Objective: To express and prepare the recombinant type II dengue virus nonstructural protein NS1, and study its immunogenicity. Methods: Type Ⅱ Dengue virus NS1 gene were cloned into plasmid pUC57 after codon-optimizing and gene synthesizing. Then the NS1 gene were subcloned into expression vector pET28a. The NS1 protein were inducing expressed by adding IPTG followed by immunoblot identification. Recombinant NS1 protein were renaturated through dialysis and then purified. NS1 polyclonal antibody were preparation by animal immunization. The titer and immunogenicity were detected by indirect enzyme-linked immunological method. Results: The recombinant type II dengue virus nonstructural protein NS1 were successfully expressed, renaturated and purified. It's proved that the recombinant NS1 protein showed good immunogenicity by Western blotting and mice immunization. Conclusion:The recombinant NS1 protein has good immunogenicity, and it's a good beginning for NS1 monoclonal antibody preparation and the development of dengue virus detection kit.
Objective: To analysis the effect of Sonic hedgehog signaling on BMP9-induced osteogenic differentiation of mesenchymal stem cells and the mechanism involved. Methods: C3H10T1/2 and C2C12 cells were treated with Cyclopamine, Purmorphamine, adenovirus Shh or/and BMP9, then the early osteogenic marker ALP activity was detected by quantitative and staining assay, the later osteogenic marker calcium deposition was detected by Alizarin Red S staining, the expressions of Shh signaling related genes and osteogenesis related genes were detected by RT-PCR, the expressions of Shh detected by Western blot, luciferae reporter assay was detected by quantitative. Results: BMP9 can promote the expressions of Shh signaling related genes. Purmorphamine and adenovirus Shh can increase the ealy and later osteogenic differentiation and expressions of osteogenesis key tromscription factors of C3H10T1/2 and C2C12 cells induced by BMP9, but Cyclopamine has the opposite effects. Furthermore, Purmorphamine and adenovirus Shh also led to luciferase activity of canonical Smad pathway stimulated by BMP9. Conclusion: Shh signaling influence BMP9-induced osteogenic differentiation of C3H10T1/2 and C2C12 mesenchymal stem cells.
Objective:To over express RAB27A by lentiviral system in human hepatocellular carcinoma (HCC) cell line HepG2 and investigate the effect.Methods:GFP-RAB27A protein coding gene sequences were amplified from plasmid pEGFP-C1-RAB27A, the PCR products were digested by double restriction enzymes and cloned into shuttle plasmid pENTR/U6, then pHAGE-GFP-RAB27A vector were constructed by Gateway technology. Recombinant plasmid pHAGE-GFP-RAB27A was mixed with the packaging plasmid(psPAX2)and the envelope plasmid (pMD2.G)and then co-transfected into HEK-293T cells. HepG2 cells were transfected with concentrated recombinant lentivirus particle. Western blot analysis were used to detect RAB27A expression in HepG2 cell lines. The proliferative activity was determined by CCK-8 and colony forming assay and the cell cycle analysis by flow cytometry. Results:The recombinant lentiviral expression vector pHAGE-GFP-RAB27A was constructed correctly. After transfected to HEK-293T, the lentivirus was successfully prepared. Strong green fluorescence was observed in transfected HepG2 cells under fluorescent microscope which showed the recombinant lentivirus expressing. The RAB27A protein was over expressed showed by Western-blot. The proliferation of infected HepG2 showed inhibited, and the percentage of S-phase cells was obviously decreased (P<0.01). Conclusion: The exogenous over expressed RAB27A was found inhibited the proliferation of transfected HepG2 cell lines. RAB27A is a key protein in the hepatocellular carcinogenesis and proliferation.
