Czech has prioritized biotechnology and bio-industry as the part of its strategic development to high-tech, low energy and raw material consumption, and efficient industry structure. The collaborative multi-ministry framework for biotechnology promotion and administration has been established in Czech to improve supporting environment, which accelerated the formation of several bio-industry clusters in Prague, Brno and South Moravia. Czech also actively involves into the Framework Programme of European Union and exploits international cooperation to serve the development of biotechnology. Favorable geographical location and skilled workforce have attracted more and more foreign investment which contributes to Czech's aim of becoming the most promising R&D outsourcing center in Central Europe.
SNX9 is a novel SNX family protein，which plays important roles in protein sorting and transporting. The prokaryotic expression vector pET-SNX9 construct was transformed into E.coli BL21(DE3) cell and induced by IPTG. The induced fusion protein (SNX9) was expressed successfully in soluble form. SDS-PAGE indicated that the molecular weight of SNX9 was about 70 kD, and the recombinant protein was confirmed by western blotting. After affinity and gel filtration chromatography purification, the purity of SNX9 could reach over 95%. Gel filtration elution volume suggested that SNX9 existed as a dimer in solution. Homogeneity was further confirmed by Native-PAGE and Dynamic Light Scattering (DLS) experiments. In the thermo stability experiment, SNX9 was stable below 15 ℃ relatively. All these information paved the way for future structural and functional study of SNX9.
Object: to obtain large quantities of β2-adrenergic receptor (β2AR) and detect β2-agonist.Method:Mouseβ2AR cDNA gene was amplified by PCR, then linked with EGFP and inserted into yeast expression vector pPICZαA. The reconstructed plasmid , PICZαDNAsGFP was transformed into Pichia pastoris KM71. After induction, a recombinant protein about 75kD in size was determined by western blot. 125ICYP was used to determine the activity of recombinantβ2AR. These results showed that the expressed protein is active.
Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60-62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. We reported here the cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/liter. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66 ?g/ml. In summary, we have developed an effective expression and purification system for recombinant CTXs in P. pastoris. This system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.
Objective Construct eukaryotic expression plasmid pVAXⅠ- P1-2A containing capsid protein precursor P1-2A gene of Asia-ⅠFMDV ,and evaluate cellular and humoral immunity level after immunization mice with pVAXⅠ- P1-2A. Methods We amplied the coding region of FMDV P1-2A by RT-PCR and inserted it to Vector pMD18-T. Eukaryotic expression plasmid pVAXⅠ- P1-2A was constructed by connecting pMD18-T-P1-2A and pVAXⅠdigested by double restriction endonuclease EcoRⅤand XbaⅠ. We transfected the right recombinant plasmid confirmed by endonuclease digestion into the Hela cell ,and perform IFA to assure the right expresstion. After this, mice serological specific antibody test ,T lymphocyte proliferation assay and IFN-γ ELISPOT test were carried out. Results The result of restriction endonuclease digestion was accordance with the anticipated objective strap size . IFA showed that there are flavovirens fluorescence in cells transfected with pVAXⅠ-P1-2A ,and confirmed the expression of P1-2A gene in the HeLa cell; The result of mice immunization test indicated that there are stronger cellular and humoral immunity response , and more T lymphocyte and the cells that produce IFN-γin immunized mice than the control groups (P<0.05). The indirect ELISA test manifested that the antibody level was higher remarkably than the control groups (P<0.05) in the 14th day after immunity. Conclusion we have successfully constructed eukaryotic expression plasmid pVAXⅠ- P1-2A ,and confirmed that specific humoral immunity and cellullar immunologic response can be induced in the group injected with recombinant plasmids with mice immunization test.
Objective To construct a DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the anti-HBV siRNA genes, and to express this DNA vaccine in HepG2 cells. Methods The HBV multi-epitope antigen gene was designed and synthesized before it was fused with enhanced green fluorescent protein(EGFP) gene, and cloned into the multi-clone site(MCS) of the eukaryotic expression vector pVAX1. The expressinig units of hIL-12 and siRNA were cloned into the BspH I and Mlu I site of pVAX1 respectively. Then the recombinant plasmid pVAX1-siHBV-HB-EGFP-hIL12 was transiently transfected HepG2 cells. The expression of HBV compound multi-epitope gene was observed through EGFP report gene. The expression of hIL-12 was analyzed by ELISA and the effects of anti-HBV siRNA was confirmed with rtPCR . Results The analysis of enzyme digestion and sequencing both demonstrated that the trible-expressing HBV DNA vaccine has been constructed successfully. The green fluorescent image was detected in the transfected cells which could confirm the expression of the multi-epitope antigen gene. The amount of hIL-12 secretion was 1289pg/ml in supernatant at 48h after transfection and 1712pg/ml at 72h after transfection. The mRNA amount of HBV S gene, which was the siRNA target, had been obviously knockdown. Conclusion The DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the siRNA genes was constructed and transiently expressed in HepG2 cells, and siRNA had shown us a good anti-HBV effect. It laid a foundation of further study on anti-HBV effect of the new DNA vaccine.
