Embryonic stem cells (ES cells) undergo selfrenewal to maintain their pluripotency. The selfrenewal and pluripotency of ES cells are under tight control of many transcription factors. Nanog is one of these transcription factors. However, the molecular mechanisms under which Nanog regulates ES cell pluripotency are not very clear. P300 is a general coactivator in eukaryotes which usually helps transcription factors to regulate downstream genes. Nanog (or Nanog mutants) and its reporter gene were cotransfeted with p300 in cells in order to see if p300 could affect Nanog transcriptional activity. The results showed that p300 enhances Nanog transcriptional activity through the homeobox domain.
Aim:To construct the short hairpin small interfering RNA(shRNA) eukaryotic expression vector specific to mouse lectin like oxidized low density lipoprotein receptor 1(LOX-1) gene and to observe its silencing effect on LOX-1 in RAW264.7 cells. Methods:(1)The pLOX-1-shRNA expression vector was constructed by gene recombination, Then transfected into the cultured RAW264.7 cells. At 48 h after Transfection, the expression of LOX-1 mRNA in RAW264.7 cells were determined by semi-quantitative RT-PCR, the expression of LOX-1 proteins examined by Western blot.(2) Oil Red O Dyeing experiment was used to show the cellular lipid droplets in lipid-loaded cells. The method of cholesterol oxidase analysis was performed to determine the content of cellular cholesterol.The ability of uptake Dil-ox-LDL in RAW264.7 cells was assayed by fluorescence microscopy. Results: pLOX-1-shRNA expression vector was successfully constructed. Transfection of pLOX-1-shRNA expression vector into RAW264.7 cells down regulaled the expression level of LOX-1 gene, as compared with the control group,transfection of the RAW264.7 cells with LOX-1-shRNA led to a remarkable reduction of the number macrophages transformed into foam cell,and could suppress the uptake of ox-LDL.Conclusion:The pLOX-1-shRNA expression vector can inhibit the expression of LOX-1 in RAW264.7 cells and the transformation of the macrophages into foam ,which may he beneficial in searching new gene therapy of atherosclerosis.
Objective:To construct eukaryotic co-expression vector pGNLY-2A-PFP and to observe its expression in A549 cells.Method : Total RNA was extracted from cultured PBMC. Granulysin and perforin gene segments were obtained with specific primers by RT-PCR and then respectively inserted into pMD18-T plasmid for sequencing.After identification,eukaryotic vectors pIRES-GNLY、pIRES-PFP and pGNLY-2A-PFP were constructed and transfected into A549 cells. The expression of target genes was detected by RT-PCR and indirect immunofluorescence. apoptosis of A549 cells was evaluated by flow cytometry (Annexin V/PI) after transfection . Result:The eukaryotic vectors pIRES-GNLY、pIRES-PFP and pGNLY-2A-PFP were successfully constructed. Target protein expression was detected after transfection.FCM results showed that the death rate of pGNLY-2A-PFP transfected cells was markedly increased (P<0.05).Conclusion: Granulysin and perforin could co-express in A549 cells and could mediate apoptosis.These findings would be help for farther study on the mechanism of cellular immunity and immune therapeutic effect of granulysin co-operating with perforin．
Objective: To study the expression, purification and physicochemical characters of serine protease domain of human plasminogen（rhPLG-SP）in yeast. Methods: A 7.5L fermenter was used for high density culture of engineered Pichia pastoris rhPLG-SP/GS115 and methanol induction was performed to express rhPLG-SP. Fermentation broth was treated using a three-stage process: ultrafiltration, Sephacryl S-100 and SP-Sepharose FF. The active fraction was dialyzed and lyophilized. The isoelectric point (IP), purity, and molecular weight (MW) of rhPLG-SP were detected by IFE, HPLC and LC-MS respectively. Its fibrinolytic activity and amidolytic activity after activation were measured with fibrin plate and peptide substrate S-2403 respectively. Results: Fermentation in 7.5 L scale yielded an expression level of approximately 400mg rhPLG-SP per liter fermentation broth. Through this three-step purification procedure, the purity of rhPLG-SP could reach above 96%. The physicochemical analysis showed that the IP and MW of rhPLG-SP was 7.5~7.8 and 27 877 Dalton respectively, its specific activity was 23.6 U/mg. Conclusion: A pilot technologies for high density culture, expression and purification of engineered yeast containing rhPLG-SP gene had been set up. The activity of intermediate production was comparable to that of plasminogen extracted from human plasma, indicating a good perspective in scaling up production and application.
