Objective To identify PINK1 interacting with α-synuclein in Parkinson's disease. Methods GST-α-synuclein fusion protein was created by inserting α-synuclein into pGEX-4T-1. The protein was expressed in Escherichia coli and purified on glutathione-Sepharose beads by standard methods. And then incubate with MN9D cells lysates to detect the interaction of PINK1 with α-synuclein. In addition, the full length of PINK1 robustly associated with α-synuclein was also determined by coimmunoprecipitation experiment in MN9D cells. The co-localization of PINK1 with α-synuclein was detected by immunocytochemical staining, and observed by confocal. Results We detected that GST-α-synuclein interact specifically with PINK1, but not GST. Coimmunoprecipitation with an anti-α-synuclein antibody and subsequent Western blotting with an anti-PINK1 antibody confirmed the specific interaction of α-synuclein with PINK1. PINK1 and α-synuclein protein partly co-localize in MN9D cells. Conclusion PINK1 and α-synuclein can interacts with each other and co-localizes partly in MN9D.
Aim The recombinant Human retinal pigment epithelium-derived factor（PEDF）protein to be obtained and the angiogenesis of the rPEDF to be identified. Method PEDF gene was amplified by PCR and cloned into pET32a, rPEDF protein was expressed in E coli BL21 and confirmed by SDS-PAGE and Western-blot. The rPEDF was purified by Ni-NTA on denature condition. The concentration of the rPEDF was determined by Bradford method. The angiogenesis of the rPEDF was determined by chick chorioallantoic membrane (CAM) method. Results The expression plasmid pET32a-PEDF was constructed successfully .The rPEDF was expressed stably efficiency in E coli BL21. The results of the CAM experiment showed that the rPEDF had notable angiogenesis effect in the concentration 0.4、0.04 ng/ml, but had no effect in 4 ng/ml. Conclusion: The PEDF gene was cloned and expressed efficiency, the angiogenesis of the rPEDF to be identified and the activity was worked in certain range. The results can facilitate studying its function and spreading its application.
IFNa-2b is used widely in clinic for the treatment of chronic hepatitis type B and C. IFNa-2b, as a therapeutic protein, is constantly eliminated from circulation within a short time by physiological processes, such as proteolysis in blood. This is often the limiting process affecting the half-life of IFNa-2b. In this study Tyr at position 89 and Glu at position 159 were substituted by His via site-directed mutagenesis in order to gain a interferon mutagenesis with higher efficiency. IFNα-2bHis89/His159 was expressed as inclusion bodies with the yield of more than 40% of total bacterial protein. The putity of IFNα-2bHis89/His159 was higher than 95% after DEAE Sepharose FF and CM Sepharose FF, and the biological activity was more than 2.8×108 IU•mg-1. The half-life of IFNa-2b was 1.72±0.23h and the half-life of IFNα-2bHis89/His159 was 2.34±0.23h, which was improved slightly in rats.
Two fragments G1 and G2 of the glycoprotein G gene of bovine respiratory syncytial virus(BRSV) were selected for expression in Escherichia coli based on the analysis of glycoprotein G by DNA Star software. Then the two fragments of glycoprotein G were amplified by PCR with synthesized G gene of BRSV as the template. The amplified fragments G1 and G2 are 570bp and 308bp in length, respectively. The PCR products were cloned into pET30a vector and expressed in soluble form in E. coli after induction of cultured E. coli with IPTG. Both of the recombinant proteins G1 and G2 were purified by immobilized Ni ion affinity chromatography under native conditions. Then the purified proteins were analysed by western blotting. The results showed that the purified recombinant protein G1 retained good antigenicity and specificity. But the purified recombinant protein G2 didn’t possess biological activity. Antibodies against BRSV were detected in suspected bovine sera by using indirect ELISA and western blotting with the purified recombinant protein G1 in China. The purified recombinant protein G1 might be used as antigen for establishing serological methods for diagnosis of BRSV infection. And the purified recombinant protein G1 might also be used for preparing polyclonal and monoclonal antibodies for research on biological functions of glycoprotein G of BRSV.
