Objective: To investigate the in vivo and in vitro killing effects of CDTK gene drove by enhanced hTERT promoter in liver cancer cell line Bel-7402. Methods: Cloned CMV enhancer, hTERT promoter and CDTK gene into hrDNA targeting vector pHrn and constructed pHr-CeTpCDTK-GFP vector. After transferred into Bel-7402 cells, CDTK gene expression and in vitro killing effects were detected by RT-PCR, HPLC and MTT. Finally, the pHr-CeTpCDTK-GFP vector mixed with liposome were injected directly into the tumor of nude mouse, which was produced by the injection of those liver cancer cells, meanwhile, transfected cells were injected into mouse, the tumor size and killing effects were closely observed. Results: The RT-PCR and HPLC results showed that the liver cancer cells transfected with the constructed pHr-CeTpCDTK-GFP vector could express CDTK successfully. The MTT showed that the vector is toxic to the liver cancer cell. Through the animal test, it was found that the concentration of 5-Fu was 7.694μg/ml in treatment group, the tumor volume of treatment group decreased 6.5 times compared with the control group. Using vector-transfected cells induce tumor, the tumor formation time of treatment group was 8 days later than control group, while the mean survival period of treatment group was 16 days longer than control group. Conclusions: The pHr-CeTpCDTK-GFP vector could kill the liver cancer efficiently and resulted in a new approach for the liver cancer gene therapy.
Objective: Isocitrate lyase (ICL) of Mycobacterium tuberculosis (Mtb) was acetylated at lysine 322. The aim was to investigate ICL Lys322 acetylation and its regulatory function in activity of ICL. Methods: The prokaryotic expression plasmids pET28a-icl was constructed and Lys322 of ICL was site-directed mutated into arginine (Arg, R) and glutamine (Gln, Q), respectively. Then recombinant proteins ICLWT,ICL322R and ICL322Q were expressed and purified in vitro. Acetylation of wild type ICL and its mutants were examined by Western blotting with anti-acetyllysine antibody, and enzyme activities of proteins were evaluated. Results: Western blotting showed that ICLWT,ICL322R and ICL322Q were indeed acetylated proteins. Compared with activity of ICLWT, mutation of Lys322 to arginine decreased the enzyme activity by approximately 50%, and glutamine substitution dramatically decreased the activity more than 70%. Conclusion: The results of Western blotting showed that the ICL and two mutant proteins can be acetylated effectively in E. coli expression system. Decreasing in ICL322Q activity suggested that Lys322 acetylation could negatively regulate isocitrate lyase of Mycobacterium tuberculosis. This provided a foundation to study an important aspect of Mtb, the metabolic regulation of Mtb, which may lead to new knowledge on latent infection.
Objective: To analysis the effect of Hey1 gene on BMP9-induced osteogenic differentiation of mesenchymal stem cells C3H10T1/2. Methods: Assemble lentivirus LV5-GFP, LV5-BMP9 and LV5-Hey1, the expression of GFP, BMP9 and Hey1 were detected by RT-PCR and Western blot in C3H10T1/2 cells. Then C3H10T1/2 cells was infected with LV5-GFP, LV5-Hey1 and/or LV5-BMP9 respectively, the early osteogenic marker ALP activity was detected by quantitative assay, the later osteogenic marker calcium deposition was detected by Alizarin Red S staining, the cell proliferation and cycles regulated by BMP9 were detected by MTT and flow cytometry assay. Results: LV5-GFP, LV5-BMP9 and LV5-Hey1 promoted the expression of GFP, BMP9 and Hey1. LV5-Hey1 can increase the early and later osteogenic differentiation of C3H10T1/2 cells induced by BMP9, and promote the cell proliferation in the early stage, but inhibit cell cycles in the later stage regulated by BMP9. Conclusion: Hey1 gene can influence BMP9-induced osteogenic differentiation and BMP9-regulated cell proliferation and cell cycles of C3H10T1/2 cells.