To develop an oral drug, ITF gene encoding ITF proein, was expressed in a live delivery vehicle lactococcus lactis. First, the ITF gene was cloned into the prokaryotic expressive vector pNICE:sec. Second, the recombinant vector pNICE:sec-ITF was transformated into Lactococcus lactis strain NZ9000 to express ITF protein. Then the recombinant ITF was induced to express and was identified by SDS-PAGE and Western blot.Rabbits are divided into blank control group,preparation group and therapeutic group which are respectively administrated wih PBS and pNICE:sec-ITF Lactococcus lactis. By grades of ulcer test whether administrated pNICE:sec-ITF Lactococcus lactis protects against HCl-induced gastric injury in rabbits. The results were described as follows. The ITF was amplified and cloned in the vector pNICE:sec successfully. The fusion protein (5.9kDa) was expressed in L. lactis by the induction of the nisin. The quantity of expression accounted for 5% of the total bacterial protein. Western bolt analysis confirmed that fusion protein could be recognized specially by Monoclonal Anti-human TFF3 Antibody. Preparation groups and therapeutic groups do good than control group. Prove that administrated pNICE:sec-ITF Lactococcus lactis is biologically active in an HCl-induced rabbit gastric mucosal injury model.
To optimize the growth condition for the established gene engineer bacteria express cardiac troponin-I( cTnI) and to obtain purified cTnI as an antigen to produce clinical assay kits used in acute myocardial injury(AMI) diagnosis. Plackett Burman Design (PBD) was applied to select the factors which effect the expression of cTnI in Escherichial coli(E.coli) mostly. Induction time, pH and KCl were proved influenced expression of cTnI notably. Afterward, Response Surface Methodology (RSM) as second step to optimize the selected three factors, an equation was deduced to predict the percent of cTnI. In the most optimized condition, the percent of cTnI can reach to 26% of total cell protein. The procedures of purification included ammonium sulfate deposition and DEAE Cellulose ion exchange chromatography. SDSPAGE shows that purified cTnI contain one band. cTnI could be used to immune animals as an antigen to produce monoclonal antibodies with high affinity and specificity. It maybe as calibrators to harmony the difference assays of cTnI measurement in clinical.
NK4 protein is considered as the best antagonist of hepatocyte growth factor in recent years. In order to produce and study the biological characteristics of NK4 protein, the complete coding sequences of NK4 gene were cloned into vector pET-26b(+) to construct recombinant prokaryotic expression vector pET-26b(+)-NK4. After induction with IPTG, the strain of E. coli Rosseta (DE3) transformed successfully with the recombinant expression vector expressed recombinant protein existed in format of inclusion body strongly. The inclusion body, 42% of the total protein in E. coli, was dissolved by guanidine hydrochloride and purified by NiNTA column. The purified protein, about 95% purity, was confirmed by Western blot as NK4 protein. These results showed that recombinant protein could inhibit adherence, migration and induce apoptosis of Hela cell, which suggested that NK4 protein acquired retained its biological activity. The findings will be helpful to further study of NK4 function associated.
The HA gene of subtype H1 swine influenza virus (SIV) A/Swine/Guangdong/LM/2004(H1N1) was amplified by RT-PCR , then it was cloned into eukaryotic expression vector pCI-neo and successfully constructed a recombinant expression plasmid pCI-HA. The pCI-HA and pCI-neo were transfected into vero E6 cells and 293T cells respectively to detect the expression of HA protein by Immunoperoxidase monolayer assay (IPMA),indirect immunofluorescence assay(iIFA) and Western blot(WB).The results showed that the HA protein expressed efficiently in vero E6 cells and 293T cells and with good biological activity. ELISA results showed that anti-H1 specific antibody response was elicited after immunizing Balb/c mice of 8 weeks old with DNA vaccine pCI-HA. This laid a foundation for H1 subtype swine influenza DNA vaccine research.