The dhaD gene encoding glycerol dehydrogenase (GDH) from Klebsiella sp. was amplified. and was inserted into expression vector pET-28a(+), the plasmid pET-28a-dhaD was constructed and was transformed into Escherichia coli BL21 (DE3). SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21. Then GDH was purified by Ni-NTA affinity chromatography, the results showed a single band about 39kDa on SDS-PAGE gel, and the specified activity was about 156U/mg. The special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%. The optimum reaction pH was 11.0, and the GDH activity have little changed when incubated in the buffer of pH7.0～11.0. The optimum reactive temperature was 30℃，and the GDH was more stable on the temperature of 25℃～45℃. The Km value was 0.54mmol/L and Vmax was 0.49 μmol/(mL·min) in the glycerol.
Four engineered zinc finger nucleases were produced for cleaving the internal transcribed spacers(ITS) of the human rRNA gene family to generate double-strand breaks, thereby increasing the efficiency of gene targeting. This study will lay a firm foundation for further in vivo studies of gene thrapy with gene targeting. First, two 9 bp-long appropriate sequences (with an internal sequence of 6 bp) within the ITS1 of the human rRNA gene family were selected as the recognition loci of zinc finger proteins. Based on the sequences of the recognition loci, two tri-zinc finger proteins were designed for each locus. The full-length DNA encoding zinc finger proteins were obtained through the gene splicing by overlap extension PCR (SOE PCR) method, then they were inserted into the expression vector pET-28a(+) to generate recombinant plasmid pET28a-ZFP. The zinc finger fusion proteins carrying a His-tag were produced by transformation of the recombinant plasmids into bacteria RossettaTM（DE3）. At the same time, the coding sequences of four zinc finger proteins were linked with the sequences encoding the DNA-cutting domain of Fok I, respectively, by SOE PCR. The resulting sequences encoding four zinc finger nucleases were then inserted into the expression vector pET-28a(+) to construct recombinant plasmid pET28a-ZFN. By transformation the recombinant plasmids into bacteria RossettaTM（DE3）, four zinc finger nuclease fusion proteins carrying His-tag were produced and purified.
The fermentation culture media for pNSR32/BL21(DE3) was investigated with shake culture method.Based on M9 medium and for the induction of lactose ,so as to determine the optimal concentration of ingredients of medium. The optimized culture medium was obtained by orthogonal and individual factor design. Optimized prescription of culture medium was investigted for the fermentation to produce the high molecular weight recombinant spider silk proteins. The optimum culture medium consisting of 0.3% glycerol,3% yeast,0.75% tryptone, 0.05%(NH4)2SO4 and little inorganic salt that was found to benefit for the growth and expression of recombinant spider silk proteins ,and to make the production level attain at 24.0%.
The ergosterols were produced from corn straw hydrolysates fermented by ergosterol yeast, which was obtained from protoplast electrofusion. The effects on the yield of ergosterol were studied in the condition of shaker, such as initial sugar concentration, nitrogen source, pH value and fermentation time. The technical conditions were optimized according to the DPS center-united experimental design principles and the method of response surface analysis with four factors and three levels. The results indicated that the four factors had significant correlation to ergosterol accumulation. The biomass and the ergosterol content could be up to 8.67g/L and 2.37% respectively after cultivated for 32h under optimal technical condition. The structure of ergosterol crystal was characterized by UV, IR and SEM. A new approach of biomass source application was presented.
The high effective somatic embryogenesis system was obtained by analyzing the different effects. The result showed that somatic embryos induced rate of ‘SC 8’ was highest. The optimum explant was Shoot-tip. The best somatic embryos induction medium: MS +0.5mg/L CuSO4 + 4 mg/L 2,4-D. The central problems about plant regeneration were also discussed，then an efficient cassava regeneration system was established.
In animal cloning, it is generally believed that the inactive diploid G0 or G1 stage of the cell cycle is beneficial to initiate cell-cycle coordination and reprogramming following transfer of the donor nucleus. In order to provide more G0+G1 stage cells for dairy cattle cloning, we carried out a flow cytometric analysis of the cell cycle of fibroblasts from the ear of dairy cow at different passage numbers after different periods of serum, after full confluence, after cycloheximide（CHX） treatment. Serum starvation effectively increased the percentage of G0+G1 sage cells after 72h. Significantly higher percentage of cells in G0+G1 was apparent when dairy cattle fibroblast cells cultured in confluence. No significant difference were observed on the 3rd passage cultures exposed to CHX, however, on the 13th passage an increase on proportion of cells was observed after all periods of exposure(p<0.05). This date indicates that high passage cells in vitro are more susceptible to serum starvation, confluence cultured and CHX G0+G1 synchronization.
