Study the protection and repairation of damaged neurons which leads to neurodegenerative diseases, neurotrophic factor Neurturin gene was transfected into Rhesus bone marrow mesenchymal stem cells (rMSC) by recombinant adenovirus. The NTN gene could be transcribed and expressed in cytoplasm of rMSC and be secreted which was detected by RT-PCR、immunofluorescence、Western blotting, and then, the culture of Chicken embryonic dorsal root ganglion test and the culture of Fetal rats midbrain dopaminergic neurons test had indicated that the NTN protein had good biological activity in vitro. The results gave a new method for stem cell transplantation to treat the neurodegenerative diseases caused by neurons damage.
The purpose is to investigate the effects and possible mechanisms on BMP9 inhibiting the bone metastasis of human breast cancer MDA-MB-231 cells. High titer adenovirus vector expressing BMP9 was used to infect MDA-MB-231 cells. Experimental group is MDA-MB-231/BMP9 cells,control groups are both MDA-MB-231/GFP cells and MDA-MB-231 cells. RT-PCR and Western blot were used to detect the expression of BMP9 and PSmad1 in recombinant MDA-MB-231/BMP9 cells; Q-PCR and Western blot were used to detect the expression of CTGF in three groups cells; At last, X Light and Immunohistochemistry were used separately to detect the osteolytic lesions and the expression of CTGF in three groups cells. BMP9 was expressed in recombinant MDA-MB-231/BMP9 cells; the recombinant MDA-MB-231/BMP9 cells exhibited higher level of phosphorylated Smad1 and lower level of CTGF than the MDA-MB-231/GFP cells and MDA-MB-231 cells, but total Smad1 protein was similar in the three groups. In comparison with the control groups, the osteolytic lesions observed were significantly reduced in MDA-MB-231/BMP9 group; The removed tumor samples were detected by immunohistochemistry, MDA-MB-231/BMP9 samples analysed showed less CTGF staining than the control samples. Taken together, BMP9 can inhibit the bone metastasis of breast cancer cells MDA-MB-231 in vivo and the downregulation of CTGF is the possible mechanism.
In order to express the central core domain of HIV-1 integrase (IN-CCD) in a soluble form and establish a method for inhibitor screening targeting IN-CCD in vitro, the gene encoding the IN-CCD protein with F185K mutation was amplified from a plasmid containing the full length of IN gene. The gene product encoding IN-CCD protein with F185K mutation was introduced into the pET28b vector to construct a pIN-CCD recombinant plasmid, which was subsequently transformed into E. coli BL21 (DE3) and induced with IPTG. After cell lysis, the soluble IN-CCD in the supernatant was purified by Ni-affinity column chromatography. DNA substrate was modified, and streptavidin-coated magnetic beads were employed as a solid support to capture the DNA reaction product. Finally, an enzyme-linked immunosorbent assay (ELISA) was developed for the activity characterization of IN-CCD and subsequent inhibitor identification. The results showed that IN-CCD was highly expressed in soluble form; the purity of IN-CCD protein was about 95% after purification and dialysis. The ELISA established in this work was sensitive and specific for the detection of IN-CCD disintegration reaction, and could be used for the high-throughput screening of IN-CCD inhibitors. Five samples to be active IN-CCD disintegration inhibitors were screened out from 100 samples.
The aim of the construction of mammary gland bioreactor is to obtain the high-level expression of exogenous proteins in mammary gland. Prolactin plays an important role in the regulation of milk protein synthesis and secretion during mammalian lactation. The previous study demonstrated that bovine prolactin can efficiently promote the expression of exogenous gene (hTF) in milk of transgenic mice. To further explore the mechanism underlying this finding, the culture protocol were developed to cultivate transgenic mouse mammary epithelia and the biological effects of bovine prolactin on these cells were studied. Mammary tissue of mouse carrying human transferrin gene (hTF) were minced, digested with collagenase. Epithelial cells were then collected and cultured, subsequently purified by timing cells adherence to flask wall. Afterwards, purifed cells were transfected with pCMV-bPRL plasmids. The synthesis ability of the cells was tested by detecting the secretion of related proteins present in supernatant. Epithelial cells of transgenic mice carrying hTF were successfully cultured in vitro, hTF was shown to be presented in supernatants of cells. The level of hTF was elevated with the presence of bovine prolactin(1 208.55±20.07 pg/ml vs 3 161.32±71.62 pg/ml). The culture protocol could be employed in the near future experiments to investigate effects of bovine prolactin and environmental factors on the regulation of protein synthesis and secretion of mammary epithelial cells.
