Biodegradable shape memory polymers integrity both excellent biodegradable and shape memory properties, as medical device implanting into human body which can be degraded after finished its function under the action of body fluids and have non-toxicity to human, that avoid secondary surgery on patient suffering and avoid security risks caused by the presence of long-term implantable medical devices. As medical device implants should have good cytocompatibility.Poly D,L-lactic acid based shapememory polymer (PDLLA-PUU SMPs) with human umbilical vein endothelial cells were cultured in vitro. Adopting the laying of the membrane method and extracting method, the ability of HUVEC proliferation was estimated by MTT assay and cell morphology was observed using HE staining method. Compared with PDLLA controls, all results showed that PDLLA-PUU SMPs had the better cytocompatibility.
To explore the activity of recombinant Methionine γ-Lyase (MGL) , the gene of MGL was prepared by reverse PCR with RNA extracted from Trichomonas vaginalis. Expression vector pET-15b-mgl1 was constructed and sequenced. Characters of the recombinant MGL purified from transformed E. coli BL21 induced by IPTG were explored. The Km of the recombinant MGL for L-Methionine, D L-Homocysteine, L-Cystein as substrate were 0.318, 2.14, 1.62 mmol/L respectively and the specific activity were 20.17, 318.69, 56.96μmol/min/mg protein-respectively. The optimal pH for recombinant MGL was pH5.0~6.0 and the recombinant MGL was stable up to 50℃. The results indicate that recombinant MGL has bioactivity and good thermal stability suggesting potential efficacy in future clinical trials.
Acidic fibroblast growth factor (aFGF) is a potent neurotrophic factor. It can stimulate the reparation and regeneration of central and peripheral nerves after various injuries. Recently, an approach to deliver therapeutic peptides to the brain is the application of fusion proteins linked to so-called trans-activator transcription (TAT) protein, which can carry the therapeutic protein to permeate blood-brain barrier(BBB) and cell membranes. In this study, aFGF was linked to TAT protein by genetic engineering, and the soluble TAT-aFGF has been expressed successfully in E.Coli. DNA coded fusion proteins Tat-aFGF14-154 and Tat-aFGF27-154 were constructed and cloned into vector pET-3c and the fusion proteins were expressed in E.coli BL21 (DE3). The fusion proteins Tat-aFGF14-154 and Tat-aFGF27-154 were puritified using the combination of CM-Sepharose FF, Heparin affinity chromatography and Sephadex G-25 and the purity were higher than 95%. The fusion proteins were confirmed as Tat-aFGF14-154 and Tat-aFGF27-154 by Western bolt and MALDI-TOF. The mitogenic activity assayed by MTT on Balb/c 3T3 cell showed that Tat-aFGF14-154 and aFGF14-154 had mitogenic activity to Balb/c 3T3 and the best concentration were 1280 ng/ml and 160 ng/ml,respectively.On the other hand, Tat-aFGF27-154 and aFGF27-154 had little mitogenic activity. Tat-aFGF14-154 and Tat-aFGF27-154 could transduce the membrane of PC12, hippocampal neurons, Balb/c 3T3 and HaCaT cells and were mainly located mainly in the cytoplasm detected by immunofluorescence. Four recombinant proteins could efficiently protect hippocampal neurons from toxicity induced by Aβ25-35.
Phage display antibody library is an important technology for generating therapeutic antibody.It was reported that the construction and characterization of a large human antibody phage display library. Total RNA was extracted from peripheral blood lymphocytes of 20 healthy donors. VH and VL gene were amplified by RT-PCR. The ScFv (single chain variable fragment) gene were assembled by overlap PCR, and cloned into a phagemid vector by electroporation of E.coli TG1. A large naive human ScFv phage antibody library containing 1.3×109 antibody members was constructed by repetitive electroporation about 300 times. The quality of the library was examined by sequence analysis and selection against 5 different protein antigens. Sequence analysis of the randomly picked 40 clones showed this library had good diversity. Panning of the 5 different antigens all resulted in successful isolation of antigen specific ScFv antibodies. The results indicate that a large nave human ScFv phage antibody library with good diversity was constructed successfully.
