Objective GFP (green fluorescence protein)-streptavidin (SA) bi-functional fusion protein was generated and characterized in order to demonstrate our novel platform for efficiently and durably modifying the cell surface with SA-tagged bi-functional proteins. Methods GFP-SA/pET24 construct was generated and expressed in BL21(DE3) host bacteria at the high level. The recombinant protein GFP-SA was purified through the Ni-NTA affinity chromatography, and then refolded. After biotinylation B16 tumor cells were modified with GFP-SA bi-functional fusion protein and then subjected to fluorescent microscopy and FACS analysis. The effect of surface modification on the viability and growth of B16.F10 tumor cells was evaluated by MTT staining. Results The GFP-SA recombinant fusion protein was expressed in BL21(DE3) at about 20 % of total bacterial proteins. The GFP-SA bi-functional fusion protein exhibited the bi-functionality, i.e., SA-mediated high-affinity binding to biotinylated cell surfaces and GFP-emitted green fluorescence. The cell surface modification with GFP-SA bi-functional fusion protein did not affect the viability and growth of the modified B16.F10 tumor cells significantly. Conclusion The GFP-SA bi-functional fusion protein was obtained and could be displayed efficiently on the surface of the biotinylated B16.F10 tumor cells through the specific and tight interaction between streptavidin and biotin, thus can be used as good trace protein and experimental control in the development of surface-modified tumor vaccine.
The endocytosis/phagocytosis of calcium alginate nanocapsules by peripheral blood derived dendritic cells was confirmed with the use of Quantum dots labeling. Results further demonstrated these nanoparticles could cause the upregulation of HLA-DR expression to induce the maturation of dendritic cells in vitro. Dendritic cells stimulated by nanocapsules covalently loaded with BSA were shown to stimulate the proliferation of self T cells with blank capsules and free BSA as controls, suggesting their potential applications in cancer cell therapy as a new antigen delivery vehicle with a strong adjuvant effect.
Abstract Background The identification of mesenchymal stem cells (MSCs) have provided exciting prospects for cell-based regeneration after myocardic infraction.However cell therapy have inherent limitations such as low sur- vival rate of transplanted cells and insufficient cell number.It is known that cell-matrix adhesion plays a key role in cell proliferation,differentia-tion and survival,and chemokine CXCL12 may involvedin these prcesses.Thus we transfected mesenchymalstem cells with CXCL12 for local secretion of CXCL12 and then explored CXCL12 triggered adhesion of mesenchymal stem cells to extracellular matrix proteins.Methods Mesenchymal stem cells was transfected with CXCL12.αV andβ3 integrins content was evaluated by Western blot analysis. Cell adhesion to extracellular matrix was examined in-vitro and cell prolife-ration after transplantation in-vivo. Results Transfection of CXCL12 resulted in increased CXCL12 in situ.Increased CXCL12 induced elevated adhesion to fibronectin in-vitro and higher survival invivo.CXCL12 mediated adhesion and proliferation was established by αV andβ3 integrin subunits.Conclusions Che- moattractive mechanisms are involved in adhesion processes of mesenchymal stem cells.Increased CXCL12 leads to enhanced expression of αV andβ3 inte- grins,which may augment cell survival,proliferation and differrentiation.
Abstract The proteomics technique platform of bovine spermatozoa was established by exploring the different sample preparation methods, the components of rehydration solution and optimizing the 2D electrophoresis protocols. The differential proteins between the fresh and frozen-thawed spermatozoa were also explored. The results indicated that the technique system with the high qualitative and stable 2D image had been obtained by preparing the bovine spermatozoa proteins with the heat TRIzol method and combining with the optimized 2D electrophoresis protocols. The differential in-gel electrophoresis showed that 20 protein spots were absent in quality, 2 spots decreased and 10 spots increased significantly in quantity after frozen-thawed. As a intermediate result, the 2D platform established in the research and the differential proteins found between fresh and frozen sperm will provide better basis for revealing the cryodamage mechanism of sperm cryopreservation and studying the differential proteomics of the X, Y spermatozoa.
The recombinant pseudorabies virus expressing ORF2 gene of porcine circovius type 2 and VP2 gene of porcine parvovirus was developed into trivalence genetically engineering vaccine to inject mice, and the result showed that the recombinant virus SA215（D）had lower virulence than velogenic strain which could resist the attack of PRV Fa strain and successfully induced humoral and cellular immune response in mice. This study established the foundation for the development of trivalence genetically engineering vaccine .