SP0306 protein is a hypothetical transcription factor of Streptococcus pneumoniae TIGR4 strains, but the protein three-dimensional structure and biological function is unclear. The bioinformatics analyses show that it may regulate the expression of carbohydrate metabolism related genes. The protein expression vector of PET28a-sp0306 was successfully constructed and a soluble form of SP0306 protein was acquired in Escherichia coli BL21 (DE3) strain. High purity SP0306 protein was obtained after Ni-NTA affinity chromatography and DEAE negative ion exchange chromatography. High quality crystal of SP0306 protein was obtained by the vapor diffusion method, then crystal X-ray diffraction was executed.The results provide a basis for helping resolve the structure of SP0306 and further function research.
Using Western blot to investigate the gene expression in R. anatipestifer is impracticable because of lacking of internal reference protein. Complete recA gene and partial recA gene harboring His-tag were amplified from the genome of R. anatipestifer and then were cloned into expression plasmid pET32a, respectively. Recombinant proteins RecA and RecAP were expressed in E. coli Rosetta under induction with 1mmol/L IPTG. Then, recombinant proteins were purified with Ni-affinity chromatography. The polyclonal antibodies against the recombinant proteins were prepared by immunizing 4 weeks old KunMing mouse with about 50 μg purified proteins. Western blot indicated that the interaction between R. anatipestifer total proteins and serum against RecAP is more specific than that between R. anatipestifer total proteins and serum against RecA. The expression of RecA protein of R. anatipestifer is stable under different condition including common medium LB and TSB, iron chelated medium TSB+Dip or sufficient hemin medium LB+Hemin. In conclusion, RecA protein of R. anatipestifer can act as an internal reference protein in iron and hemin metabolism researches.
As a linker of proteins, 14-3-3γ plays critical roles in cell signal transductions, cell growth,differentiation and protein synthesis. Based on cultured dairy cow mammary gland epithelial cells in vitro, the impact of 14-3-3γ on the physiological function of dairy cow mammary gland epithelial cells were ivestigated. Expressing plasmids of 14-3-3γ gene were constructed, then stably and transiently transfected into dairy cow mammary gland epithelial cells. The effect of over-expression on dairy cow mammary gland epithelial cells was analyzed by CASY. Triglyceride was detected by triglyceride GPO-POD assay and beta casein was detected by high performance liquid chromatography. Expression of mTOR and p-mTOR was detected by Western blot. Experimental results show that the viability of dairy cow mammary gland epithelial cells, the expression of triglyceride and β-casein, the expression of mTOR and p-mTOR was identified up-expressed(P<0.01). All above results indicated that 14-3-3γ regulates the physiological function of dairy cow mammary gland epithelial cells through mTOR signaling pathway. The result enriches the lactation signaling pathway, which provides theoretical basis for improving the quality of milk.
The effects of chitin on the production of transglutaminase (TGase) in Streptoverticillium mobaraensis were tested. It indicated that enzyme activity had increased extremely significantly with the adding of 0.5% chitin compared with control, but 2% of chitin inhibit strains from producing TGase. Next, the mechanism about the enhancement of TGase production by chitin from four aspects,which was the pH changes during the process of metabolism, the production of TGase, the protein content and total nitrogen content in the fermented liquid were discussed. Studies show that chitin produce a certain stress for the growth of strain,thus stimulate the produce of secondary metabolites, so as to improve the production of TGase. Microscopic observation of strain fermentation process shows that it could be improving of the production of TGase by dispersing strains.
Objective: To achieve an effective capture efficiency of breast cancer cell (MDA - MB-231) by the microfluidic chip, and then analyse the changes of the target genes FN1, ITGA6 and LAMB3 expressing level in the tumor cells. Methods: The substrate of microfluidic chip was coupled with MUC1 antibodies, which could capture the tumor cells by the antigen on the cell surface. The conditions of cell capture were optimized to gain high cell capture efficiency. The captured cells were released by trypsin digestion and then the released cells were collected and re-cultured. The normal and re-cultivated cells were incubated with 1μmol/L doxorubicin for 24h respectively. Then the RNA in the cells was extracted and reversed to synthesize DNA and the target genes FN1, ITGA6 and LAMB3 were amplified by reverse transcriptase polymerase chain reaction (RT-PCR). Results: The tumor cells can be effectively captured by the microfluidic bio-chip after MUC1 antibodies were modified on the surface of chip and the capture rate can reach 80%. The cells release efficiency was as high as 98% and the released cells still had high viability which could be re-cultured. Doxorubicin could inhibit the expression quantity of FN1, ITGA6 and LAMB3 in normal and re-cultivated MDA-MB-231 cells. Conclusion: The tumor cells can be captured effectively by the microfluidic chip and re-cultured. The expressions of genes of the cells were not interfered remarkably for pre or post-captured. All of these would lay the foundation for the following related research such as biochemistry and molecular biology research, the efficacy evaluation analysis of antitumor drugs and so on.