Objective: To investigate the effect of IQGAP1 knockdown by RNA interference (RNAi) on homotypic cell-cell adhesion of esophageal carcinoma (EC) cells. Method: Western blot analysis was adopted to detect the protein expression of IQGAP1 in KYSE150 and EC9706 cells. Slow aggregation assay and cell dissociation assay were performed to detect the homotypic cell-cell adhesion properties of tumor cells. Further, KYSE150 and EC9706 cells were transfected with RNAi plasmid for targeting IQGAP1 gene or negative control, and then the cell lines of IQGAP1 knockdown were screened with G418. Level of IQGAP1 protein in interference group and control group was measured by Western blot, and the changes of homotypic cell-cell adhesion were observed by slow aggregation assay and cell dissociation assay. Result: Compared with EC9706 cells, level of IQGAP1 protein in KYSE150 cells was lower, while the homotypic cell-cell adhesion in KYSE150 cells was stronger. Further, Western blotting analysis showed that IQGAP1 protein expression was efficiently downregulated by stable transfection with IQGAP1 siRNA expression vector. The homotypic cell-cell adhesion was obviously enhanced by IQGAP1 knockdown. Conclusion: These data suggest that downregulation of IQGAP1 expression could significantly enhance homotypic cell-cell adhesion and decrease malignant phenotype of EC cells.
Objective: Construct recombinant lentivirus NUP88-shRNA mediated by the carrier, by RNAi technology respectively to observe silence after NUP88 MCF-7 proliferation, adhesion, invasion and metastasis of influence, looking for new targets for clinical gene therapy of breast cancer. Methods Build NUP88 Lentivirus vectors, packaging test after drop, to MOI cell transfection breast cancer MCF-7, using RT-PCR and Western-blot test each mRNA and protein expression in MCF-7 cell efficiency; Determined by MTT method and flow cytometry assay, after testing NUP88 gene is interference effects on MCF-7 cell proliferation and apoptosis; Cell invasion after experimental detection NUP88 gene is interference effects on MCF-7 aggressivity. Results Four groups of virus and a negative control group are building a successful, drop degrees are 4E+ 8 Tu/mL; RT-RCR and Western-blot test, the results show that the NUP88-shRNA transfection of MCF-7 cell group NUP88 mRNA and protein expression and the negative transfection group and blank MCF-7 cell group compared with significant difference statistically significant (P<0.01); Determination of NUP88-shRNA1 group of silence, the most efficient silence rate can reach 86%; Determined by MTT method, the results show that the experimental group after NUP88-shRNA1 Lentivirus transfection cell proliferation significantly reduced, compared with the blank group and the control group with significant difference (P<0.05); Flow cytometry instrument detection of three groups of MCF-7 cell apoptosis results show that the experimental group after lentivirus transfection cell apoptosis rate increased significantly, compared with the control group and blank group had significant difference (P<0.05); Cell invasion experiment show that the tumor cells to conventional training after 24 h, compared with the blank group and the negative control group, experimental group in membrane cells decreased significantly, with significant difference (P<0.05). Conclution NUP88 recombinant lentivirus can suppress the MCF-7 by RNAi successful NUP88 gene expression, and can significantly inhibit the proliferation and invasion ability in the distance.
Objective: To observe the effect of combined-silencing MMP-9 and FAK on invasion and migration of mouse melanoma highly metastatic B16F10 cells in vitro. Methods: Construct recombinant plasmid vectors pGV102-MMP9-siRNA and pGV102-FAK-siRNA respectively. Culture mouse melanoma B16F10 cells, then the plasmid vectors were transfected into cells using Lipofectamine TM2000 method. This experiment was divided into 5 groups of blank control group, negative control group, Anti-MMP-9 experimental group, Anti-FAK experimental group, and Anti-MMP-9 &FAK experimental group. G418 was used to screen those GFP+ cells. In addition, the transfection efficiency were assayed with flow cytometry, and the cellular morphology of transfected cells were observed by confocal microscopy. mRNA level of MMP-9 and FAK of all groups B16F10 cells was detected by RT-PCR. Finally invasion and migration of transfected B16F10 cells in vitro were tested by transwell experiment. Results Transfection efficiencies of the three experimental groups were to 92.41±1.64%, 95.72±0.21%, 91.52±0.11% respectively by flow cytometry via G418 screening. Moreover, screened cells morphology was good. Compared with the blank control group, MMP-9 and FAK mRNA level of three experimental groups' cells was significantly lowered (P<0.01); their invasion and migration also weakened dramaticly (P<0.01), and Anti-MMP-9 &FAK experimental group was very distinctly lower than Anti-MMP-9 experimental group and Anti-FAK experimental group. Conelusion Combined-silencing MMP-9 and FAK could obviously inhibit invasion and migration of mouse melanoma cell B16F10 in vitro.
