STAT3 (signal transducer and activator of transcription 3) is a dual functional transcription factor with functions as signal transduction and transcription regulation. There are reports that the expression of STAT3 in breast cancer is significantly higher and STAT3 can facilitate breast cancer metastasis. To clarify the definite effect and molecular mechanism of STAT3’s involvement in breast cancer metastasis, STAT3 was silenced by RNAi in the murine highly metastatic 4T1 cells. The silencing of STAT3 didn’t affect the proliferation rate of breast cancer 4T1 cells in MTT assay. However, the migration activity of STAT3 knockdown cells was strongly reduced in Transwell assay. The real-time quantitative PCR results indicated that STAT3 silencing significantly suppressed the expression of VEGF, IL-6 and mosin, while increased E-cadherin expression. Western Blot assay showed that phosphorylation level of MAPK was reduced in STAT3 knockdown cells. The results of this study demonstrate that STAT3 plays an important role in breast cancer metastasis and provide experimental evidences in therapy with STAT3 as a target.
To express and purify herpes simplex virus type I (HSV I) glycoprotein B (gB) in eukaryote cells with the purpose of analyzing the antigenicity and immunogenicity. At the beginning, the extracellular domain fragment gene of gD1 was synthesized by chemical method and cloned into eukaryotic expression vector pCEP4 to construct the recombinant plasmid pCEP4-gD1. After transfection of HEK293 cells with the recombinant plasmid, the expressed protein was characterized by Western blot and purified through Ni affinity chromatography. Then antigenicity of the protein was detected by ELISA. Finally, the purified protein was used to immunize Kunming mice in 1, 3, 5 weeks respectively, and antiserum were collected in 3, 5 and 7 weeks. The antibody titer was detected by indirect ELISA for immunogenicity analysis. Gene sequencing analysis demonstrates that the recombinant plasmid pCEP4-gB1 was constructed successfully. Western blot analysis indicated one major protein band, which molecular weight is approximate 85 kDa corresponding to the truncated forms of gB1 protein, was observed. In addition, ELISA detection showed that expressed gB1 has good antigenicity. After the third immunization, antibody titer of the mouse anti-gB1 was 5×103. The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.
Objective: To investigate the mechanism of LINE-1 encoded ORF-1p in the tumorigenesis of lung cancer. Methods: The down-regulation of ORF-1p was conducted by RNAi approach and the features of transfected A549 cells were characterized by the approaches of MTT，flow cytometry and soft-agar anchored colony formation. Results: With the constructs of recombinant plasmids, LINE-1 encoded ORF-1p was down-regulated significantly in transfected A549 cells. The characters of transfected A549 cells were appeared to change as follows: Cell proliferation was significantly reduced, which was probably attributed to up-regulation of p21 and p15; cell cycle exhibited S phase arrest (P<0.05); colony formation was significantly attenuated. Conclusion: The down-regulation of ORF-1p can attenuated the A549 characters significantly upon its tumor formation ability.
As one of the important histone acetyltransferases, PCAF(P300/CBP associated factor) mainly mediates the acetylation of core histones and hence plays a vital role in the transcriptional regulation. Several cellular proteins that interact with PCAF directly, such as P300/CBP, p53 and MDM2, have been reported localized in nuclear dot 10 (ND10), an intranuclear structure with a typical discrete punctuate distribution. ND10 contains more than 70 kinds of proteins and has been implicated in the regulation of gene transcription. The most key component of ND10 is PML (promyelocytic leukemia protein). PML is well known to be essential for the maintenance of the normal structure and function of ND10. By the use of yeast two-hybridization, GST pull-down, co-immunoprecipitation and fluorescence colocalization assays,the evidence for the direct interaction of PCAF with PML and the localization of PCAF in ND10 was provided, which will be beneficial for the further research on the biological function of PCAF.
Objective: To observe neointima formation in rabbit artery injury-block model which were transferred by pcDNA3.1/hTM plasmids. Methods: After transferred pcDNA3.1/hTM plasmid into rabbit artery by high-pressure injection, rabbit common iliac artery were cut and anastomosed again. The expression of hTM protein at vessels wall were examined with immunohistochemistry at 3d,7d,14d and 28d after operation. At the 14d and 28d after second operation, the inside diameter of anastomotic stoma and blood flow velocity were checked by color Doppler. The treated artery were sliced and stained by Verhoeff. Local neointima formation and the ratio stenosis of intervascular was calculated by computer. Result: The expression of hTM was at higher level in hTM gene treated group than vector treated group and control group at 3day, 7day, 14day, 28day after second operation. At 7day, the level was highest. At 14day and 28day, Color Doppler showed that the stoma bore were 1.93mm±0.34mm(14d),1.89mm±0.28mm(28d) in hTM gene treated group；1.59mm±0.43mm(14d),1.38mm±0.28mm(28d) in vector treated group；1.46mm0.25mm,1.44mm±0.32mm in control group. The ratio stoensis were 32%±23%(14d),37%±14%（28d） in hTM gene treated group；58%±21%(14d),63%±17% in vector treated group；58%±19%(14d),61%±23%(28d) in the control group，respectively. Conclusion: Human thrombmodulin gene could reduce the neointima formation and vascular stoensis in rabbit artery injury-block model.