Objective To construct the lentiviral-vector encoding human interleukin-10 protein(LV-hIL-10) and to observe the effect of LV-hIL-10 on controlling neuropathic pain via intrathecal administration in CCI rats. Methods hIL-10 gene fragment was isolated and amplified from pCYIL-10 plasmid by PCR, and was cloned into pWPXL. The recombinant plasmid pWPXL-IL-10、envelope plasmid pMD2.G and packaging plasmid psPAX2 were cotransfected into 293T cells, LV-hIL-10 is prepared by concentrating the collected supernatant .At the same time, the empty plasmid pWPXL-GFP、pMD2.G and psPAX2 were cotransfected into 293T cells, LV-GFP is prepared for contrast.135 sheer breed pathogen-free adult male Sprague-Dawley rats divided into 9 arrays at random: CCI models 4 arrays (C0、C1、C2、C3), sham operatived rats 4 arrays (S0、S1、S2、S3) and a normal contrast array (N), each respectively intrathecal injection LV-hIL-10 (C1、S1)、LV-GFP (C2、 S2)、isotonic Nachloride (C3、S3) and control (no implanted catheters and no administration, C0、S0), the pain threshold of each array and the expression of mRNA and protein of IL-10 in spinal cord、pallium and hippocampus on different time were observed after intrathecal administration LV-hIL-10 in successful CCI model rats . Results The hIL-10 gene fragment was obtained from pCYIL-10 plasmid, pWPXL- hIL-10 was recombinated successfully. the cloned gene segment was validated by DNA sequencing .High titer（2x1010）and highly purified LV-hIL-10 particles were obtained by three plasmids were cotransfected into 293T cells. The mechanical allodynia and thermal hyperalgesia were alleviated via Intrathecal injection LV-hIL-10 in CCI rats. The overexpression of IL-10 were detected in spinal cord、pallium and hippocampus , especially in the spinal cord .Conclusions The mechanical allodynia and thermal hyperalgesia can be relieved by Intrathecal injection LV-hIL-10 in CCI rats
Granule-bound starch synthase and soluble starch synthase are the key enzymes in starch synthesis. Their activity would decide starch quality via affecting the chain length, the ratio of amylose content to amylopectin and the structure of starch. We performed homology analysis on gbss, ssII and ssIII with software Blast and DNAStar followed by using 261bp fragments from gbss（1～261），244bp fragments from ssⅢ（2164～2407）and 281 bp fragments from ssⅡ（161～441）to make the fusion gene gbs3s2 with overlap PCR. Then we constructed the promoter CaMV 35S driven, containing 'forward gbs3s2 fusion fragments-reverse pdk intron-reverse gbs3s2 fusion fragments', plant siRNA expression vector, which lay the foundation for breeding a low gelatinization temperature potato.
The cloning of promoter is important for studying the genetic engineering and the regulation of gene expression in plants. In this paper, we cloned two promoters Os772 and Os359, which are predicted to be highly expressed in the endosperm of rice from the EST database. After construction of the Os772::GUS and Os359::GUS expression vectors, they were transformed into rice. X-Gluc staining of transgenic plants showed that Os772 and Os359 can promote GUS gene expression in matured endosperm but not in root, stem, leaf and flower. This result indicates Os772 and Os359 are two rice endosperm-specific promoters.
The single preimplantation embryo differential display reverse transcription polymerase chain reaction(SPEDDRT-PCR) was used to identify differential genes in 2-cell,4-cell,8-16-cell porcine parthenogenetic activated embryos.A total of 8 ESTs(expressed sequences tags) were found using SPEDDRT-PCR and reverse northern dot blot, one of which in 2-cell,four in 4-cell,and the other three in 8-16-cell embryo.All 8 ESTs were compared with nucleotide sequences deposited in the nr databasse and the dbEST database of Genbank using BLAST.DD3,DD4,DD5,DD6,DD7 and DD8 ESTs had their highly similar nucleotide sequences with ESTs existing in nucleotide databases but with unknown function.DD1 and DD2 ESTs had no significant similarity with exisiting genes or ESTs and were regarded as new ESTs. The two new ESTs were submitted to GenBank(accession numbers: EU545158, EU545159).This lays a foundation for further study on the mechanism of porcine preimplantation embtyo development.