Abstract:Enormous economic loses were caused by FMD which was a kind of virulent infections disease.This study was carried out to research the molecular immune mechanism of inactivated FMD vaccines, lay the foundations for invention of anti-FMDV medicine. In the research, mRNA differential display method was used to compare gene expression between PK-15 cells stimulated by Swine Foot and Mouth Disease and normal controls. 30 ESTs were obtained by extraction , amplification and purification. The 30 ESTs were identified by reverse northern dot bloting .8 positive DNA fragments were cloned and sequenced.All 8 ESTs were compared with nucleotide sequences deposited in the nr database and dbEST database of GenBank via BLASTn tool. E1 was found highly similar to Heat shock 60kDa protein 1, E2 was found highly similar to Sus scrofa MHC class I SLA genomic region, haplotype H01,clone. E7 were extended and separated by searching database. The ORF database were used to analyse the extened bands. The result indicated that E7 was highly similar to arginase I of Sus scrofa. heat shock 60kDa protein 1, Sus scrofa MHC class I SLA genomic region haplotype H01 clone, arginase, type I of Sus scrofa and the other genes with unknown functions were regarded as candidate genes in anti-FMDV research, and their specific functions need to be explored in further study.
The high conservative sequence of Canine Distemper Virus (CDV) N gene was amplified with RT-PCR, cloned into pMD18-T vector , and then constructed into upstream site of His Tag coding sequence in prokaryotic expression vector pET24b. The N fusion protein was highly expressed when the recombinant plasmid was transformed E coli Rosetta 2(DE3) strain and induced with IPTG. SDS-PAGE and Western blot showed the 15kD expressed products reacted with standard positive serum against CDV. An indirect ELISA demonstrated recombinant expressed protein had good antigencity and can distinguish effectively anti-CDV positive serum and negative serum. The results showed expressed CDV N protein had similarity of antigencity with natural N protein, and can be used as diagnosis antigen. It provided evidence for establishment of an indirect ELISA method for CDV detection.
VHA-c3 gene is one of the five homologous genes which belong to the subunits of vacuole H+-ATPase in Arabidopsis thaliana. Former reports indicated that it is related with resistance to abiotic stress in plants. In this article, different fragments of the upstream regulation sequences of Arabidopsis VHA-c3 gene were cloned, and fused with GUS reporter gene, to study the localization of VHA-c3 gene. The results showed that: The essential element for the basal activity of VHA-c3 is located within the -722bp fragments upstream of the translation start codon of VHA-c3, it can guide genes' constitutive expression in leaf blade, trichome, petiole, root, stamen, stigma and sepal in Arabidopsis; And there is two negative regulatory elements located in -2812bp to -2234bp and -1496bp to -772bp respectively, and an up-regulating cis-element from -2234bp to -1496bp fragment of VHA-c3 gene, which may control the gene's expression in guard cell.
OsRacD is a critical molecular switch involved in photoperiod fertility conversion of photoperiod sensitive genic male sterile rice. Taking OsRacD as bait, a new rice myosin heavy chain partial cDNA was isolated from the rice panicle by yeast two-hybrid,and designated OsMY1.In this study, the interaction between OsMY1 and OsRacD had been testified by yeast two-hybrid and pull down assay, the results showed that OsMY1 could bind with OsRacD, Our study suggested that OsMY1 maybe the target of OsRacD. The results also provided important evidence for further study on the functional relationship between OsMY1 and OsRacD in vivo.
An α-type cyclodextrin glucantransferase was purified to homogeneity by ammonium sulfate precipitation, HiTrap-phenyl column chromatography and HiTrap-Q column chromatography. Molecular weight of the α- cyclodextrin glucantransferase was 75 kDa with an isoelectric point of 5.3. Purified α- cyclodextrin glucantransferase exhibited optimal catalytic temperature of 50°C and optimal catalytic pH of 6.0. Ca2+、Zn2+、Fe3+、Cu2+、Fe2+、Ag+ strongly inhibited enzymatic activity. Km and Vmax values were 50mg/ml and 6.07 mg/ml/min, respectively, using amidulin as substrate. Tryptophan residue was necessary for the catalytic activity. N-terminal amino acid sequence was N-SPDTSVDNKV-. Potato starch was the most suitable substrate for the formation of α-CD. Optimal conditions for preparing α-CD were as follows: 200u/g starch of CGTase, 40°C, 24 hours of incubation. Total conversion yield attained 41% and α-CD occupied about 78% in the products. This work did benefit to the development of α-CD.