Abstract HSA-NIFN gene was got by total gene synthesis technology in vitro. The NIFN-HSA gene was inserted into Pichia pastoris express vector pPIC3.5. The recombinant plasmid pPIC3.5/HSA-NIFN was linearized by restriction enzyme Sal I and then transformed into Pichia pastoris GS115 by electroporation. The recombinant strains confirmed by PCR analysis and were induced by methanol to express fusion protein HSA-NIFN. SDS-PAGE , Western blot and MALDI-TOF-MS analysis of the fusion protein showed that the expressed fusion protein HSA-NIFN with 86369Da molecular weight had the antigenicity of HSA.The putity of HSA-NIFN was higher than 90% after Blue Sepharose FF and CM Sepharose FF, and the biological activity determined by WISH-VSV system was more than 7.75±0.39×106 IU/mg.
Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. It’s difficult to be applied because of its extremely short half life time. Modifying the dipeptidase IV recognition amino acid can prolong its half life time. Different GIP mutant genes that were obtained by artificial synthesizing or site-directed mutagenesis were cloned into the vector pET32a(+). These plasmids were expressed in Escherichia coli. The 6-histamine fused proteins were purified by NTA-Ni2+ affinity chromatography, respectively. Purified fusion proteins were then digested with enterokinase and purified with another affinity chromatography and desalting. The biological activities of GIP analogs were tested in vivo in Kuming mice or diabetic db/db mice at a dose of 48nmol/kg by intraperitoneal injection associated with injection of glucose at 18mmol/kg. The results showed that the half life time of analog mGIP243 in Kunming mice was extended to 90 minutes (p<0.001), which was much longer than those of ala to ser analog mGIP2 (30 minutes) or native GIP(shorter than 30 minutes). The oral glucose tolerance tests in Kuming mice and db/db mice both supported the results with a magnificent glucose lowering effects (p<0.01 VS saline group) by the end of a 60 minutes test. Interestingly, the novel GIP analog mGIP243 played good glucose lowering effect in diabetic mice. In conclusion, the novel GIP analog mGIP243 is a good drug candidate for diabetes.
The human soluble receptor for advanced glycation end products (hsRAGE) fused with the immunoglobulinin G Fc fragment (IgG Fc) was expressed in Rosetta(DE3) by IPTG induction.The result of SDS-PAGE analysis indicated the hsRAGE-IgG Fc fusion protein was expressed in the form of inclusion body.The expression level of the fusion protein was about 40% of the total cellular protein.After renaturation and purification,a purity of about 96% of the fusion protein was obtained.Result of Western blotting indicates the fusion protein can specifically combine with sRAGE antibody and significantly inhibit the over expression of NF-κB p65 of ECV-304 cells induced by AGEs.
A homologue of DAHP（3-deoxy-D-arabino-heptulosonate 7-phosphate synthase）, designated StDAHP(GU479467), was isolated from Solanum torvum by homology cloning and RACE. Bioinformatics analysis showed that the full length of the cDNA contained an open reading frame of 1683 bp coding a protein of 560 amino acids, corresponding to a 62.53 kDa polypeptide with an isoelectric point of 9.1. Alignment of the amino acid sequence of StDAHP and that of other homologues showed that StDAHP had a high identity with DAHP from S. tuberosum, S. lycopersicum, N. tabacum, and O. sativa). StDAHP protein was more conservative near its N-terminal and C-terminal. Analysis of StDAHP mRNA level by semi-quantity RTPCR showed that its expression was induced by Verticillium dahliae infection.
To study N-linked glycosylation of the phytase from Aspergillus niger 963 in the significance of its enzymatic properties, we constructed two N-glycosylation mutants by Megaprimer PCR-mediated gene site-directed mutagenesis techniques , Make the codon of N87 and N102-asparagine was replaced by the codon of glutamine. according to the location ,the two mutants were named N87Q、N102Q, sequencing alignment and analysis showed that site-directed mutagenesis was achieved in the nucleic acid level,we also constructed expression vector pPIC9-N87Q, pPIC9-N102Q,The recombinant protein were expressed in Pichia pastoris GS115 by fermentation,The N87Q retained more than 50% of its initial activity after incubation at 60℃ for 1h, while the activity of N102Q was lost completely after ten minutes,The N87Q retained more than 70% of its initial activity after being incubated under varying pH conditions (pH 1.0～10.0) at 37?C for 1h,however,The N102Q was not active above pH 8.0.