Phosphoethanolamine N-Methyltransferase (PEAMT) is a key enzyme that catalyzes the synthesis of phosphocholine, which is an important precursor of phosphatidylcholine and glycine betaine. In this study, a 1249bp 5'-flanking region of phosphoethanolamine N-methyltransferase gene was isolated by anchored PCR, based on the cDNA sequence of PEAMT from halophyte Salicornia europaea. The transcription start site was identified as A and localized at 301bp upstream of the ATG according to the results of RLM-RACE. In SePEAMT promoter region, many potential cis-acting elements were predicted by PlantCARE and PLACE programs. Aside from the basal transcriptional elements TATA-box and CAAT-box, some stress-responsive motifs such as ABRE, HSE and LTR were found. In addition, some pollen-specific activation-related elements were also present in this region. Binary expression vector was constructed by fusing SePEAMT promoter with GUS gene and designated as pPro. The pPro was transferred into tobacco by Agrobacterium-medicated transformation and transient GUS expression analysis indicated that SePEAMT promoter could drive strong GUS expression.
Nicotiana tabacum is an important and classical model plant which can respond to the change of environmental conditions by accumulating osmoprotectants, such as glycerol and proline which contribute to the re-establishment of homeostasis when exposed to various adverse environmental stresses, such as drought, salinity, high and low temperatures. In this paper, the optimization of ultrasonic extraction (UE) conditions of glycerol-3-phosphate dehydrogenase (GPDH) of tobacco leaf have been built by orthogonal test. It showed that optimum of the powers, treatment times, slot times and leaf-to-solvent ratios of UE was 75w, 2h, 2s, and 1:12 g/mL, respectively. Under these conditions, the activity of GPDH has been tested as 0.3937U/mg protein, which was higher than other extraction methods such as liquid nitrogen and grinding on ice bath. To our knowledge, it is the first description of determination of content of GPDH with ultrasonic in tobacco. It could provide basis for the further research in the relation of content of glycerol and osmotic pressure in tobacco.
Previous results indicated that the abnormal development of haploid goldfish embryos is due to the expression obstruction of some regulation proteins.In search of the mechanism of abnormal development in goldfish gynogenetic haploid embryos, differential display proteomics techniques was employed to study protein changes among three different develomental stages time of haploid embryos called HE-1, HE-2 and HE-3. Proteins were sepatated by two dimensional electrophoresis, then image analysis was carried out using PDQuest image analysis software. 15 proteins were identified by MALDI-TOF/TOF. These results may be helpful in further studing developmental mechanisms of goldfish gynogenetic haploid embryos.
Three purple non-sulfur bacterial strains were enriched and isolated from the fish pond water and sediments in Hangzhou, they were named HZ-3,HZ-4,HZ-5 respectively. Among the three purple nonsulfer photosynthetic bacterias , the strain HZ-5 can purify the aquaculture water from fish pond and shrimp pond more efficiently . After 5 days treatment by the strain HZ-5, the COD of fish pond water was decreased by 24.87%; the COD of shrimp pond water was decreased by 36.99%; the Nitrite-Nitrogen of fish pond water was decomposed by 97.79%. The strain HZ-5 was identified as Rhodopseudomonas palustris according to its morphological characteristics, absorption spectrum scanning of live cell and physiological and biochemical properties, and the sequence analysis of 16S rDNA.
SOE PCR, which employs chimeric primers to generate PCR products with complementary ends in amplifications. This method can assemble DNA fragments without the treatment of restriction endonucleases and T4 DNA ligase. Five site-directed mutagenesis of chitinase (chi58) were obtained successfully with SOE-PCR. The mutation of codon-modifications of chi58 was constructed. The results showed that SOE-PCR have the characters of simplification and high-efficiency. Other mutations were not caused during the progress of amplification. The mutation of chi58A can be used at following research.
The technical requirements of in-situ microscope mounted in biochemical reactor were proposed based on biochemical reactor application. A novel in-situ microscope was designed and manufactured with dark field principle after all principles of in-situ microscopes used currently had been reviewed in detail, the designing principle and structure of a new in-situ microscope was then described. In addition, it was applied to monitor growth of baker yeast and mammalian cell HEK293, respectively. It indecated the cell amount obtained by in-situ microscope was a good agreement with dry cell weight and that determined using haemocytometer，suggested that the designed in-situ microscope could satisfy the requirements of high performance used in bioreactor .