In order to reduce acetate production and improve succinate production and explore the main acetate formation pathways of C.acetoacidophilum, gene of pta, ackA, ctfA and aceE, which encoded phosphotransacetylase, acetate kinase, CoA-transfease and pyruvate dehydrogenase complex in C.acetoacidophilum were disrupted respectively by the means of homologous recombination. In C.acetoacidophilum ΔldhAΔpta-ackA, the titer of acetate, gluocse consumption rate, and both yields of acetate and succinate were found to be similar to that of C.acetoacidophilum ΔldhA. Furthermore, the titer and yield of acetate decreased by 81.4% and 77.2%, respectively, the glucose consumption rate decreased by 28.3% and the yield of succinate increased by 25.3% in C.acetoacidophilum ΔldhAΔctfAΔpta-ackA. In addition, when gene aceE was deleted, C. acetoacidophilum ΔldhAΔaceE almost produced no acetate, the glucose consumption rate decrease by 35.6% and the yield of succinate increased by 34.7%. Under oxygen deprivation, almost all acetate formed through acetyl-CoA in C.acetoacidophilum. Gene pta, ackA and ctfA were the main genes of acetate formation pathways from acetyl-CoA. Disrupting gene aceE could cut off acetate synthesis in C.acetoacidophilum, and effectively increase succinate production.
The gene of α-amylase was amplified by through RT-PCR with Solanum tuberosum cv.Shepody cDNA as template, which was subcloned into vector pPIC9k and the recombinant plasmid pPIC9K-amy was linearzed with Sac II, then transformed into P.pastoris GS115 by electroporation. The high expressed amylase active strain GSamyA5 was attained by Trypan blue-BMMY medium screening and induced to express the enzyme with 0.5% methanol for 3 days under the 30℃, and fermentation broth was purified by Ni2+-NTA agarose. the enzyme characterization was studied. The result showed that The enzyme activity was stable between 40~50℃ with maximum activity at 45℃ and the activity hold 92.6% keeping 50min. The enzyme activity was stable between pH6.0~7.0 with maximum activity at pH6.0. Ca2+ and K+ had a stimulating effect on the recombinant amylase. The relative enzyme activity was up to 125% with Ca2+, Cu2+, Fe2+, Fe3+ and Zn2+ had an significantly inhibitory effect on the recombinant amylase.
Myxobacteria are known to be the third category of bacteria, which produce metabolites of potential anticancer effects. Here the lab-stored Myxococcus macrosporus STXZ54 was cultured, and components of its supernatants were attained by ammonium sulfate or acetone precipitation. Cytotoxic effects of the sediments were evaluated through an in vitro method. Further, the unique protein, WGF5, was successfully separated by high performance liquid chromatography from the myxobacteria. Its inhibitory effects on cancer cells were also analyzed by MTT assay. Simultaneously, confocal microscopy was employed to observe the alterations of B16 subcellular structure after treatment by WGF5. The results demonstrated that WGF5 was a very stable protein under room temperature and could efficiently inhibit the growth of B16, Hela, MCF-7 and Hep-3B cancer cells. The IC50 of WGF5 after 48 h incubation with the cancer cells was 2.767 μg/ml, 2.204 μg/ml, 3.758 μg/ml and 3.073 μg/ml, respectively. Moreover, MTT assay and confocal microscopy observation found that WGF5 probably restrains the proliferation of the cancer cells through changing the cell morphology. Briefly, this study indicates that the WGF5 of Myxococcus macrosporus STXZ54 has an broad-spectrum anticancer effects and could be used for cancer therapy in the near future.