A deeper analysis of the role of anaerobic ammonium oxidation (ANAMMOX) bacteria will help to understand its application in the biological nitrogen removal. In this study, using the single-stage autotrophic nitrogen removal system with stable operation and 90% of ammonia conversion rate and 80% of total nitrogen removal rate, the total DNA of cultivated sludge taken from the bottom of the system was extracted by molecular biology methods. With the total DNA partial 16S rDNA sequence of ANAMMOX bacteria was amplified by polymerase chain reaction (PCR) with a pair of specific primers Pla46rc/Amx820. Amplified product was cloned, sequenced and BLAST analysis and the result indicated that the sequence has 99% identities with ANAMMOX bacteria Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anammoxidans. The phylogenetic analysis showed that the anaerobic ammonium-oxidizing bacteria in the single-stage autotrophic nitrogen removal system have closer relationship with Candidatus Kuenenia stuttgartiensis evolutionarily.
A new type of flocculant which modified chitosan magnetic microscopic particles were prepared by inverse suspension method in Lab.And based on this, the flocculability experiment on papermaking wastewater was carried out . Relations of flocculability with the dosage of flocculant, pH, agitation speed, agitation time and settling time were discussed. The results indicate that the removing efficiency for CODCr are 56.52% under the best conditions.It also had some advantages such as little dosage, wide pH and shorter settling time.So it is a good method for treatment of pulp and paper wastewater by modified chitosan magnetic microscopic particles.
Through gradient acclination, a strain that could use four kinds of dyes (Congo Red , Malachite Green, Methylene Blue, and Reactive Blue) as sole carbon source were screened from dye waste contaminated soil and named as strain XSMR. It was identified as Achomoobacter sp. by its morphological, physiological and biochemical characters, and 16SrDNA sequence analysis. Strain XSMR had strong decoloring capability for four kinds of dyes. Moreover, strain XSMR reproduced and grew while decolorizing dyes. Bacterium dryweight was more than CK after 24h. The results from the study on the decoloring condition of strain XSMR showed that the optimum temperature for decolorization was 35 ℃, optimum pH was 7.5, and optimum inoculating dose(v/v) was 4%. The best decolorization rate could be obtained at the initial concentration with Congo Red , Methylene Blue, Malachite Green, and Reactive Blue less than 200mg/L, 200mg/L,150mg/L, and 150mg/L respectively. Under the optimum conditions, the strain could remove more than 98% of the color of four kinds of dyes in 14 h treatment. The products of four kinds of dyes degraded by XSMR were determined by UV-Visible spectra assay, and they were found that the dyes were completely degraded by XSMR. The mechanism of decolorization of dyes by XSMR includes biodegradation and biosorption.
Objective:To study the optimum protocol for analyzing proteins from cells by SELDI-TOF-MS.Methods:Proteins were extracted from cell samples by ultrasonic disruption, U9 cell disruption buffer and cell disruption buffer were established respectively. The concentrations of proteins were detected by BCA method, then protein samples were applied directly to the ProteinChips arrays using WCX magnetic bead and the bioprocessor, respectively, and protein samples were captured by Chips of WCX2, SAX2, IMACCu and H50, respectively, their results were compared, and detailed protein expression differences were detected using the chip of WCX2.Results:The concentrations of proteins isolated from cells using 3 different ways are: 0.25±0.034μg/μl，0.6±0.06μg/μl，1.02±0.077μg/μl,respectively. The method of Biochip Processor is simple, which requires less sample size and time. There is a good relationship between the protein peaks and protein concentrations in SELDI protein spectra. There are differences in the type of proteins captured by chips of WCX2, SAX2, H50, IMAC-Cu. In the limit of 1000~300 000Da, 87 protein expression difference peaks were detected by WCX2 chip, amount of which, 17 show a tendency variation.Conclusion:Using selfprepared cell disruption buffer receive highest protein concentration and which is better for further test.Bio-Chip Processor does better in application of sample.SELDI can not only detect protein expression differences but also reflect the protein concentration relationship; Selecting an appropriate chip or combining use of different chips to detect the protein expression differences can obtain more satisfactory results.
