached 339U/ml; By screening severn different fermentation media, the result showed that 4# medium is favorable to producing penicillinase by the recombinant cells than the others, with the yield of the enzyme being 1580U/mL; when the recombinant cells was cultured in 7 liter fermentor for 24h ,the penicillinase activity reached 1255.8U/ml．
Chromhalobacter sp.NJS-2 was isolated from Antarctica deep-sea sediment. The ectABC gene from this strain was amplified by PCR，consisting of 2,378bp. Analysis of the OMIGA software showed that the whole sequence involves three open reading fragments, which were 576bp、1272bp and 393bp. They encoded L-diaminobutyric acid transaminase (EctB)，L-diaminobutyric acid acetyl transferase (EctA), and ectoine synthase (EctC), respectively. The amplified fragment of ectA was cloned into the expression vector pET-his. The insert position, the size and the reading frame were identified by PCR, restriction digestion and the sequence analysis of the recombinant plasmids. SDS-PAGE showed that the relative molecular mass of the expression product was 21 KD as predicted, which indicated that the recombinant plasmids could express the gene of L-diaminobutyric acid acetyl transferase. Enzyme activity detection of purified EctA partially elucidated the biosynthetic pathway of ectoine.
Fungal diseases of plant reduce yield and productivity of many economical crops. Chemical control of disease is costly both to the environment and to human .Therefore, screening antifungal bacteria for use in biological control becomes more important. Bacillus amyloliquifaciens isolated from the compost showed strong antifungal activity against a lot of plant pathogens. By ammonium sulphate precipitation, the crude extract was obtained from the culture medium of Bacillus amyloliquifaciens. Then it was isolated and purified by gel filtration on Hiprep 26/10 Desalting column，ion-exchange chromatography on HiLoad 26/10 Q Sepharose column. There are four fractions after the two steps above, and the third one was collected to be analyzed by HPLC. Eventually, three peaks were obtained, the second one which showed strong antifungal activity was collected as a pure substance. The molecular weight of it is 1498 Da as determined by ESI mass spectrometry. The pure substance shows strong inhibitory activity against Fusarium oxysporum f. sp. spinaciae O-27, Fusarium graminearium，Ramularia tulasnei Sacc and Coniothyrium diplodiella. The abnormal hyphal growth of Ramularia tulasnei Sacc by the active substance such as twisted hypha, swollen tips was observed, In addition, the number of the conidia is less.
One strain Bacillus subtilist B-115 was screened, which can use crop materials to product γ-Polyglutamic acid. The high productive strain B-115 of γ-PGA was obtained from the primitive strain Bacillus subtilis B-1through UV mutagenesis. The yield of γ-Polyglutamic acid increased from original 12.5g/L to 19.5g/L by flask fermentation. In order to enhance the production of this strain, we used Response Surface Methodology (RSM) to optimize fermentation medium ,such as carbon source, nitrogen source, concentration of sodium glutamate, metal ions, the final yield of γ-PGA was upto 40.98g/L.
ep8, a thermostabilized mutant of Penicillum expansum lipase obtained in a previous study, contained a single amino acid substitution. To further improve the thermostability of the lipase, the Lys of wild-type (lip07) and mutant (ep8) in 202 were separately substituted by Ala using the Overlap extension PCR technique. The mutant genes (lip07-K202A and ep8-K202A) were subcloned into pAO815, and then transformed into the Pichia pastoris GS115 for extracelluar expression, separately. 15% SDS-PAGE analysis indicated that the molecular mass of PEL-ep8-K202A and PEL-lip07-K202A are both about 28KD, which are just the same with the wild-type lipase. The comparison experiments showed that: The Tm of PEL-ep8-K202A is 41.66℃，2.63℃ higher than that of the wild-type (39.03℃) and 1.21℃ higher than the random mutant(PEL-ep8:40.45℃); the Tm of single mutant (PEL-lip07-K202A) is 37.08℃, 2℃ lower than that of the wild-type lipase.