Gastrointestinal complications are common in patients with diabetes mellitus which involve the acute gastritis and gastric ulcer. Because gastric dyskinesis, delayed gastric emptying, gastric bacterial over-growth, these will further lead to intestinal diseases. Research on the change in diabetes stomach content bacterium group structure, and it’is important for the study of diabetes pathogenesis and the treatment of complications. Bacterial DNAs from 10 type 2 diabetes mice and 10 normal control mice were extracted from gastric contents and gastric mucosal samples and then characterized by PCR-denaturing gradient gel electrophoresis (DGGE). After DGGE profilings was obtained,the diversity and similarity analyses were carried out by the number of band,Shannon-Weaver(H’), Margalef index(R), Pielou index(E) and Simpson index(D),cluster analysis and cloning sequencing. The results show that there was no significant difference in Shannon-Weaver diversity index、Margalef index 、Pielou index、and Simpson index between experimental group and control group. There is no significant distinction in the Dice similarity coefficient (Cs) between the two groups. The result of sequencing illustrates that Lactobacillus is in the gastric of normal control mice, but type 2 diabetes mice. These results suggest that lactobacillus is closely related to type 2 diabetes.
Signal recognition particle receptor (SR) is important in secretive protein synthesis and secretion process. Tubulin is essential in various life process inside cells. Comparison between proteins encoded by ESTs of α-tubulins and SR in GenBank database of Aspergillus Flavas resistance(C20R) and sensitive(TFR) cultivars reveals that for two types of α-tubulins α and α7, there are 3 ESTs each in TFR early seed developmental stage 5, there is only one EST each in C20R seed developmental stage 5 and 7. For SR, only in C20R seed developmental stage 6 and 7, there is one EST in each stage, none in any seed developmental stage of TFR. QPCR assay on expression of α-tubulins and SR in different organ and developmental stage of peanut fruit of Aspergillus Flavas resistance cultivar KB153 and sensitive cultivar JH1012 indicates that in small fruit stage of fruit early developmental stage, SR and two types of α-tubulins all up-regulated in Aspergillus Flavas resistance cultivar KB153 than sensitive cultivar JH1012. This suggests that protein transport via ER intermediated by SR is related to Aspergillus Flavas resistance. SR is also up-related in cotyledons of Aspergillus Flavas resistance cultivar KB153. This is corresponds to the higher content of storage protein including proteinase inhibitor in Aspergillus Flavas resistance cultivars.
Upstream sequence and partial coding sequence (CDS) of the phosphoglycerate kinase (PGK) gene were cloned from oleaginous yeast Trichosporon sp. by degenerate PCR and chromosome walking. The computation analysis revealed that a promoter-like sequence in the upstream region of the PGK coding region including essential elements such as TATA BOX and CAAT BOX was cloned. The promoter-proximal region cloned was fused with the coding region of the hygromycin gene, and then cloned into the expression vector pTF-PHT. The recombinant vector was transformed into Trichosporon fermentans, and the transformed yeast was able to grow on the selective agar containing hygromycin. The results indicated that the promoter-proximal region of the phosphoglycerate kinase gene of Trichosporon sp was able to drive the expression of heterologous genes in the Trichosporon fermentans, which provides new avenues for developing novel engineering yeast or yeast expression system.