In order to get high quality RNA from mangrove plants rich in tannins and polysaccharides，based on summing up and improved our predecessors, the conditions of extraction buffer was optimized to inhibit the oxidation of tannins by the conditions of alkaline conditions, PVP and β-mercaptoethanol. Most of the polysaccharjdes and tannins extracted with nucleic acids are removed by taking advantage of differences in solubility of these compounds in the solvent 2-butoxvethano. Moreover, LiCl was used to selectively precipitate RNA and obtained higher purity of the RNA which can used for operation of molecular biology such as cDNA synthesis, cDNA library construction and RACE technique.
A gene expression system were constructed, driven by a heat-shock gene promoter (AtHsp17.6-C2), to control the expression of reporter gene (GUS) in transgenic plants. The expression of the AtHsp17.6-C2 chimeric gene in the stable transformants of ⊿F122-13 was hardly detected in culture at 22℃. However,the expression increased dramatically at the transcriptional level when the incubation temperature was shifted to 34~37℃. The optimal temperature for heat (shock) induction was 37℃. After a 2h incubation at 37℃ and 2h recovery phase at 22℃, GUS activity was about 80 fold greater than that before heat shock. Multiple heat-shock treatments showed that this system was heat reinducible. This new system can be applied in numerous and various applications.
Vibrio vulnificus is an important foodborne pathogen, which mainly exists in estuaries and the marine environment and seriously harms aquaculture development and human health. Therefore, the development of a technique that can identify the pathogen quickly, accurately and protect aquaculture development and enhance the food safety is of great importance. Here a rapid method for detecting Vibrio vulnificus with loop-mediated isothermal amplification (LAMP) using the vvHA gene of Vibrio vulnificus as target gene were established. Forty-six bacterial strains were selected to test specificity of the LAMP method and only Vibrio vulnificus strain was LAMP positive result, and indicating the high specificity of the LAMP method. The LAMP method could be completed in 40~60min and showed a good sensitivity of 15 CFU/ml for cultured Vibrio vulnificus and of 20 CFU/g for its contaminated food. Finally, 25 samples in detected 278 practical samples of shrimps, crabs, oysters and artificial contamination ones were positive results using the LAMP method, which accorded with the detection results according to SN/T 1870-2007. The LAMP assay is a sensitive, rapid simple tool for the detection of Vibrio vulnificus.
Genome shuffling was an important technology of strain improvement. The effects of glycin concentration, cell age, lysozyme concentration, the operational time and temperature were conducted to optimize the conditions of protoplast formation and regeneration of Actinobacillus succinogenes CGMCC 2650. The effects of the different osmotic stabilizing agents on regeneration were also examed. The protoplast yield was the highest under these conditions: The bacteria was cultivated in Tryptic Soy Broth (TSB) medium containing 0.6 mg/ml glycin for 5 h, then collected and diluted with SMM until the optical density (660nm) reached 1.0. The diluent was treated by 0.025 mg/mL lysozyme at 37℃ for 45 min and plated on the TSB medium with 0.3 mol/L sucrose as osmotic stabilizer. The regeneration rate was up to 40.9%. This study provides the optimized conditions of protoplast preparation and regeneration for genome shuffling of A.succinogenes.
Acidithiobacillus ferrooxidans (A.f) could synthesize intra-cellular nanometer magnetic particles. With the development of study on their culture conditions, the research of magnetotaxis bacteria will get more benefit from it. By fed-batch fermentation magnetic A.f could be batch cultured in the laboratory. In the shake-flask fermentation, the final volume of liquid was 2 L. After 40 h in the fermentation Fe2+ (5%) was added in the fermentation liquid. The max cell density could reach 2.33×107cells·ml-1. Compared with control experiments, it increased by 60.69%. At the same condition, the completion 9K culture medium was added in the fermentation liquid. The max cell density could reach 2.47×107cells·ml-1. Compared with control experiments, it increased by 70.34%. And atom force microscopy and magnetic force microscopy showed that cells in the fermentation were magnetic.