Objective: To compare the effect of three ways of acellular process on biocompatibility and immunogenicity of the small intestinal submucosa, preparing for the scaffold of tissue engineering skin. Methods: Fresh jejunum of pig was prepared by mechanical method, mechanical-chemical method and machanical-enzymicmethod into SIS, kept as groups A,B,C, respectively. Fibroblasts were seeded on the SIS scaffolds to construct the derm substitute. The comparative examinations were performed by histological observation, MTT assay to observe the structure of the scaffolds, proliferation and adhesion of the cell on the scaffolds. The inflammation cause by the scaffolds after subcutaneous implantation for 1,2,4 weeks were also analysed. Results: Histological observation shows that there were no residual cells in groups B and C, but residual cells in group A. The proliferation and adhesion test indicated that group A was better than the other two(P<0.05).The subcutaneous implantation analysis showed that group B caused a less serious inflammation, and the vascularization capacity of group C was greater than groups A and B. Conclusion: Mechanical-chemical method and machanical-enzymic method were better ways to prepare SIS as a scaffold for tissue engineering skin.
Hpt as one of the most commonly selective marker gene was used in transgenic plants. To establish immunoblotting method for detectting HPT protein expression profiles in transgenic plants has important theoretical significance and application value. HPT recombinant protein was expressed in E. coli, and used to prepare specific monoclonal antibody. Western blot was used to detect the expression of HPT protein in transgenic rice of 4021, the results showed that in 10% of single grain of rice (about 1.5mg) contained HPT protein can be detected. Quantitative analysis revealed that the content of HPT protein in rice seedling account for about 0.01 ‰ of fresh weight. The expression profiles of HPT protein under CaMV 35S promoter-driven start in transgenic rice showed that the abundance of HPT was regulated higher levels in the leaves of the seedling and adult, showing a rising trend in the seed filling period, but HPT showing low abundance in the stems, tillers, node, shears, cushion, roots and other tissues. In short, an applicable immunoblot method for detectting HPT protein was established, while the HPT protein expression profiles were analyzed in transgenic rice.
Agarose, which is resourceful and low-cost, is one of the natural high polymer materials with biosecurity and biodegradability. It can be made into gel with three-dimensional network structure and plastic shape. Comparing with other natural materials, agarose have advantages in mechanical properties, such as tensile strength, resistance to compression, viscoelasticity, rheological property and so on. On account of the property of anti-adhesion, agarose tissue engineering scaffold, which is for filling, repairing or regenerating defective tissue, must be composited with other materials or custom-made in suitable ways, in order to enhance its histocompatibility. Research progresses have been made in tissue engineering, although the research history of agarose is not so long. The properties of microstructure and mechanics make it a suitable material for tissue engineering of cartilage, nerve, bone, cornea, oral mucosa, especially in the field of cartilage. So far, there is a long distance between research and clinical usage, and scaffold preparation and mechanism study may be the research hotspots of agarose in the future.
Basic fibroblast growth factor(bFGF) is part of the fibroblast growth factor (FGFs) family.bFGF is unique in that it is expressed both in the animal and human model but has also garnered attention for its extensive physiological functions and important clinical applications. Current evidence suggests that bFGF may play a prominent role in nerve cell regeneration, due to its pluripotent effects and neurotrophic action. Although it is already extensively studied in various disease states, there still remains much to be discovered about the functions and mechanisms in nerve regeneration of bFGF. The following review summarizes the current knowledge on bFGF with a special emphasis on central nervous system and peripheral nervous system disease.