Objective: Bacillus amyloliquefaciens Q-426 could produce varieties of antifungal lipopeptides during its stationary growth process, including bacillomycin D, fengycin A and B. Effects of diketopiperazines (DKPs) as signal molecules of quorum sensing (QS) on the biosynthesis of above antifungal compounds were studied through real time fluorescent quantitative polymerase chain reaction (Real-time Q-PCR). Methods: DKPs at a final concentration of 5 mg/l were added to the culture broth of strain Q-426 which was incubated at 30℃ for 12 h. After continuing cultivation for 48 h, quantitative analysis of mRNA expression levels was carried out by Real-time Q-PCR. Result: DKPs could inhibit the expression of some genes related with the biosynthesis of antifungal lipopeptides.
The essential oil of C. reticulate Blanco was extracted by hydro-distillation and analyzed by GC-MS. Forty six components were identified. The major components were limonene (57.67%), followed by β-linalool (5.36%), 2-carene (4.47%), β-pinene (4.31%), γ-terpinene (4.08%), α-pinene (3.27%), Cyclobutane,1,2-bis(1-methylethenyl)-(2.87%), β-myrcene (2.77%), α-thujene (2.40%), β-phellandrene (2.24%), decanal(1.80%) and citronellal (1.49%) etc. The effects of C. reticulate essential oil and pure compounds in varying concentrations on the growth of Penicillium sclerotiorum by poisoned food technique and liquid culture were also elucidated. Results showed that C. reticulate essential oil has significant inhibitory effect on the growth of P. sclerotiorum in varying concentrations, with the increasing concentration positively correlated with inhibitory efficiency, and 20μl/ml of the essential oil can inhibit the growth of P. sclerotiorum absolutely. Results by poisoned food technique showed that the antifungal activity of the essential oil at a concentration of 20μl/ml became weaker with time progressed. The study of seven pure compounds against P. sclerotiorum by poisoned food technique indicated that the growth of this strain was effectively inhibited by 0.04μl/ml citral and 1.07μl/ml β-linalool, while no obvious inhibition effect was observed by the remaining pure compounds. The present study suggested that the significant inhibitory effect of Ponkan essential oil on the growth of P. sclerotiorum could be attributed a lot to citral and β-linalool in it.
Objective: Aims to explore the effect of the incubation time in the enrichment of the phosphopeptides with Titanium dioxide. Methods: Firstly, the optimum ratio between beads and peptides based on the preliminary study is ascertained, and then compare the effect of the incubation time on the enrichment with Titanium dioxide. Results: The optimal ratio between bead and peptide is 3 :1. Under this optimized ratio, the longer incubation time decreases bead selectivity and 5 minutes is the optimal incubation time. Conclusion: The incubation time has the negative effect on the phopshopeptide enrichment which indicates that the optimal time depends on sample species.
Objective: Explore the effect of a new airway miniaturized nebulizer on the plasmid DNA (pDNA) integrity and animals gene transfection efficiency.Methods: In the first experiment, the effect of the new airway miniaturized nebulizer on pDNA integrity was tested.2ml pDNA(20μg/ml) was put into the sample pool of the new airway miniaturized nebulizer and a clinical commonly used jet nubulizer respectively. Aerosol samples were collected at the 1st min, 3rd min, 5th min from the nozzle of the two nebulizers respectively and investigated with agarose gel electrophoresis.In the second experiment, the effect of gene transfection efficiency of the new airway miniaturized nebulizer on animals was studied.3.3μg PEI/pEGFP (polyethylenimine and plasmid DNA of green fluorescent protein complex formation which contain 3.3μg pEGFP) was aerosolized to rats via the new airway miniaturized nebulizer and a clinical commonly used jet nubulizer respectively.Rats were sacrificed 24h after aerosol treatment. The mRNA was abstracted from the lung of those rats, reverse transcribed into cDNA and determined by real-time PCR. PCR products were also investigated with agarose gel electrophoresis.Result: At the 1st min, 3rd min, 5th min, the proportions of integrity pDNA of the new airway miniaturized nebulizer were (99.6±0.7)%,(100±1.2)%,(99.6±0.7)% respectively, and compared with the control group. The result was no significant difference between the two group(P>0.05, n=3). The effect of the new airway miniaturized nebulizer on plasmid DNA can be neglected.At the 1st min, 3rd min, 5th min, the proportions of integrity pDNA of the clinical commonly used jet nubulizer were (70.3±1.5)%, (49.3±1.5)%,(32.7±0.6)% respectively, and compared with the control group. The results was significantly different between the two group(P<0.05, n=3). The proportion of integrity pDNA of the jet nubulizer was decline graduately accompany with time.The new nebulizer yielded 382.1±101.1 fold higher expression levels than the ordinary jet nebulizer(P<0.01, n=3)Conclution: The new airway miniaturized nebulizer provided an excellent protection on the plasmid DNA, increased the efficiency of gene transfection significantly, and provided a suitable device for aerosol gene therapy.
