The DNA sequences, which originated from gene COL1A1, optimized for E.coli codon usage and contained cell adhesion domains of GER units, was amplified by RT-PCR technology from human tissue. The recombinant expression vector pET28a(+)-COL encoded the recombinant human-derived collagen (RHDC) was constructed and transformed host E.coli strain BL21(DE3)pLysS. The RHDC production was induced by the addition of IPTG and approximately 40% of the total strain proteins. RHDC could form a triple-helical structure suggested by the SDS-PAGE and Western Blot analysis. Cell adhesion assay indicated that RHDC had high cell adhesive character, which makes it suitable for use as biomaterials. analysis. Cell adhesion assay indicated that RHDC had high cell adhesive character, which makes it suitable for use as biomaterials.
Objecive: To study the effect of antizyme1 on the cell proliferation and cell apoptosis induced by As2O3 in human leukemia K562 cells. Methods: The base of frameshift was deleted by site-directed mutagenesis assay and the mutation antizyme1 gene was then cloned into vector of pEGFP-N1. Then the recombination DNA was transfected into K562 cells and stable transferctants were isolated after selection with G418. Cell proliferation was checked by MTT assay, and the cell cycle was detected by FCM. The level of cyclin D1 mRNA was measured by RT-PCR assay. After treated with various concentration of As2O3, apoptosis rate of K562pAZ1m was detected by FCM.The level of survivin mRNA was measured by RT-PCR assay also. Results: The mutation antizyme1 gene was successful cloned into pEGFP-N1. When transfected into K562 cells, the cell proliferation was inhibited and cell cycle was arrested at G0/G1 stage according with down regulation of cyclin D1 expression. After treated with As2O3, apoptosis rate of K562pAZ1m rising is coincident with the decrease of survivin expression. Conclussion: antizyme1 can inhibite proliferation in K562 cells and arrest cell cycle by down regulating cyclin D1 expression. And antizymes also enhance the apoptosis rate induced by As2O3 partly via decreasing survivin expression
Objective To inhibit the expression of Vascular Endothelial Growth Factor (VEGF) by RNA interference in HT29 cells. Methods Using the service of E-RNAi, we designed and constructed two kinds of shRNA expression vectors which were aimed at the VEGF gene, then transfected them into colon cancer HT29 cells by lipofectamineTM 2000. The level of VEGF mRNA was determined by RT-PCR and Northern blotting. The protein expression of VEGF was examined by Immunofluoresence staining and Western blotting. Results The two kinds of VEGF specific shRNA expression were found to efficiently inhibit the expression of VEGF in HT29 cells, the inhibition rate showed by RT-PCR, Northern blotting, Immunofluoresence staining and Western vectors blotting were 42%, 88%,73% and 82%, respectively. Conclusion The expression of VEGF could be inhibited by RNA interference in HT29 cells.
The thalassemia is a heterogeneous group of inherited disorders. The main pathological mechanism which causes thalassemia is the imbalance in the synthesis of αandβglobin chains, due to mutations in the globin loci. The C→T transition at nucleotide 654 of intron 2 inβglobin gene is one of the most frequent βthalassemia mutation(β-IVSⅡ-654) in GuangDong China. The expression of human β- globin gene cluster is definitely regulated with strict developmental stage and tissue lineage specificities as well as the delicated control of the biosynthetic equilibrium. It was all know that the expression of human β- globin gene cluster concerned with the β locals control region(βLCR) in human being. Purpose：We wanted to identify that what contribution wouldβLCR give toβ thalassemia gene (β-IVSⅡ-654) in the transgenic mouse. Methods: To prepare two genes, one was the merelyβthalassemia gene, the other was a modified βthalassemia gene contains βLCR. To made up two lines of transgenic mouse by use these two genes. Then, detect the expression of two genes in the two transgenic mouse lines by fluorescent quantitation RT-PCR. To compared the two expressions of two genes by statistical analysis. Result: Two transgenic mouse were established. The statistical analysis shows that the expression of the merelyβthalassemia gene was more lower than the modified βthalassemia gene contains βLCR in the transgenic mouse. Conclusion: We therefore conclude that a combination ofβLCR is necessary to highly expressionsβthalassemia gene in the transgenic mouse.