Magnetic chitosan microspheres (M-CS) were prepared and used for yeast alcohol dehydrogenase (YADH) immobilization. The optimum technology and the properties of immobilized YADH were studied. The optimal immobilization conditions for YADH were: 20mL of 0.25mg/mL of YADH in phosphate buffer (0.05mol/L, pH 7.0) reacted with 50mg of magnetic M-CS at 4℃ for 2h.The microspheres were characterized by transmission electron microscopy, the results showed that M-CS were regular sphere with an average size of 30nm and had magnetic response characteristic. The M-CS suspended in H2O solution was easily precipitated and separated by magnetic field. Mechanical strength and crosslinking degree of M-CS were influenced by the ratio of carrier and immobilized YADH, ion concentration in phosphate buffer and pH of the solution. The immobilization was slightly influenced by the reaction temperature. The immobilization would improve its thermal, basic resistant and acid resistant stability. After the immobilized enzyme was kept between 35℃ to 75℃ for one hour , it still had 70 % of initial enzyme activity , when it was kept pH between 5.0 to 7.4 for one hour , it still had 80% of initial enzyme activity. The optimal reaction temperature of the immobilized enzyme was 40℃ compared to 30℃ of the free YADH, the optimal reaction pH of the immobilized enzyme was 6.8 as same as one of the free enzyme. Storaged at the temperature of 4℃ for 30 days without any protection by reagent，the free enzyme only kept 26% of the original activity but the immobilized enzyme still retained 60% of the activity .The immobilized enzyme maintained 70% of the activity after circular use 5 times. The Km value of immobilized YADH for Pyruvate was 2.58mmol/L compared to 3.31mmol/L of the free YADH, it would reduce its appetency for the substrate.
Objective：To observe the effect of retrovirally mediated RNA interference targeting CXCR4 of human cervix carcinoma Caski cells．Methods：Construct the fragment of CXCR4-siRNA, insert it into a retroviral vector which contain Green Flu Protein, named pSOS. Then, recombinated it by transfecting in PT67 cells, collect the supernatant of culture fluid, and use the supernatant transfect Caski cells. The expression of CXCR4 mRNA and protein were detected by Real-Time PCR and Western boltting. The abilities of invasion was detected by Matrigel invasion assay. Results: The retrovirus vectors pSOS-CXCR4 were constructed successfully. Compared with the negative control group, the inhibition rates of CXCR4 mRNA on the 24th, 48th and 72th hour were 29.9%, 56.8% and 62.8%, respectively. The inhibition rates of CXCR4 protein on the 24th, 48th and 72th hour were 43.6%,49.6% and 62.9%, respectively. And the the abilities of invasion was weaken when CXCR4's expression was inhibited. Conclusion: Retrovirally mediated RNA interference could effectively inhibit the expression of CXCR4 in human cervix carcinoma cells and weaken the the abilities of invasion.
Bioimprinting is a new developed technique to improve the characteristics of enzymes. In this study, bioimprinting by lauric acid was conducted to improve the esterification activity of lipase PS in sol-gel immobilization process with methyltrimethoxysila (MTMS) and tetramethoxysila (TMOS) as the precursors. Results generated by checking the esterification activity and scanning electron microscope showed that bioimprinting can enhance the specific activity and thermal stability of lipase PS. The bioimprinting system was optimized by orthogonal experiment, and the optimal condition for lipase bioimprinting is water/silane molar ration (R) 12, polyethylene glycol (PEG) 120μl, and lauric acid 0.15 mmol. Compared with the free enzyme and the non-imprinted enzymes, the specific activity of imprinted enzymes has been improved 44.3 fold and 2.4 fold, respectively. Imprinted lipase show better thermal stability, and the relative activity is 58% after incubated in 80 ℃ for 0.5 h, while no activity was detected for the free enzyme.
Methods for studying the population diversity of microorganism in activated sludge usually require enrichment of bacterial genome. The efficient information on microbial species composition provided and shifted in diversity revealed are dependent on the effective DNA recovery technique. The method was based on washing by alkaline phosphate buffer and digestion with extended heating of the activated sludge suspension in the presence of lysozyme and freeze-thawing in high-salt-SDS buffer. The extraction was tested for four activated sludge differing in places and dates. The DNA fragment from all sludge was integrity. DNA yields ranged from 105 to 823 μg/g sludge and were of sufficient purity for PCR-based 16S ribosomal DNA analysis and restriction digested. In general, all methods produced DNA pure were not enough for PCR amplification and libraries construction et al. As basis of experimental goals, this study provides an appropriate extraction method of microbial DNA in sludge.