miR-17-92 cluster is highly conserved and involved in the development of diverse organs and solid tumors in mammal. Several transcriptional factors and target genes are found, which regulated and be regulated by miR-17-92, formed some feed-forward and feed-back loops via different online databases. After functional cluster analysis of genes involved in the circus regulated by miR-17-92, the regulation network responses to miR-17-92 has been drafted out. The anlysis indicates that the jointargets of miR-17-92 and its upstream transcriptional factors may play important roles in the biological processes such as cell cycle, metastasis, apoptosis, hormone responses and the development of immune system. KEGG pathway analysis also implied that it was related to many signalling pathways in tumors. The study will be a good guideline for the further research and provide great facility for understanding the mechanism of cell development and tumorousgenesis.
Neurospora crassa is the native degrader of cellulose, it has capability to produce the mainly components of cellulases, such as CBHs, endoglucanses and β-glucosidases. It has some studies focus on the mechanism or cellulases productivity in Neurospora crassa, but the cellulases fermentation by this fungus is far from optimized. Here, The Response Surface Methodology(RSM) to optimize the medium for cellulased production in FGSC.2489. Combined the Plackett-Burman and Central Composite Design design, the peptone, yeast extract have significant effects on enzyme activity were found. The optimum amount of the Peptone is 7.27g/L and Yeast extract is 5.51g/L in medium. Based on the optimized medium, the cellulases production have significantly improved near 2 folds, from 0.64FPU to 1.27 FPU.
A mutant database has been built and mutants with acid tolerance or thermostability or high activity which would be taken as parent gene of DNA shuffling was also screened. Then mutants with higher thermalstablility and acid tolerance from DNA Shuffling database was searched by facilitation and 96 deep wall plate culture screen. Futhermore, some information about bioinformatics of β-mannanase by sequence comparison and homologous model was also been got. Through two cycles of DNA shuffling, a mutant database was built. Then one optimum 1108 was screened from about 104 mutants. The evoluted β-mannanase displayed both higher thermalstability and acid tolerance than wide type. The evoluted enzyme 1108 retained high activity after treatment at 90℃ for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of 1108 under 80℃ and acid treatment was 10 times and 5 times as wide type respectively. The sequence comparison illustrated that there were three nucleotide substitutions T289A, A535T, T1085C which carried corresponding amino acid changes Ser97Thr, Val362Ala, Ile179Leu. According to homologous modeling by SWISS-MODEL Repository and amino acid analysis, There is a foundation mutations locate near the catylyze core, So there is a speculation Ser97Thr, Val362Ala, Ile179Leu have something with acid tolerance activity and thermalstablility respectively.