Objective：Evaluate the immunogenicity of an recombinant fowl-pox virus rFPV-3C-P1-2A-IL-18. Methods：Mice were inoculated with the recombinant FPV(rFPV) rFPV-3C-P1-2A-IL-18 、rFPV-P1-2A-3C vaccine, 282E4 FPV and PBS was set as the control groups.The FMDV specific antibody、number of T subtype 、cytotoxicity of special CTL were detected. Results：Compared with other control groups, the mice inoculated with rFPV-3C- P1-2A-IL-18、rFPV-P1-2A-3C vaccines were induced higher anti-FMDV antibody titer, alarger number of T subtype, and higher cytotoxicity of special CTL. Compared with rFPV-P1-2A-3C vaccine，rFPV-3C-P1-2A-IL-18 can induce larger number of T subtype and higher cytotoxicity of special CTL. Conclusion：It is evident that the recombinant fowl-pox virus rFPV-3C- P1-2A-IL-18 show excellent immunogenicity in mice , thus provide valuable support for further development of FMDV genetic engineering vaccines.
The internal ribosome entry site (IRES) element from FMDV was amplified by RT-PCR, and was cloned into pcDNA3.1(+) vector, resulting in a bicistronic vector. To determine whether the bicistronic mRNAs from the vector can be translated, the enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, then BHK-21 cells was transfected by the recombinant plasmid. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results showed that the IRES element from FMDV has the role of initiating CAP-independent translation, and laid foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the bicistronic vector.
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is the only antioxidant enzyme which could reduce membrane-bound hydroperoxy lipids. In this report, Circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) were employed to investigate the effect of temperature on enzymatic activity and the conformational change of a recombinant Oryza sativa PHGPx (OsPHGPx). PHGPx activity was increased slowly from 10 to 27.5°C and reached the maximum at 27.5°C, then descended rapidly from 27.5°C to 45°C. At temperatures exceeding 45°C, the enzyme activity was completely lost. A classic two phase thermal unfolding of OsPHGPx was found with temperature increasing. First, from 20 to 40°C, Far UV CD, intrinsic fluorescence and DSC spectra did not change obviously with increased temperature, indicating the whole structure nearly maintained integrity. Second, from 40 to 55°C, an abrupt secondary level change was found in the far UV CD spectra, indicating the secondary structure started unfolding; the change of intrinsic fluorescence spectra revealed that the tertiary structures began partial unfolding and the region near active site was buried into the unfolding structure. The DSC indicated OsPHGPx exhibited thermal transitions at around 42 °C. Third, at over 55°C, the whole conformation of the protein kept unchanged.
To study the function of the starch synthase III (SSIII) gene in synthesis of wheat starch. SSIII partial cDNA sequences (GenBank No. EF466009) from common wheat (Triticum aestivum, Yujiao 2 cultuvar) grains was cloned with RT-PCR. The result demonstrated that the cloned SSIII gene sequences were identified to97% to the reported SSIII genes in GenBank previously. Its antisense expression vector was constructed with pWM101plastid, and RNAi vector was also constructed with pFGC5941 plastid. These constructed vectors would further be transferred into wheat grains through gene engineering way to study the function of SSIII in starch biosynthesis of wheat plants.plants.
Fusion PCR, which employs chimeric primers to generate PCR products with complementary ends in its amplifications, is a rapid and flexible method in joining different DNA fragments. This method can assemble DNA fragments without the treatment of restriction endonucleases and T4 DNA ligase. It offered a shortcut for the construction of homologous recombinant fragments. Through assembling three recombinant fragments successfully, fusion PCR procedure was improved and manipulation essentials of fusion PCR were described in detail. Conclusions indicated that the improved procedure can assemble three or four fragments simultaneously, and the length of each fusion product is above 4.5 kb. The result recombinants were proposed to be use in further experiments, which structures had been confirmed by sequencing.