In order to obtain high yield of the HrpNEcc protein with a lower total cost, fermentation and lactose induction conditions for recombinant E. coli BL21(DE3)/pET30a(+)hrpN Ecc were optimized in flasks and the recombinant E.coli was fermentated in 7L fermenter. The optimized incoulum concentration was 5% and the optimized nutrient medium was TB medium. The HrpNEcc protein yield reached 417.60mg/L by adding 3g/L exogenous inducer lactose in the growth prophase of log-phase for the recombinant E.coli. The HrpNEcc protein yield was higher 36.73% than that of the case of no any inducer, and was higher 16.85% than that of the case of adding IPTG. The wet weight of cell pellet of the recombinant E.coli reached 57.24g/L after fermentation in 7L fermenter, the HrpNEcc protein reached 3.29g/L, about 50.2% of total cellular protein.
The embryo of japonica cultivar Zhonghua 11 (ZH11) after flowering fifteen days was used for the callus inducing. This embryogenic callus was inoculated onto four kinds of mediums N6D，N6B5D，MsD and B5D and compared. It suggested that all of the four kinds of mediums could be used in the inducing and culturing of rice callus, but N6B5D was more appropriate than N6D, N6D and N6B5D were better than MsD, MsD was better than B5D. The N6D medium was used to the sensitivity testing of embryogenic callus to Hygromycin concertration. Embryogenic callus was sensitive to the concertration of 30 mg/L, but was thoroughly restrained from the concertration of 50 mg/L. This concertration was suggested to be used in the screening of embryogenic callus resistant to Hygromycin. Using above results, it was validated in rice genetic transformation.
The artificially synthetic gene calb(only mature peptide sequence) was inserted into a yeast constitutive vector, pGAPZαA. Under the control of the promoter GAP(glyceraldehydes 3phosphate dehydrogenase)CALB was expressed constitutively. The yeast transformants still had the foreign gene after ten times of passage in YPD medium.The activity of CALB in shake flask cultured 4 days achieved 14.5U/ml.And after two steps of purification (the desalinization and DEAE column), purity of the final protein achieved 95% and protein concentration has achieved 220mg/L.
ACEI,ACEII and Xyr1 are transcriptional factors that regulate cellulase gene expression in Trichoderma koningii. In vitro experiments have shown that ACEI and Xyr1 bind to the cbh1 promoter fragment (-304 to -18) and regulate the gene transcription. However, whether ACEII binds to this 287bp fragment is still unclear. To further elucidate the regulatory mechanism of ACEII for cellulase, DNA-binding domains of ACEII from T. Koningii were expressed in E. coli. It could not show binding to the cbh1 promoter fragment (-304 to -18) by electrophoresis mobility shift assays,suggesting that it is Xyr1 but not ACEII binds playing an essential role during induction of cbh1 gene transcription.
The IgG binding domain of Streptococcal Protein G which can selectively immobilizes the Fc regions of immunoglobulin G (IgG) is a kind of good material for oriented immobilization of antibodies in antibody microarrays. Here, genetically engineered three glutathione S-transferase (GST) fused proteins ,bearing one, two and three B-Domains respectively(GST-GBx). The IgG-bindding ability of GST-GBx was investigated by ELISA. The date revealed that when the B-domain’s quantity of GST-GBx is identical, the GST-GB3 is the most efficient protein among three GST-GBx protein both the capacity and sensibility of binding IgG. The GST-GB2 is the next one and GST-GB1 is the least one. Thus,the GST-GB3 has significantly predominance in comparison to GST-GB2 and GST-GB1.
A model for screening the yeast which can ferment xylose to produce the ethanol was constructed. An ethanol yeast was obtained using the lignocellulose as substrate production the ethanol. By malt extract medium preculturing, soil samples use the plate with xylose as sole carbon source as the primary screening, then finally screen by the potassium dichromate color-displaying method. A strain named Y2-3 was screened from the soil. Phenotypic analysis including morphology and physiology and biochemical characteristics and 26D1/D2 sequence analysis were carried out. Based on taxonomy results, the Y2-3 was identified as Pichia caribbica. The strain Y2-3 ferments using xylose as sole carbon source: biomass 23.5 g/L，xylose utilization rate 94.7 %，ethanol final yield 4.57 g/L；using mixture sugar：biomass 28.6 g/L，xylose utilization rate 94.2 %，glucose utilization rate 95.6%，ethanol final yield 20.6 g/L.Pichia caribbica is a yeast which can utilize xylose and mixture sugar as substrate.It established the foundation for further research fermentation of ethanol by yeast using lignocellulose.