RNAi is a common specific sequence homology-dependent gene silencing phenomenon in eukaryotes, it is an important way to regulate gene expression for resistanting to nuclease invasion. Since being found, RNAi is widely used in gene function identification, and has been widely applied in crop and antiviral improvement as a kind of reverse genetics methods. In recent years, with the deepening understanding of RNAi, RNAi showed good promising prospects as a new strategy to enhance plant resistance to nematodes, herbivorous insects, fungi and other harmful eukaryotes and achieved some results. RNAi and its research progress in enhancing plant resistance to harmful eukaryotes, and the prospect of persistent pest resistance breeding strategies for RNAi were summarized.
Natural competence is a physiological state in which bacteria develop a capacity to take up exogenous DNA in the natural environment. Most bacteria have similar DNA-uptake mechanism which mainly includes two processes. Firstly the exogenous DNA is degraded into single-stranded DNA (ssDNA) with the coordinated effect of competence protein. Then ssDNA are integrated into the chromosome under the protection of DprA, SsbB and RecA. Nowadays, the competence regulation in Streptococcus mutans is extensively studied. S. mutans combined with other bacteria were cited as the examples to introduce the advances of natural competence. The ComX in S. mutans works as a core regulatory protein during natural competence. It could activate a large set of late competence genes required for exogenous DNA uptake and integration. The comX in S. mutans are focused on. The regulatory mechanisms of ComDE, ComRS, HdrRM, BsrRM and RcrRPQ on comX were summarized. Besides, ComX stability and ComX-regulated genes were elucidated. Meanwhile, the intimate linkage between natural competence and biofilm formation, acid resistance as well as bacteriocins synthesis was analyzed. Finally, the potential research trends of natural competence were put forward.
Bioactivity determination of antibody-based therapeutics is essential in quality control.Presently, the main method to determine bioactivity is in vitro assay.Cell-based determination is widely used because of its advantages, such as simple operation, short period, good specificity and small variation, etc.In order to obtain the cell lines with ideal reactivity and simple detection method,construction of transgenic cell lines become one of the common strategies.In addition, some new technologies have rapidly been applied to evaluate the bioactivity of antibody drugs.Combined with the current research status and trend in development of bioactivity determination method, three aspects of antibody bioassay methods, which are based on cells, genetically modified cells and new technologies were described,thus providing new ideas for drug development and quality control.
Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system is a novel tool for targeted genome editing and regulation of gene expression. Cas9 offers several potential features, including the case of customization, higher targeting efficiency and the ability to facilitate multiplex genome editing, which suitable for large-scale gene editing in a variety of type cells. The CRISPR/Ca9 system with the background of investigation, the mechanism of reaction and its progress was described. The application of CRISPR/Cas system in transgenic animals was summarized in the gene knockout, knock-in and knock-down. CRISPR/Ca9 system was compared with Zinc finger nucleases and transcription activator-like nucleases, and demonstrated obviously advantages. CRISPR/Ca9 technology has great prospects for application in transgenic animals.
Based on ISI Web of Science, the published articles on field of transgenic rice during 1988 to 2013 are analyzed with Thomson Data Analyzer,Histcite and visualized by Citespace. These analysis can show important countries,important orgnaziton,important journal in the field of transgenic rice.These co-author maps and co-term clustering analysis can reveal co-author phase and hotspots in this field. These analysis can help the researchers get an overview of this field quickly.
Starting from the practice of China's science communication of GMOs, systematically investigates the international and domestic empirical research in this field. From the perspective of communication studies, psychology, and sociology, analyzed the difficulties faced by China's science communication of GMOs and trace the polarization and solidification of the public attitudes to GMOs. As a whole, scientific knowledge has a low correlation with the acceptance of GMOs, while trust, value and scientific institutionalism have deeply influenced the public attitudes towards the controversial technology. Based on the analyses, this paper proposes a set of possible routes that might be adopted in China's science communication of GMOs.