Fermentation for Eicosapentaenoic Acid (EPA) production by Nitzschia laevis at various temperature between 10℃ and 30℃ was investigated and the dynamics characteristics during fermentation process were also analyzed. Based on the results, a varying temperature nursing method of two stage control strategy is proposed: During the firs stage, which comprises the delay phase and the initial index phase, the temperature is maintained at 25℃; then the temperature is shifted to 20℃ and kept up till the end of the fermentation process. By this method, a EPA content of 6.0% and a yield of 291.60 mg·L-1 have been gained. These are 24.07% and 18.81% higher than that of fixed temperature (25℃) fermentation, respectively.
The oxidation of nitrite to nitrate by nitrite oxidizing bacteria (NOB) in aquaculture systems was an important process, while it was difficult to get the commercial NOB in the market, the one of reasons was that NOB degraded quickly during the course of conservation. But the studies on the decay of NOB were few. Therefore，the influencing factors of the decay of NOB and degradation kinetics was studied. The results showed that the temperature, pH, ionic strength and osmotic pressure conditioner were important influencing factors, the anoxic decay rate of nitrifying bacteria reduced from 0.25 to 0.015 under 4℃, the half life was extend to 55d.
Vibrio parahaemolyticus has been considered as one of the most important foodborne bacterial pathogens. In this paper, the loop-mediated isothermal amplification (LAMP) that amplifies DNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of this pathogen for the first time. A set of four primers, two outer and two inner primers, was designed specifically to recognize the thermolabile hemolysin gene (tlh) of V. parahaemolyticus in this study. The LAMP reaction mix was optimized. The most optimal reaction temperature and time of the LAMP assay for the tlh gene were 60 ℃ and 60 min, respectively. Genomic DNAs from 28 bacterial strains including 14 V. parahaemolyticus strains were amplified using LAMP, and no amplicon was observed in other bacterial strains. The detection limit of this LAMP assay was around 90 fg of V.parahaemolyticus genomic DNA and 24 colony forming units for pure cultures. In addition, this method was applied to detect artificially contaminated food samples, and the detection limit was 89 cfu/g for non-cultured artificially contaminated food samples. These results suggested that detection of V. parahaemolyticus by LAMP is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid detection and identification of V. parahaemolyticus.
In this paper, we have analyzed and discussed some aspects in the 2-dimensional gels electrophoresis such as the serum sample solubilization, the gel isoelectric focusing program ,the gel equilibration, the gel stain etc, generalized some problem in operation which must be stressed, and obtained the high resolution and good repetition results. It is referred to the serum sample analysis in 2-dimensional gels electrophoresis technology.
Statistic-based experimental design (RSM) was used to optimize the culture conditions for Pseudomonas aeruginosa ME16 strain which capable of utilizing methane. Effects of inoculum volume, temperature, methane content, and initial pH of media on cell growth were studied with the medium of mineral salt and methane gas, then the response surface analysis was used to optimize culture conditions, The optimum conditions found are: temperature 29.4℃, inoculum volume 1.8% and methane content 25%. The optimized culture conditions allowed the production to be increased 0.8 times and cultivation time to be decreased. The removal of methane gas was preliminary studied by ME16 , the removal rate for methane gas reached 65.7%. The results indicated that the strain capable of removing of methane.
Abstract To explore the plasticity of human amniotic mesenchymal cells (hAMCs) into cardiomyocyte-like cells. hAMCs were isolated from human amnion with collagenase digestion. Phenotype of the isolated cells was analyzed by flow cytometry (FCM). hAMCs were treated with 5-azacytidine and basic fibroblast growth factor(bFGF) to investigate their ability of differentiation into cardiomyocytes. The induced differentiated cells were evaluated by immunofluorescence for desmin and α-actinin expression and by RT-PCR for Nkx2.5, GATA-4 and alpha-myosin heavy chain (α-MHC) mRNA expression. The results showed that, after primary culture, hAMCs could reach a confluence of 80% with swirl like growth at 6 days. The cells proliferated rapidly by passaged with a 100% confluence at 3~4 d. hAMCs was positive expression of CD44 and vimentin, but negative for CK19. After induced differentiation at 8~10 days, the differentiated cells have close-up arranged with long spindle-shape. At 2 weeks and 4 weeks, induced cells expressed α-actinin and cardiac-specific transcription factor Nkx2.5. Expression of GATA-4 and desmin can be detected but α-MHC can not both in before and after induced hAMCs. In conclusion, hAMCs have a ability of differentiation into cardiomyocyte-like cells which mean that hAMCs can be regarded as candidate cells for cellular cardiomyoplasty (CCM).