Object: The bicistronic recombinant adenovirus containing enhanced green fluorescent protein (EGFP) and transcription factors Lmx1A (LIM homeobox transcription factor 1-alpha) was constructed and packaged. The feasibility of dicistronic construction methods was evaluated. An experimental basis provides for differentiation potential of mechymal stem cells into neurons. Methods: The CDS of EGFP of Lmx1A, which was obtained by PCR, was inserted into the bicistronic expression plasmid pcDNA3.0BA. The dicistronic expression cassette which obtained by PCR, was inserted into the adenovirus shuttle plasmid pShuttle-CMV and followed homologous recombination with backbone plasmid pAdEasy-1.The adenovirus Ad-EGFP-Lmx1A was packaged in HEK293 cells. Human umbilical cord mesenchymal stem cells(hUC-MSCs) were infected with Ad-EGFP-Lmx1A.The expression and location of Lmx1A and EGFP was identified by RT-PCR, immunofluorescence and Western blot technology. Results: The packaged adenovirus showed typical morphology of an adenoviral particle, about 75nm, through TEM examination. The expression product s of EGFP and Lmx1A could be detected independently of each other. There was no significant difference in the expression strength. In conclusion, two genes can be expressed simultaneously in bicistronic eukaryotic expression plasmid pcDNA3.0BA.Two genes were transmitted into mesenchymal stem cells through adenovirus infection. Adenoviral expression efficiency can be observed in real time by observing the EGFP. These findings establish a rapid and effective method for constructing any two-gene expression. The construction of Ad-EGFP-Lmx1A provides an important basis for the role exploration in mesenchymal stem cells differentiating into neurons.
Chromatin Immunoprecipitation which is an important method in gene expressing regulation has been widely used recently. However, the application of ChIP for mammary gland has not yet been reported. When the traditional tissue-ChIP kit was used in mammary gland, it had some problems, including that the low sample recovery rate, hybridization efficiency disturbed by butter fat/lactoprotein and still-accumulating heat in the sonication. In order to solve these problems, in this study, the ChIP method was improved for mammary gland. In the improved protocol, the step of homogenizing the tissue was omitted, sonication was replaced by enzyme digestion-sonication, and the washing procedure was added between swelling and enzyme digestion. The results showed that the mammary gland was not suitable for homogenizing compared with the spleen. Enzyme digestion-sonication was more suitable for shearing the chromatin than sonication. Compared with the traditional CHIP method, the target sequences were significantly enriched in the DNA sample, and the DNA sample reached the demands for sequencing by using the improved method. It indicated that the improved ChIP method was suitable for mammary gland, and it would help for the research of gene expression regulation in the mammary gland.
The ovine myostatin gene was subcloned into the pET-32a(+),and transformed into E.coli BL21.The MSTN recombinant fusion protein was purified and the healthy Bactrian camel was selected for immunization with the MSTN protein.Total RNA was extracted from peripheral lymphocytes for the amplification of VHH fragments by RT-PCR.Primers designed according to the single domain gene sequences of the antibodies and the variable camel IgG2, IgG3 genes were amplified by Nested PCR.PCR products were then purified and inserted into phage mid vector pCANTAB5 E. The VHH antibody gene library was obtained by electroporating recombinant pCANTAB5E VHH vectors into E. coli TG 1 cells.The first set of antibody gene library was composed with 9.5×105 individual colones after cloning VHH genes and the library we possessed high diversity and good capacity,which provides a platform for panning VHH camilid antibody aimed at ovine MSTN.