Tumor necrosis factorrelated apoptosisinducing ligand(TRAIL) is a novel anticancer therapeutic agent. In order to reduce the incubation time and enhance the production efficiency, firstly the response surface method was used to optimize the fermentation and expression condition of TRAIL in shake flasks. As a result, the expression of the total TRAIL protein reached 25.7%. Based on this, the influence of fermentation condition to the soluble TRAIL protein expression was studied. The recombinant strains were cultured in 3.7L fermenter and the expression of soluble TRAIL protein per gram cell enhanced 67%. That realized the high soluble expression of TRAIL protein per gram cell in high cell density and the enhancement of volume productivity.
Objective: To study the feasibility of using the bionic technology to construct small-caliber vascular prostheses with modified SIS . Methods: The vascular endothelial cells and smooth muscle cells were separated from canine saphenous artery, the cells blended with collagen gel, which were planted respectively on the SIS films, these films were rolled into the biologic three-layer prostheses around a 3mm diameter polyethylene tube; one-layer prostheses without these cells and collagen gel served as control. These 2 types of prostheses were implanted into the defect of bilateral canine femoral by anastomosis in 15 dogs. doppler colour ultrasonic, histology detection and electron microscope examination were done postoperatively. Results: By 12 weeks postoperatively, 14 biologic vascular prostheses had kept well patency, the patency rate being 93.3%, the biologic structure like blood vessels had formed, the inner surface of the vessels had been covered with full endothelial cells, a lot of smooth muscle cells had been found in the media of vascular walls in regular line; the patency rate in control group was 60.0%, the endothelial cell coverage was incomplete. Conclusions: The bionic vascular prostheses showed potent blood compatibility, which could keep long-term patency in vivo.Curative effect of repairing the small-caliber vessel defect was well satisfactory.
Microarray technology are being performed more widely than ever before on many areas in lifescience, although the technology is still evolving, the challenge of performing a microarray experiment is no longer in the data generation, but in extracting useful information and utilizing it to get the results with biological meanings.Some methods and tools used for expressional microarray data mining based on previous work were summarized. These methods include gene clustering ,GO analysis ,regulating pathway analysis, and related algorithm. We hope this can be helpful for those researchers who are implementing expressional microarray for biological analysis.
DNA shuffling is the most convenient and effective strategies for directed molecular evolution. Together with erro-prone PCR，DNA family shuffling and single-gene DNA shuffling, through iterations of random mutations, recombination and high-throughout screening, can accumulate beneficial mutations while simultaneously removing deleterious mutations, increase the abundance of mutant libraries, create novel genes and obtain desirable functional proteins. Furthermore, DNA shuffling procure broad application in many aspects, especially in pharmaceutical production, and drive the development of biological sciences and biotechnology. Moreover, taking advantage of the tremendous data analysis power, DNA shuffling as a postgenomics technology platform is being increasingly recognized.
Recently, zebrafish，as a new model species,has been used widely in the study of developmental mechanism of the embryo, a model of human disease and the drug screening. Zebrafish has been applied widely in the study of the drug for tumor antiangiogenesis with the development of the advanced technology of the mutagenesis and the confocal microscopy using for observation.Zebrafish applied in the screening of tumor antiangiogenesis drug and the mechanism of tumor angiogenesis are summered.
Ginsenosides, which belongs to triterpenoid saponins of plant terpenoids, are the main active components of the valuable medicinal herbs ginseng and American ginseng. Recent studies show that ginsenosides have a variety of beneficial effects, including anti-inflamatory, antioxidant, and anticancer effects. Ginsenosides are synthesized by complicated modification of triterpenoid skeleton after cyclization of 2,3-oxidosqualene through triterpene saponin synthesis pathway in which they share common early steps with phytosterols synthesis.It outlines recent advances for the biosynthesis of ginsenosides, the cloning and characterization of genes encoding key enzymes in the pathway and the basal framework of ginsenosides biosynthesis pathway. The prospects of secondary metabolism engineering in the biosynthesis of plant natural products and its application in ginsenosides biosynthesis are also discussed.
Clostridium acetobutylicum and C.beijerinckii are extensively studied strains for producing ABE (Acetone, Butanol and Ethanol). Both acetone and butanol from ABE fermentation are widely used as important chemicals and raw materials. Butanol has been also recognized as a kind of potential biofuel or fuel additive with better performances than ethanol in the car engine tests. Genetic engineering has been broadly applied in the modification of these strains based on current industrial requirements and development of new biological technologies.The genetic features of C. acetobutylicum and C. beijerinckii, and progressive achievements on genetic manipulations of these strains were reviewed.