CAPN1 was investigated as a potential candidategene for a quantitative trait locus (QTL) affecting meat tenderness.According to the CAPN1 gene sequence of Cattle in GenBank,the specific primers Were designed.Using the Tianzhu White yak cDNA as template,PCR amplifying,cloning and Sequencing for every fragment.Using the BioEdit to splice sequences and the a 2267bp yak cDNA of CAPN1 gene was obtained.Including a complete open reading frame and partical sequence of 3′ end and 5’ end UTR. Analysis of the gene revealed that the yak CAPN1 gene is encoded by 2151bp and the 716 AA potein predicted from this sequence. The nucleotide sequence and amino acid sequence of yak calpain were compared with those o f cattle, pig, human,and mouse calpain. Identities for nucleotide and amino acid sequences were 99.3 and 99.4% for cattle,93.9 and 96.1% for pig, 90.0and 94.6% for human, and 85.5and 89.0% for mouse calpain. Search using NCBI BLAST of yak four domains respectively found the four domains in the above 4 species have shown good conservative, the most conservative in domain IV (> 96%). There are 14 nucleotide mutations between yak and cattle and three of that were predicted to alter the protein sequence,both of which occurred in the domain III. Construction of molecular phylogenetic tree shows : The cluster results coincided with the traditional taxonomy .
Gentamicin was a broad-spectrum aminoglycoside antibiotic. It not noly was used on the clinic to cure, but also applied to the stockbreeding widely. By reason of fermentation cycle long, production rate low and production costs high, so amelioration production method was imperative under the situation. The paper reported that added a little quantity of lysine, glycine, tyrosine, methionine into the medium of gentamicin producing Micromonospora purpurea during inoculation could improve the metabolic cabability of microbe, shorten fermentation cycle and increase production rate.of gentamicin. The new method was successfully developed in shaking flask test and applied to 5L glass fermentor for one month. Average data of one month showed that the fermentation time per cycle was shortened about 40% and the yield of gentamicin per fermentor increased about 14% in comparison with the conventional technique. The production rate of gentamicin was increased 30% ~950% (according to the capability of strain). The quality of the end product ——gentamicin conformed to CP2000, BP2000 and USP26 pharmacopoeia. Therefore, the new method remarkably may reduce production costs of gentamicin, show strong competitiveness in the marketplace.
A review of studies on the influence of impurities on protein crystallization is given . The possible sources of impurities and its effect on the protein crystallization are presented. The effects of impurities on protein crystallization, including nucleation, macroscopic morphologies, microscopic surface morphologies, growth rates, kinetics, quality, and repartitioning of impurities are reviewed.
3-Dehydroshikimic acid (DHS) is an intermediate in the pathway of aromatic amino acid biosynthesis and is shown to serve as a suitable starting compound for the renewable production of a variety of industrial chemicals . The biocatalysis using renewable material as feedstocks has less environmental pollution compared with traditional chemical synthesis. In addition, DHS can be used as a potent antioxidant. Chemical synthesis and fermentive method is the major industrial way to produce DHS, and microbial rational modification based on the development of metabolic engineering made the fermentive method more widely used. In this paper, metabolic engineering in improving DHS-producing microorganisms is reviewed ,involving the regulation of genes and enzymes in biosynthesis pathway of DHS，reconstruction on central metabolism and modification in DHS biosynthesis branch，moreover ,the prospect of DHS production is also mentioned．