Interferon alpha are used clinically to treat a group of viral diseases and cancer because of its broad anti-viral，anti-tumor and immune regulation activities. Nevertheless, the clinical usage of interferon-α can still be improved in many ways, such as increasing activity and so on．In order to obtain a new type of molecule with higher potency, human interferon α1b mutant IFNα1b/31K was constructed. ATG was substituted by AAG at 31 of IFN α1b through PCR site-directed mutagenesis in vitro. The amplified fragments were inserted into pET-23b expression vector, and the recombinant plasmid was transformed into Escherichia coli BL21(DE3)．After being cultured in complex auto-inducing media and purification process, IFNα1b/31K was expressed as soluble protein with the yield more than 30% of total bacterial protein. The purity was more than 95%, the anti-viral activity was 1.7 times comparing with IFNα1b, anti-tumor activity was lower than IFNα1b, and the p-value of cytotoxicity & acute toxicity was more than 0.05．
Objective:To construct a prokaryotic plasmid expressing the recombinant proteins of porcine FcγRIII and prepare the polyclonal antibodies of mouse anti porcine FcγRⅢ. Methods The fragment of gene encoding porcine FcγRIII was amplified from pTG19-T-FcγRIII plasmid by PCR. The fragment was then inserted into the prokaryotic expression vector pET-32a to construct the recombinant plasmid pET-FcγRIII, and expressed in E. coli BL21 (DE3). After induction with IPTG, the fusion protein was obtained , and then purified with Urea by washing . A polyclonal antibody , analyzed by Western blotting and ELISA staining , was developed by immunizing mouse with the purified recombinant protein. Results: The recombinant plasmid was constructed successfully ; the fusion protein was successfully expressed and purified. Western blotting and ELISA staining demonstrated that the polyclonal antibody was obtained by immunizing mouse with the purified recombinant protein. The results of ELISA and Western blotting indicated that the prepared polyclonal antibody had high titer and specificity.Conclusion: The preparation of the polyclonal antibody against porcine FcγRIII lays a foundation for the further study on the function of porcine FcγRIII protein.
Ribozymes and DNAzymes have a function of hydrolysis to mRNA molecules. They have become important tools for blocking gene expression and anti-virus. In recent years, Ribozymes and DNAzymes have been used in inclinical studies, and there have got many successful examples. This revew compares their differences and characteristics between Ribozyme and DNAzyme, and summarise the progress in anti-virus, anti-cancer, treatment of genetic diseases, treatment of nervous system diseases in pre-clinical researches. It was supposed to give a prospective remarks.
Retroviral vector was a virus expression vector which was designed by some characteristics of retrovirus. Because it can integrate into the chromosome of host cells and stably express the interesting gene. It was considered as the most efficient means of delivering a interest gene into human cells. Presently, retroviral vector was extensively used for gene therapy, exogenous gene expression and genetically engineering vaccine. The use of retroviral expression vector in genetically engineering vaccine and its applied perspect were mainly discussed,which provide valuable reference for the application of biomedicine.
Adipose derived-mesenchymal stem cells (ad-MSCs) with high proliferative ability have been concerned about due to their steady bionomics, sufficient source and a common cultured condition in vitro. Their potentiality of multi-directional differentiation becomes the important origin of pool of human stem cells. Nowadays, ad-MSCs are successfully induced into endothelial cells, osteoblasts, chondroblasts, fat precursor cells, smooth muscle cells, myocardial cells, nerve-like cells and so on. A description of the basic concepts and currently research advance of the adipose derived-mesenchymal stem cells that can be differentiated to vascular wall cells in vitro was provided.
One of the major keys for successful gene therapy is to developing safe and high effective gene delivery vectors. Multifunctional envelope-type nano-device（MEND） has shown a promising perspective in gene delivery systems, because of its easy cellular uptake, low-toxic, non-immunogenic, non-oncogenic, easy-to-produce and high transfection activity properties. Recent progress of MEND were reviewed.
With the applications of cyclodextrins expanding in the industries related to food, pharmaceuticals, etc, cyclodextrin glycosyltransferase (CGTase), the essential enzyme for the production of cyclodextrins, has become the focus of scientific research nowadays. Especially in recent twenty years, CGTase has been studied extensively, leading to increasing knowledge of its structure and function. Firstly the function and structural characteristics of CGTase were focused on. CGTase is a multifunctional enzyme. It can catalyze three transglycosylation reactions (disproportionation, cyclization, and coupling), and a hydrolysis reaction. The specific CGTase reaction is the cyclization reaction, which can result in the formation of a cyclodextrin. CGTase is a member of the α-amylase family of glycosyl hydrolases. α-Amylases generally consist of three structural domains, A, B, and C, while CGTases show a similar domain organization with two additional domains, D and E. In addition, substrate binding of CGTase and mechanisms of transglycosylation and cyclization reactions catalyzed by CGTase were discussed in detail. Domain B contributes to substrate binding by providing several amino acid side chains alongside a substrate binding groove on the surface of the CGTase protein. Maltose binding site assists in guiding the linear starch chains into the substrate binding groove containing at least nine subsites. After the glycosidic bond cleavage reaction has taken place between subsites ＋1 and －1, the non-reducing end of the oligosaccharide at the groove is transferred to the reducing end of the same oligosaccharide chain, leading to cyclic products.