HIV-1 envelope glycoprotein gp41, which is a hopeful target for HIV-1 fusion inhibitors, plays a critical role in the fusion of viral and cellular membranes. In order to build up the screening assay of HIV-1 fusion inhibitors targeting gp41, HIV-1 gp41 5-helix and 6-helix were expressed in prokaryotic cells. Gp41 5-helix and 6-helix recombined plasmids were constructed by using PCR, enzyme digestion and ligation taking the clade B HIV-1 genome as a template. The plasmid was transferred into E. coli BL21（DE3）and then induced by IPTG. The expressed protein was purified by affinity chromatography after denaturation and renaturation. The SDS-PAGE analysis was used during expression and purification. Native-PAGE was used to identify the interaction between gp41 5-helix and T-20. It will be helpful to build up the screening assay of HIV-1 fusion inhibitors targeting gp41.
CP10A, an anionic antimicrobial peptide designed from Indolicine, has a strong resistance to Gram-positive bacterial, which usually cause clinical infections. The DNA sequence of this 13 amino acids peptide was designed according to the reported CP10A peptide sequence and synthesized by PCR amplification. The sequence was cloned into the E.coli expression vector pET32a (+) and constructed the recombinant expression vector pET32a (+)-CP10A. The recombinant plasmid was transformed into E.coli AD494 and induced with IPTG. The Expression product was detected via 15% SDS-PAGE. The fusion protein was about 50% of total cellular protein and expressed as inclusion bodies. We got the pure fusion expressed CP10A protein via degeneration, Ni-NTA affinity chromatography and refolding of the complex. The purity of the refolding protein was more than 95%. It could be of great value for providing some basis to study biological activities of CP10A. It also provides some approach to study the expression of antimicrobial peptides.
The zinc chloride and cystine resistant strain of S. cerevisiae YZM-14(ZnCL2r, Cysr) was screened with the mutant processing of the protoplast of S. cerevisiae by combinative mutagens of ultraviolet and nitrite. The glutathione (GSH) production (84.72 mg/L), dry cell weight (7.63 g/L) and the intracellular GSH content (11.10 mg/g) of the mutant strain were about 2.79, 1.63 and 1.71 times than that of the initial strain. The biosynthetic process of GSH was divided into three phases according to the time course of the specific cell growth rate and GSH yielding coefficient. In the second phase, the metabolic flux of the pentose phosphate pathway and the GSH precursors biosynthetic pathway of the mutant strain increased by 8.1%, compared with that of the initial strain. Furthermore, the metabolic flux of the organic acids secretion of the mutant strain decreased. Through these mechanisms, the utilization efficiency of the carbon sources was enhanced and high production of GSH was obtained.
The assessments of oocyte quality were usually needed in assisted reproductive technology and reproduction science. Based upon the changes of structure and biochemistry in the development of oocyte, assessments of oocyte quality mainly comprised the oocyte morphology and its maturity. This paper discusses the assessment method of oocyte quality from nondestructive manner and invasive technique to provide multi-pathway for evaluating the oocyte quality reasonable.
SUMO(small ubiquitin-related modifier) is an important member of the small ubiquitin-like protein (Ubl) family, which functions in diverse cellular processes by becoming covalently attached to other proteins. SUMO proteins are structurally and mechanistically related to ubiquitin. However, unlike ubiquitylation which primarily targets a substrate for degradation, sumoylation participates in a number of biological processes, including transcriptional regulation, nuclear transport, maintenance of genome integrity, and signal transduction, as an important post-translational modification. Dysregulation of sumoylation has been associated with a number of diseases.
Selective protein degradation by the ubiquitin 26S proteasome pathway has emerged as a key regulatory mechanism in a wide variety of cellular processes. The ubiquitin/26S proteosome pathway mainly consists of ubiquitin activating enzyme (E1),ubiquitin conjugating enzyme (E2),ubiquitin protein ligase (E3), and 26S proteasome. In an ATP-dependent reaction,uibquitin (Ub) is conjugated to E1,the activated Ub is then transferred to an E2. Finally,the Ub-E2 intermediate delivers the Ub to the target protein by E3 recognition. Polyubiquinated proteins are eventually degraded by the 26S proteasome.In plants,regulated protein degradation by /26S proteasome pathway contributes significantly to development by affecting a wide range of progress, including hormone signaling, photomorphogenesis, self- incompatibility and cell cycle.This review highlights the recent progress towards understanding the role of the Ub/26S proteasome pathway during plant development.
Vitreoscilla Hemoglobin (VHb) was discoved in the late 1970s, it is an element that can ensure Vitreoscilla survive and improve growth in an environment that contains insufficient amount of oxygen. Along with further mechanism research on VHb, this protein get more and more application on biotechnology fields. In this review we summary main results of research on VHb in recent years, including protein structure, intracellular localization, DNA shuffling and mechanisms of VHb function. Finally, we inllustuate the application of VHb on microorganism fermentation industry.