Chick embryos have become a great experimental model system for developmental biology, immunology and cancer,etc. The chicken embryo and myostatin are used as model system and target gene respectively. The process of transferring gene into chicken embryo and detection are researched using retrovirus-mediated gene transfer and in situ hybridization. The experimental results show that target gene myostatin can be integrated into the embryonic genome, and has transcriptional activity;Whole-mount in situ hybridization can accurately reflect the virus integration sites and target gene expression levels.The study not only provides a base for further study myostatin functions in embryonic development, but also is a methodology attempt.
Objective: To screen the binding sequences of ComE from the genome of Streptococcus mutans. Methods: ⑴The genome of Streptococcus mutans UA159 was extracted and sheared by sonication, and then incubated with two different random primers successively. Finally, the extention product was purified from gel extraction and amplified by PCR, and the genomic library of Streptococcus mutans was constructed.⑵Genomic SELEX for eight times to obtain the binding sequences of ComE. Selected DNA fragments were cloned into the pGEM-T Easy Vector and sent to sequence analysis. The sequences were further analyzed by bioinformatics programs. ⑶Two obtained sequences were cloned into pFW5-luc,and then the corresponding recombinant plasmids were transformed into Streptococcus mutans UA159 and Streptococcus mutans comE mutant respectively. Finally, the expression of luc were tested by RT-PCR. Results: ⑴The genomic library of Streptococcus mutans UA159 was obtained with the sequences ranging from 100 bp to 300 bp. ⑵54 clones were selected randomly and sent to sequencing. Two candidates were identified through bioinformatics analysis. Through primary validation, one possible binding sequence was obtained. Conclusion: According to genomic SELEX, eight selection cycles were finished and one possible binding sequence was obtained, which laid the basis for the explanation of the regulation mechanism of ComE in Streptococcus mutans.
Objective: To construct trivalent-epitope DNA vaccine encoding SjFABP, SjGAPDH and Sj26, and evaluate the protective immunity efficacy against Schistosoma japanicum by pharmacological trials. Methods: The monovalent antigen gene SjFABP, SjGAPDH and Sj26 were obtained by RT-PCR. Sj26 and SjGAPDH are spliced into covalent fusion gene of Sj26.SjGAPDH by recombinant PCR. pVIVO2-SjFABP/Sj26.SjGAPDH is constructed by subcloning the SjFABP and Sj26.SjGAPDH into mcs1 and mcs2 of pVIVO2-mcs respectively. The expression of fusion antigen protein in MCF-7 cell line transfected with pVIVO2-SjFABP/Sj26.GAPDH was determined by indirect immunofluorescence assay (IIF) and reverse transcriptase-polymerase chain reaction (RT-PCR). The evaluation of vaccine’s immuno-protection efficacy is tested by pharmacological trials. Results: The trivalent-epitope DNA vaccine pVIVO2-SjFABP/Sj26.SjGAPDH was successfully constructed. In the trivalent-epitope DNA vaccine group, the worm reduction rate and the egg reduction rate were 58.6% and 59.8% compared with control group. Conclusion: The trivalent-epitope DNA vaccine pVIVO2-SjFABP/Sj26.SjGAPDH could induce higher protection efficiency than both monovalent and bivalent vaccine, and paves the way for development of DNA vaccine against Schistosoma japanicum.
Elevent's MHC class I alleles around 3000bp have been identified from 10 Macaca fascicularis. BLAST analysis showed that the novel alleles include 7 Mafa-A alleles and 4 Mafa-AG alleles, which had been submitted to GenBank. Different Mafa-A alleles have been identified in each sample, it can be inferred that Mafa-A alleles have been duplicated and Mafa-AG alleles are derivative of Mafa-A alleles. Based on the new alleles, we analyze the alleles distribution among the samples and the phylogenetic tree.