In recent years, the emergence of drug-resistant RT gene mutations in HIV-1 strains has become the major reason for AIDS treatment failure. The detection of drug-resistant mutations is important for the treatment choice. In this work, a novel assay for the detection of HIV-1 RT drug-resistant mutations was developed. Drug-resistant and wild strains of HIV-1 B subtype were investigated based on Two-Step MS-PCR. Magnetic beads associated DNA isolation technology and ELISA were employed in this assay to detect point mutations including Y181C and T215F. As a result, this novel assay gave a good P/N ratio and as less as about 5% of mutant type template could be detected, indicating that this assay is highly sensitive and specific. The assay can be completed in relatively less time consumption and applied in high-throughput screening. We suggest that it will be used in clinic assay for the detection of HIV-1 drug-resistant mutations as well as other point mutations.
Because the biology characteristic of HIV is extremely different from that of other microorganisms, HIV vaccine research has being faced with unprecedented difficulties and challenges. In the last 20 years, HIV vaccine research has been carried out mainly with two strategies, i.e., neutralizing antibody based and cellular immunization based; however, substantive breakthroughs haven't been achieved until now. Inducing effective neutralizing antibody is always an important strategy in traditional vaccine research, but this strategy is much less efficient in HIV vaccine research because of high variation and much subtypes of HIV. In recent years, the discovery of broadly neutralizing monoclonal antibodies and elucidation of their corresponding antigen epitopes have brought new hopes for the development of neutralizing antibody based HIV vaccine. Reviewing these advancements will be helpful for reconsidering HIV vaccine development with a better strategy.
From genomics to proteomics, and then to mini RNA, all without exception showed that proteins are the substance which directly regulate life. Comparing with genomics, study of proteins is more difficult for its great variety, complicated modification and complicated construction. Recently, some new techniques for protein assay and research have provided us, e.g. Fluorescence labeling, SELDI, SPR and optical protein chip, of these, SELDI SPR and optical protein chip are label-free.
Embryonic stem (ES) cells derived from the inner cell mass(ICM) of the blastocyst, which can proliferate indefinitely in vitro(self-renewal) and can differentite into cells of all three germ layers(pluripotency). These unique properties make them exceptionally valuable for cell replacement therapies drug discovery and regenerative medicine. However, as for organ transplants, tissue rejection remains a significant concern for ES cell transplantation. Another concern is the use of human embryos. One possible means to avoid these issues is by reprogramming the nuclei of differentiated cells to ES cell-like, pluripotent cells. Several extrinsic signals such as LIF, BMP and Wnt can support the self-renewal and pluripotency of embryonic stem (ES) cells through regulating the "pluripotent genes." A unique homeobox transcription factor, Nanog, is one of the key downstream effectors of these signals. Elevated level of Nanog can maintain the mouse ES cell self-renewal independent of LIF and enable human ES cell growth without feeder cells. In addition to the external signal pathways, intrinsic transcription factors such as FoxD3, P53,Tcf3 and Oct4 are also involved in regulating the expression of Nanog. Functionally, Nanog works together with other key pluripotent factors such as Oct4 and Sox2 to control a set of target genes that have important functions in maintenancing ES cell pluripotency. These key factors form a regulatory network to support or limit each other's expression level, which maintains the properties of ES cells.
piRNA(Piwi-interacting RNA) is a novel class of small single strand RNA that were recently isolated from testes of the mammals, associate with PIWI proteins, and are organized into distinct genomic clusters. These RNAs are are typically ~30 nt long, strikingly different from microRNAs in their length, expression pattern, and genomic organization. piRNA has a role in RNA silencing via the formation of an RNA-induced silencing complex (RISC) with Piwi proteins, these piRNA complexes (piRCs) have been linked to transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, particularly those in spermatogenesis. In this paper, recent researches and progresses of piRNAs are reviewed.
N-3 polyunsaturated fatty acids (PUFAs) is an important component of the fatty acid. It is necessary to maintain the appropriate proportion of n-3 PUFAs physiological. However most animals have to obtain n-3 PUFAs from diet,because they can not synthesize them by themselves.Fat-1 gene, translating into ω-3 PUFA desaturase, can convert PUFAs from n-6 to n3 form. The main functions of n-3 PUFAs, and the applications and potential of the fat-1 in n-3 PUFAs research are reviewed in this paper.
The production technology for the first generation of biofuels has been made great achievements and some countries, such as USA, EU countries, and Brazil, have formed successful biofuel industrial chains. Cellulose ethanol, a representative of the second generation of biofuels and a kind of more promising alternative fuels, has not yet got enough key technological breakthroughs and its large scale commercial production still needs rather some time to realize. Now it is in the early stage of transition to the second generation of biofuels. Taking the development of second generation of biofuels as their national policies, many countries have developed long-term development plans and objectives, and provided a good policy environment and strong support for it. Thus institutes and enterprises are engaging and successing in addressing key issues relating to the development of biofuels. At the mean time, some important issues relating to the sustainable development of biofuels have attracted people's attention.