As a real-time, fast and label-free instrument for biomolecular interactions, surface plasmon resonance biosensor has been widely used in biomedical analysis and research. Carboxymethylated dextran modified CM5 sensor chip is the core of the Biacore instrument. CM5 sensor chips are now mostly purchased from Pharmacia Biosensor AB (Uppsala Sweden), which are expensive and once the covalent immobilized receptor molecules have lost bioactivity, it cannot be reused.An easy, cost-effective method for regeneration, characterization and preparation of carboxymethylated dextran modified (CMD) sensor chips for SPR biosensor was described. The prepared CMD surface was well characterized by cyclic voltammetry and atomic force microscopy and successfully used for the detection of prostate-specific antigen in solution and the measurement of the constants of affinity and kinetics of the binding of PSA with PSA antibody.
Receptors play a crucial role in determining the host specificity and tissue tropism of virus. Foot-and-mouth disease virus（FMDV）has been showed to use four integrins, αvβ1, αvβ3, αvβ6 and αvβ8 as receptors to initiate infection and αvβ6 functions as the major receptor. In this study, we first cloned and sequenced the cDNA encoding bactrian camel integrin β6 from the lung tissue. The 2367bp cDNA of bactrian camel integrin β6 encodes a polypeptide of 788 amino acids consisting of a 26-residue putative signal peptide, a 681-residue ectodomain with 8 potential N-linked glycosylation sites and 58 cysteine residues, a 29-residue transmembrane domain, and a 52-residue cytoplasmic domain with a NPLY motif and 1 potential N-linked glycosylation site. The nucleotide sequence similarity of integrin β6 between bactrian camel and cattle, pig, sheep, human, mouse, Norway rat is 91.1%、91.8%、90.6%、90.5%、83.7%、84.1%, and the amino acid sequence similarity is 94.3%、93.4%、93.4%、93.7%、88.7%、88.6%, respectively. The bactrian camel β6 gene exhibited the higher sequence homology with the β6 gene of cattle, pig and sheep, indicating their close genetic relationships. It is possible that host tropism of FMDV may related to divergence in β6 receptors among different species.
he fermentation conditions of lipase production by Geotrichum candidum Y162 were optimized. Initially, the most suitable carbon olive oil , nitrogen source soybean flour and NH4Cl , salt BaCl2 and MgCl2 were selected according to single factorial experiments respectively. Based on the result, screening methodology Plackett-Burman design was used to evaluate the effects of twelve factors related to lipase production and three statistically significant factors olive oil , BaCl2 and NH4Cl were selected. The path of steepest ascent was used to approach the optimal region of lipase production subsequently. Then, the optimal combined concentration for maximum enzyme activity were further optimized by response surface methodology and determined as follows : olive oil 2.35％, BaCl2 0.36％，and NH4Cl 4.69％.The optimization of culture conditions of G. candidum Y162 led to a 2.25-fold increase in lipase production relative to initial result 14.16 U/mL, which indicate that single factor in combination with response surface methodology is an effective method for optimization of lipase production conditions by G. candidum Y162.
Recent studies have identified the existence of adult stem cells in nearly all tissues, and enormous reports showed that adult stem cells have a greater differentiation potential than previously expected. Adult stem cells have been suggested to contribute to the repair and regeneration of injured lungs and ameliorate pulmonary fibrosis evidently in mice by homing to the injured tissue immediately after injury. Stem cells likely play key roles in the repair of diverse lung injuries. However, due to very low rates of cellular proliferation in vivo in the normal steady state, cellular and architectural complexity of the respiratory tract, and the lack of an intensive research effort, lung stem cells remain poorly understood compared to those in other major organ systems. The goal of this review is to provide an overview of the stem cells within the lung and the adult stem cells that are believed to take place in the lung regeneration, and describe potential clinical applications with respect to human pulmonary disease.
Site-saturation mutagenesis is a newly-developed technology in protein engineering. By manipulating the encoding genes, it can rapidly obtain the mutants of desired proteins whose target residues are substituted by 19 other common amino acids. Site-saturation mutagenesis could serve not only as a powerful tool in protein engineering, but also as an important method in exploring the structure-function relationship of proteins. In this review, we summarize several techniques to achieve site-saturation mutagenesis and introduce their application status in the protein engineering. The problem and promising future of its application were also discussed.