Cadmium is a kind of highly toxic heavy metals. Even very low concentration of Cd2+ in soil solutions can result in toxic effects to plants. To survive, plants must change their metabolism to cope with cadmium exposure. Before that a lot of genes had changed their expression. A novel differential display PCR method was adopted that is based on annealing control primers (ACPs) to identify up-regulated genes of Arabidopsis by Cd2+ exposure. Nineteen differentially expressed bands were isolated and sequenced. They represent eighteen genes. Among them, six genes were identified by RT-PCR that they were really induced by cadmium treatment, including LEA(late embryogenesis abundant protein), AtGSTF2 (Glutathione S-transferase 2), AtGSTF6(Glutathione S-transferase 6), HSP70(heat shock protein 70), sHSP17.6B-CI(17.6 kDa class I small heat shock protein) and sHSP17.6-CII(17.6 kDa class II small heat shock protein). The results will help us to understand detoxification mechanism of plant to cadmium. And promoters of these three HSPs could be used for phytoremediation of cadmium pollution.
In order to analyze transcriptional regulative mechanism of PpMADS1 gene, the PpMADS1 promoter was obtained using Genome Walking method from peach (Prunus persica) genomic DNA. Sequence analysis indicated that there exist TATA-box, CAAT-box, two CArG box, one G-box, one TGA-element and a large number of regulatory elements involved in light response, such as GT-1, Sp1 and as-2-box, these results indicated that the promoter may be regulated by light and hormone. The PpMADS1 promoter was truncated according to the prediction of putative of cis-acting elements and fused with GUS reporter gene and transferred into Arabidopsis thaliana. Histochemical staining of different organs of the transgenic plants showed that the region between -197 to -454bp specified GUS expression in flower primordium and the region between -454 to -678bp in sepals and petals. A negatively regulatory element was shown to be present between -678 to -978bp that repressed GUS expression in filament.
The genes encoding the mature both types of thioredoxins, f and m type, were cloned by RT-PCR respectively using the total RNA from maize young leaves. The second conservative cystine residues in the catalytic site from two types of the proteins were mutated into serine and alanine residue respectively. The wild type and mutated thioredoxins with histidine-tag were overexpressed in Eecherchia coli and purified. All of them displayed one band on SDS-PAGE. The molecular weight was estimated 18 kD for f type thioredoxin and 14 kD for m type thioredoxin. Both thioredoxins with SUMO fusion tags were also purified and the tags were removed with the SUMO protease Ulp. The proteins displayed pI values of 4.6 for Trx-m and 5.9 for Trx-f. The reduction of insulin suggested that the m type thioredoxin has more active than f type thioredoxin. Both mutated proteins hardly reduced insulin. The modification of the proteins by the specific cystine reagent AMS revealed that the purified wild type thioredoxins displayed the redox states, but the mutated thioredoxins showed the reduced state, suggesting that there is no disulfide bond in the mutated thioredoxins. The entrapped proteins from young leaves of maize by the mutated f type thioredoxin with histidine-tag immobilized on the Ni-NTA resin were more diverse than those by the mutated m type thioredoxin, as shown by SDS-PAGE.
Large-scale culture has a high level requirement for the strain of microalgae, so screening of high quality microalgae strain has always been a problem to be settled. By researching three strains of biodiesel-producing microalgae, this article has built a two level estimate system which use 18 indicators such as the growth rate, oil content rate and oil composition. Property of biodiesel-producing microalgae was analyzed using the method of two -level fuzzy comprehensive evaluation. And the result of the evaluation: Chlorella vulgaris LICME001, Nannochloropsis oculata LICME002 and Botryococcus Braunii LICME003. By the biggest membership principle, Chlorella vulgaris LICME001 were finally determented as high quality biodiesel-producing microalgae strain which is suitable for the requirement of biodiesel production technology. Nannochloropsis oculata LICME002 and Botryococcus Braunii LICME003 were determented as good quality biodiesel-producing microalgae.