With the development of the new generation of biomass energy, it has become a popular topic that to use fermentable sugar and Clostridia to produce butanol. The study is going to use Clostridium acetobutylicum AS1.7,Clostridium acetobutylicum AS1.132,Clostridium acetobutylicum AS1.134 and Clostridium beijerinckii NCMIB 8052,which can produce butanol in various sources of sugar by fermentation. By comparing growth behaviors, ratio of using sugars, yield of butanol and by-product, and endurance of butanol and xylose, we are going to find out the most suitable Clostridium, which can be used to the industrial. In the experiment, NCMIB8052 is the most outstanding Clostridium as it has the highest yield, relative high endurance and the ability using multiple sugars.
Genome shuffling is a new-type technology based on protoplast fusion and recursive protoplast fusion. With the development and mature of genome shuffling, many new metabolites have been obtained through this technology, which demonstrates a promising prospect for genome shuffling to be a way to develop new metabolites. The new metabolites developed by genome shuffling technology are focused, including metabolites from activation of silent genes, new antibiotics from introduction of single enzyme gene, hybrid antibiotics from exchange of gene modules and new materials from replacement of precursor gene.The prospect of this technology is also discussed.
Akirin is a recently discovered gene, which plays important roles in skeletal muscle development and immune response.The relationships between akirin gene and immune response, development and regeneration of skeletal muscle, myostatin gene and NF-κB factor are mainly summarized, and the research progress of poultry akirin2 gene. Additionally, the using prospects of akirin gene were also discussed briefly. is reviewed provide References for further studying and use of akirin gene in medical and animal husbandry fields are provided.
Banana is one of the most important tropical fruits and the fourth grain crops in the world. The research of banana functional genomics involved in stress resistance has always been the hot spot and core in all banana researches. Novel researches on the banana genome sequencing, isolation and identification of functional genes involved in resistance are reviewed, which will help us to investigate banana originally and provide theoretical basis for banana genetics improvement and new varieties breeding.
Map-based cloning refers to a technique for the isolation of genes characterized by a phenotypic alteration usually caused by variation in its DNA sequence. The central procedure used in map-based cloning is the genetic mapping of the gene of interest at extremely high resolution. The high resolution mapping results in the identification of a small interval harboring the gene. Further intermediate steps such as the establishment of clone-based physical maps, the identification of expressed regions and extensive sequencing and sequence annotation may be necessary. Finally, several approaches may be used for the verification of the identified gene harboring a sequence polymorphism. The progress in Map-based cloning of the function genes in rice(Oryza sativa L.) was reviewed.
L-amino acid oxidase is dimeric flavoprotein, and each subunit contains a non-covalently bound FAD molecule as cofactor. It is able to catalyze the stereospecific oxidative deamination of L-amino acids to the corresponding a-imino acids which are then hydrolyzed to corresponding a-keto acids with release of NH4+, along with two electrons transferring from the amino acid to the flavin cofactor which subsequently reduces molecular oxygen to H2O2. This enzyme is widely distributed in nature. So far snake venom LAAO is the best characterized member of this enzyme family. Recently, non-snake venom LAAOs have increasingly been found. Current researches show that different LAAOs have different physiological properties, including substrate specificity, pI value, and storage stability. Little is known about its structure, but the structures of both snake venom and non-snake venom LAAOs indicate that it all consists of FAD-binding domain, substrate-binding domain and helical domain. LAAO has various biological functions which are found to be probably related to the produced-H2O2. Probably due to post-translational modification of LAAO, only some heterologous expression systems have been reported hitherto.
As the number of biological patents are about to expire, with the affection about drug cost reducing, availability increasing and a huge market space and so on, there showed great interest on developmenting biosimilars,major companies have set foot in this area,however because of specificity of biosimilars, success is not so easy. Based on the above background, the external environment, the macroeconomic environment, policy environment, legal and regulatory environment of the domestic and international bio-pharmaceutical industry are analyzed, then the current key technology problems of biosimilar developed are described.Based on this, the main ga Pabout biosimilar companies between demestic and abroad is proposed.Based on the above analysis,SWOT analyze the strategy and proposes that China should be carry out SO strategy mainly and supplements with SW strategy is used. Finally, some suggestions are for reference.