An efficient alginate-degrading strain isolated from rotten seaweed was identified. With the results of morphological properties, physiological characteristics as well as 16S rDNA sequencing, the strain was identified as Halomonas sp. WF6. The fermentation medium component was optimized by both single factor and orthogonal test, which was composed of sodium alginate 6.0 g/L, peptone 5.0 g/L, yeast extract 2.5 g/L, NaCl 30 g/L, K+ 5 mmol/L. This strain was cultivated at 25℃ for 39 h with optimized conditions of initial pH 8.0, inoculum size 2% and liquid volume 30 ml/250 ml, the alginate lyase activity was increased to 117.66 U/ml, which was 2.1-fold compared with the control. The enzymatic hydrolysates of sodium alginate mainly included di- and tri-alginate oligosaccharides.
Hepatitis B is one of the highest incidence and mortality diseases and caused mainly by the acute and chronic infection of hepatitis B virus (HBV). Nowadays, the treatments of HBV-infected patients are mainly dependent on the antiviral drugs, such as nucleotide analogues and interferon. However, analysis of viral kinetics during therapy revealed that the virus replication was not completely inhibited and the rate of clearance of infected hepatocytes was slow during therapy. Recently, large amount of experiments showed that RNA interference (RNAi) were capable of reducing HBV gene expression and replication in vivo when HBV genes were delivered simultaneously with small interfering RNA (siRNA) or siRNA expression constructs. RNAi-based therapeutics has the potential to treat chronic HBV infection in a fundamentally different manner than current therapies. The main target HBV gene to be silenced, siRNA delivery strategy, the therapy efficiency and the application prospects have been discussed in this review.
Bacterial ghost (BG) is an empty bacterial envelope of gram-negative bacteria devoid of cytoplasmic content,and produced by controlled expression of bacteriophage PhiX174 lysis gene E. BG keeps intact bacterial outer membrane structure, such as flagella, LPS and outer membrane proteins, making bacterial easy to adherence to the human or animal cell receptors, so it can be used as vaccines without adjuvant to induce strong humoral immune response and cellular immune response. BG has many spaces, such as the outer membrane, the periplasmic space and the inner membrane to load other substance. BG also can be used as DNA, antigen and drugs delivery vector as it exhibits potency with regard to target antigens and large capacity compared to conventional approaches. In addition, BG can be provided with a foreign antigen, nucleic acid or other ingredients to construct combined vaccine for human or animal health services. BG has wide application prospect in the prevention and treatment of disease.
Biocatalysis is the process of applying enzymes or biological organisms in useful chemical conversion. In the context of concerns about the environmental aspects of the traditional chemical catalysis, biocatalysis provides an attractive alternative. In the past few decades, with a great progress on the study of biocatalysts, there will relevantly be a development wave on biocatalysis. So, the discovery and protein engineering of biocatalysts are becoming a hotspot in current researches. The emergence of the metagenomic library technology overcomes the barriers that many microorganism can not be cultured, hence more and more potential biocatalysts can be gained from natural resources. Owing to the development of the molecular modification technologies based on the rational design, rapid and efficient protein engineering of potential biocatalysts can be done to meet the need of industrial production. With the progress of the discovery and protein engineering method of biocatalysts, more and more excellent biocatalysts have been widely used, and biocatalysis have further application in the industrial production. The latest progress on the discovery and protein engineering of biocatalysts based on the research work were summarized to provide the theoretical foundation for getting more excellent biocatalysts which can be applied on industrial scale.
Bio-imprinting technique is a straightforward technique for enzyme modification, which is utilized in organic synthesis, preparation of biosensor, bioseparation. It has a potential application in green synthesis of chemicals. A few recent literatures were reviewed in the occurrence and development of "bio-imprinting" conception, classification, application, strategy, and so on. Aiming at the problems occurring over the course of the study of bio-imprinting technique, its development trend in the future was outlooked. It is expected that this work will provide helpful references for relevant researchers.