"CapFinder" technology, which can be used to clone the full length of 5' UTR sequence of mRNA, was described. This technology used the terminal transferase activity of certain MMLV RT variants that added 3-5 residues (predominantly dC) to the 3'end of the first-strand cDNA exhibited when MMLV RT reached the 5'cap structure of mRNA. In the reverse reaction system containing GGG oligo, the terminal transferase activity was harnessed by the GGG oligo whose terminal stretch of dG residues can anneal to the dC-rich cDNA tail and serve as an extended template for RT. After RT switch templates from the mRNA template to the GGG oligo, a complete cDNA copy of the original RNA was synthesized with the additional GGG oligo sequences at the end. 5'UTR of mRNA can be amplified with GGG oligo as forward primer and a gene-specific reverse primer. 5'UTR of Bt toxin receptor E-Cadherin gene in midgut of cotton bollworm was cloned.
Protein extracting method、protein dissolution、protein loading amount and gel strip transfer were tested for the protein extracted from seedling rhizome of winter wheat cultivar Dongnong Winter Wheat No.1. The results were showed that the loss of low abundance protein in TCA/acetone protein extracting process (T process) was less than that in urea/thiourea protein extracting process (N process) and the spots in protein map in T process were more than that in N process. The purer protein dissolved two times in the hydrated liquid could maintain higher voltage of 8000 volt by Isoelectric Focusing and Electrophoresis. The protein loading of 10mg by two times dissolution in hydrated liquid could obtain clear map with more protein spots and better separation effect. After adding 400ul 0.3% common agarose solution to glue surface and washing support film of gel strip with 200ul electric buffer, the gel strips could be transferred easily to second dimension glue surface and no air bubbles appeared between gel strips and glue surface.
The feasibility of embryonic stem cells to combine with silk fibroin for neural differentiation was invaluable and unknown. We suspended the ES cells into EBs and then transferred them to three different substrates-coated 35 mm dishes including gelatin, Bombyx mori silk fibroin (SF) and Tussah silk fibroin (TSF) to identify the adherence and proportion of ES cells-derived neurons under these three substrates. The results showed that silk fibroin as a reliable biomaterial which can support EBs’ adherence and differentiated into euron-like cells compared with control group, all the proportion of β-Ⅲ-Tubulin positive cells is approximately 40%. The results further proved that the adhesion ability of cells on SF is poorer than on the control group and TSF group, which also showed that TSF is an excellent biomaterial for the neural differentiation of ES cells. This study may provide important experimental information for tissue engineering, in which ES cells-derived neuron cells and silk fibroin materials are scaffolds, and also offer a source for cell therapy research of neurodegenerative disease.
To increase the biotransfomation efficiency from the orotic acid to the Uridine 5’-monophosphate(UMP), URA5 gene encoding Orotate phosphoribosytransferase was amplified from Saccharomyces cerevisiae BY4742 by PCR, then it was inserted into the expression vector pYX212(contained orotidine monophosphate decarboxylase gene URA3)and the pYX212-URA5 was transformed into Saccharomyces cerevisiae BJX12 by electroporation. The recombinant strain was elementarily used to convert orotic acid to UMP. The results showed that pYX212-URA5/BJX12 could accumulate 7mM UMP from 32mM orotic acid in 26h, significantly higher than both control groups pYX212/BJX12 ( 2.7mM ) and BJX12 ( 2.4 mM )
The limited number and low immunogenicity of tumor antigen make it difficult to screen out valuable phage antibody by panning on purified immobilized antigen because of the alternation of protein conformation. Most of these antigens have complex structure and the transporting and localization are depended on the cell types and microenvironment. Cell panning is applicable to complex antigen in its natural state without an additional purification procedure, or the exact antigen is not even known. So it is widely used in selection of tumor specific antibody, especially internalizing antibody and antibody specific targeting to vascular endothelial antigen. The major problem encountered in cell panning is related to the high background binding of non-specific antibody. Many efforts had been made to improve the specificity and sensitivity of cell panning. The combination of high throughout flow cytometry, combinatorial chemistry and proteome research will make this strategy more practical and fruitful. Here we outlined the progress of panning on intact cells and tissue sections to provide beneficial reference for understand and design of cell based panning.