Studies were carried out to isolate Acid Scarlet GR resistant and decolored bacteria from sludge of a wastewater treatment plant, Zhejiang Province. Through dyeing decoloring tests, one strain, named Z1 was found to have a better decolorization performance at the high pH 12 and identified as Staphylococcus pasteuri by 16SrDNA gene sequence analysis. A strain named Z1 was screened through dye decolorization test and was identified as Staphylococcus pasteuri by 16SrDNA gene sequence analysis. And then the decolorization properties of Z1 were investigated. The results showed that the decolorization rate of 50mg/L Acid Scarlet GR could exceed 90% within 40h under anaerobic conditions at the range of pH 7~12, and the decolorization rate of 300mg/L Acid Scarlet GR even reached 93% within 48h. Moreover, the strain could effectively degrade other azo dyes with broad spectrum and could be applied for removing the azo dyes from the industrial wastewater.
Objective: A high throughput screening system of mGluR5 was established by Ca2+ mobilization detecting method. Methods: The human mGluR5 cDNA was cloned using gene synthesis technique and subcloned into the pCNDA3.1 mammalian expression vector. The recombinant plasmid was then transfected into HEK293 cells. The transfected cells were stably selected by G418. Single cell clones with high Ca2+ inducibility and low background were isolated. Results: Assay conditions were optimized, under which more robust signals were observed. Further validation results indicated that in this mGluR5 cell line, the potencies of mGluR5 agonists, antagonists were comparable with the published data. The rank order of agonists potency for mGluR5 was L-Quisqualic acid (EC50=67.8nM) > L-Glu (EC50=2.73μM). The rank order of antagonists potency for mGluR5 was MTEP (IC50=3.3nM) >Fenobam (IC50=23.5nM). The cell line was stable for at least 15 passages. The results of functional assay indicated that a HTS system for mGluR5 antagonist screening had been established successfully with a Z’ Prime of 0.68.By using this assay system, several positive compounds were identified which represented strong inhibition effect. Conclusion: The recombinant cell model is suitable for mGluR5 receptor targeted high throughput screening.
Objective:To generate the transgenic mice that express human Asialoglycoprotein receptor(ASGPR),which is considered as one of the hepatitis B virus (HBV) receptors. Methods: Both cDNA of the Human ASGPR H1&H2 subunits were cloned and the expression vector PCAGGS-ASGPRH1&H2 were constructed, Both of the 3.9kb DNA fragments were introduced into the fertilized eggs of mice by microinjection. The genotypes of the transgenic mice were identified by PCR and Southern-blot methods, and the expression of the exogenous ASGPR gene was confirmed by the RT-PCR and Western blot. Result and Conclusion: One transgenic mouse line that express the human ASGPR was obtained. It can be used as an animal model for the research of HBV infection.
A method for the determination of content and uniformity of recombinant human interferon α1b dry powder inhalation by HPLC was developed. A good linearity was obtained in the range of 92.50~462.50μg/ml(r=0.999 9).Good peak shape of interferon α1b was achieved. The impurities and flow phase have no interference to the interferon in this system. This method was approved to be precise, accurate, stable and rapid for the determination of content of recombinant human interferon α1b in medicinal product.
The transposon donor vector harboring both polyhedrin gene droved by BmA3 promoter and zeocin resistant gene controlled by IE1 promoter from OpNPV co-transefeced with a lepidoptera transposition helper plasmid pie2piggBac into BmN cells. A polyhedrin expressing stable cell line was constructed by screening with the 200μg/ml zeocin selection medium for one month. Occlusion bodies (OB) were rescued successfully in the polyhedrin expressing BmN cells infected with recombinant BmBac-gfp budded virus without polyhedrin gene. The amount of rescued OB produced in polyhedrin expressing cells is only 8% of wild type BmNPV OB produced in normal cells. The silkworm larvae were infected efficiently when they were feed by the rescued OB sprayed mulberry leaves. Foreign GFP was expressed efficiently and no OB was observed in the hemolymph of silkworm larvae. This rescued OB oral infection method is benefit to promote the industrial progress of silkworm bioreactor by eliminating the tedious injection budded virus solution by hand.