EST (expressed sequence tag，EST) is approximately 150～500 bp sequence of the gene expression exogenous sequence fragment，and randomly selected from large-scale cDNA sequencing of the genome of the cells or expressed sequence tags. EST represents a certain period of a biological cell or a gene expression. This paper reviews the technical principles EST, early mammalian embryo research and the theoretical foundation of early embryo research EST in the application, EST to discuss the study and analysis of the development trends.
MicroRNAs (miRNAs) are a class of small RNA molecules which play a pivotal role in the regulation of genes involved in diverse processes. Recently, many viral-encoded miRNAs have been discovered, which suggests that viruses also use this fundamental mode of gene regulation. Although the functions of most viral- encoded miRNAs are unknown, some of them are involved in evading CTL，mediating latent infection,apoptosis suppression,etc.Uncovering the role of viral miRNAs in the pathopoiesis offers an immense opportunity not only to develope effective antiviral therapies，but also to identifying novel molecular targets for developing antiviral reagents.Therefore，this review discusses recent progress on vmiRNAs.
Data mining of gene expressions using high-throughput methodologies has become very popular in recent years．Data generated through techniques such as microarray hybridization allows the simultaneous quantification of tens of thousands of gene transcripts．The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) is the largest fully public repository for high-throughput molecular abundance data．The database has a flexible and open framework that allows the submission，storage and retrieval of many data types．These data include microarray-based experiments measuring the abundance of mRNA，genomic DNA and protein molecules，as well as non-array-based technologies such as serial analysis of gene expression (SAGE) and mass spectrometry proteomic technology． GEO currently stores approximately a billion individual gene expression measurements，derived from over 100 organisms，addressing a wide range of biological issues． Features are provided to examine data from both experiment and gene-centric perspectives using user-friendly Web-based interfaces which are accessible to those without computational or microarray-related analytical expertise．Here，we review the recent database developments and its future directions，while introduce some tools that allow effective exploration，query and visualization of millions of gene expression profiles through GEO enabled data-mining procedures．The GEO database is publicly accessible through the World Wide Web at http://www.ncbi.nlm.nih.gov/geo．
With the development of functional genomics, expressed sequence tags (ESTs) has become an important resource of PCR-based molecular markers for plants. This paper briefly reviews advancement in functional molecular markers for plants in the functional genomics era, including EST-SSR, CAPS, SNP, SRAP and TRAP. Compared with conventional markers, the EST-derived markers are a novel type of molecular tool with remarkable advantages, such as being easily to develop, more informative, and highly transferable, especially for preferential targeting ORFs. Applications of the new functional molecular markers in plants genetics and breeding are summarized in this paper, such as construction of genetic linkage maps, important trait gene tagging location, comparative mapping, genetic diversity, cultivars identification and marker-assisted selection breeding.
Plant transgenic system secures a safe, economical and reliable supply of recombinant proteins. Plant oilbody expression system simplifies the downstream purification steps and reduces capital investment based on the nature of oleosin including high expression and easy extraction. The structures and characteristics of seed oil body and oleosin were reviewed. And the research progress and industry of the seed oil body expression system, as a new bioreactor, to produce valuable recombinant proteins were discussed. The benefits and questions of the oil body expression system were also set forth. Our laboratory is developing new medicine haFGF based on the oilbody system,analyzing its biological activity. As a new resource for medicine protein, oilbody expression system will be perfected and applied broadly.
Heparinases, a kind of polysaccharide lyase, can degrade heparin and heparin sulfate to low molecular weight polysaccharides. It has been noted that many bacteria have heparinases although only few of them have been purified and characterized. Heparinases I, II and III from Flavobacterium heparinum have been extensively studied for many years and been commercialized recently. Heparinases have some important applications in the industry and clinic as well as in the determination of heparin structure, which is a very important anticoagulant drug used world-widely. In this paper, we will review recent progresses in isolation of heparinase-producing bacteria, purification of the heparinases, the study of biochemistry and regulation of heparinase production as well as the recombinant expression of these enzymes. Important applications of heparinases and their potential applications in the future will also be highlighted.