As a new recombination system, Red recombination has been widely used in E.coli gene knockout. Comparing with Rec recombination, Red recombination has the advantages of exploring short homologous arms and high recombination rate.The length of homologous arms usually are 36~60 nt.The function of Exo、Beta and Gam proteins in Red recombination, three kind of plasmids and their functions in E.coli gene knockout system were reviewed in this paper. The three kind of plasmids are plasmid which has exo、bet、gam for Red recombination, plasmid which has resistance tag and FRT site for PCR template and plasmid which has FLP recombinase for eliminating resistance tag. We also summarized the concentration and inducing time of L-arabinose and the length of homologous arms by literature mining, and proposed the optimal parameters. The optimal concentration and inducing time of L-arabinose are 100 mmol/L and 90 min, and the optimal length of homologous arms is 56 nt.
MicroRNAs (miRNAs) are endogenous non-coding RNAs, about 20 nucleotides in length. They play a pivotal role in the regulation of genes involved in diverse biology processes such as cell development, proliferation, differentiation and apoptosis by the translation repression or mRNA degradation. Recent evidence has suggested that miRNA alterations are involved in the initiation and progression of various human cancer including hepatocellular carcinoma (HCC), and miRNA-expression profiling of HCC has identified signatures associated with diagnosis, staging, progression and prognosis．As a novel molecular target, miRNAs holds great promise in diagnosis and biotherapy of HCC．
Actinobacillus succinogenes was isolated from the bovine rumen and has a great prospect on the succinic acid fermentation. Due to its high yield and fermentation with a broad range of carbon source, the metabolic pathway and fermentation of Actinobacillus succinogenes has become the research hotspot in succinic acid fermentation, recently .The research on Actinobacillus succinogenes is reviewed in this paper, which includes the development of metabolic pathway, the kinetic model of succinic fermentation, the novel and economic mediums and the screening of high yield strains. These results significantly increase the productivity according to the strain reconstructing and the reasonable fermentation technologies designing.
Alzhimer's disease (AD) is the most prevalent neurodegenerative disease in the elder. The disease is characterized by memory impairment and cognitive obstacle. The current treatments of AD provide only symptomatic relief and are practically inefficient. With the advancing ageing population in the world,AD is becoming one of the most severe diseases that damages humankind's health. Now, anti-Aβ antibodies have been suggested as a potential therapeutic strategy for the treatment of AD. In this paper, we summarize the recent progress in pathogenesis based on the Aβ hypothesis, possible mechanisms of Aβ-clearance by anti-Aβ antibody, and potential methods to prepare anti-Aβ antibody, etc.
There is immense interest in the long-chain polyunsaturated fatty acids (LC-PUFAs) on account of their health-beneficial properties. There has been considerable interest in the production of LC-PUFAs in transgenic plants to provide a cheap, sustainable and clean source of these fatty acids. In this paper, several main LC-PUFAs, their roles and metabolic pathway in plant are introduced simply, progresses on biosynthesis of LC-PUFAs in transgenic plant are not only reviewed, strategies for improving the yields of LC-PUFAs in plant are also discussed.
Abstract The Saccharomyces cerevisiae cell-surface display expression system is one kind of eucaryotic display system which expresses heterogeneous protein on the cell surface by immobilization. The target protein is immobilized with GPI anchor and expressed on the cell surface after fusion gene that combines gene encoding protein and the specific carrier gene is introduced into yeast cells. It can be utilized in the fields of biocatalyst, cell-absorbent, live vaccine, environment management, protein library screening, high affinity antibody, biosensor, the antigen/antibody library construction, cancer diagnosis and so on. This mini-review describes molecular display using Saccharomyces cerevisiae and its basic principles, present research situation, various applications and development prospects.