By acetone precipitation, SP-Trisacryl cation-exchange chromatography, Sephadex G-75 gel filtration chromatography and C8 reversed-phase chromatography, a single component ribonuclease is purified from oocytes of Chinese Northeast frog (Rana dybowskii). SDS-PAGE electrophoresis shows a single band with relative molecular weight of 13kDa. Its optimum temperature is 65℃, and optimum pH is 5.5~6.0. Its Michaelis constant is 4.11μmol/L, and maximum reaction rate is 2.82pmol/s. In vitro cytotoxicity experiments the IC50 value for HeLa，K562 and MCF-7 tumor cells is 0.6μmol/L, 0.8μmol/L and 4μmol/L separatedly, while for the normal human fibroblasts cells, even up to 8μmol/L, no cytotoxic effects were observed. This low molecular weight ribonuclease with selective cytotoxicity for tumor cells isolated from oocytes of the Chinese northeast frog(Rana Dybowskii) will join the candidate proteins of drug molecular for the treatment of cancer.
Varicella-zoster virus (VZV) is a ubiquitous human alphaherpesvirus that causes varicella (chickenpox) during primary infection, can establish latency in sensory ganglia, and may reactivate to cause herpes zoster (shingles). Though VZV possesses the smallest genome among all human herpesviruses，little was known about its gene function and the attenuation mechanisms of VZV vaccine due to the difficulty of generating mutants to investigate gene function and the long period of time needed to establish pure clones using conventional techniques with mammalian cells. Recently，a completely new approach for full-length infectious clones of VZV based on bacterial artificial chromosomes(BACs) has been developed．This technique allows the maintenance，propagation and genetic modification of the viral genome as a BAC plasmid in E．coli，thus making the procedures fast，safe and effectivein prokaryotic cells．This technique also makes it possible for the reconstitution of viral progeny or mutants by transfection of the BAC plasmid into eukaryotic cells，thereby facilitating the an alysis of viral gene functions in the context of genome．The VZV BAC system also was useful as a vector for construction of recombinant live vaccines.
New blood vessel growth via angiogenesis is a fundational process in both physiological and pathological conditions. Physiological angiogenesis is critical during embryogenesis and placental development, whereas pathological angiogenesis plays an important role in the progression of many disease, most notably tumor growth, progression and metastasis. As “antiangiogenesis therapy for tumor” was proposed, concentrated efforts was leading to the discovery of a growing number of pro- and anti- angiogenic molecules, both of which regulated the “angiogenic-switch”. Arresten, Canstatin, Tumstatin and Hexastatin are newly discovered endogenous angiogenesis inhibitors, which derive from non-collagenous(NC1) domain of the α chain of type Ⅳ collagen. They are similar in structure and molecular weight. Furthermore all of them have been shown as novel integrin ligands and exhibit an ability to inhibit endothelial cell proliferation and migration and lower the density of tumor microvessel. Tumor growth and metastasis are halted for lack of nutrients and oxygen supply. Advances in the understanding of their molecular mechanism may contribute to the discovery of kinds of pharmaceutical agents.
Interferons are a class of cytokines that play key roles in the regulation of cell growth and differentiation via activation of a cascade of intracellular pathways and represented the functions of antiviral, immunomodulatory, and antiproliferative effects，which are synthesized and secreted by somatic cells of all mammalian species. Interferon alpha (IFN-α) are used in clinic to treat a variety of viral diseases and cancers. Howerver，the short circulating half-life of IFN-α necessitates frequent administration to patients which limits the broader usage. The methods of prolonging the half-life of protein drug in common use are chemical modification, albumin fusion technology, analog construction and combination with drug delivery system. Herein the method of prolonging the half-life of IFN-α in the above-mentioned of the latest research methods were reviewed.
Elastin-like polypeptides(ELPs) were a family of artificial, genetically encodable polypeptidesthey are primarily composed of the repeating pentapeptide sequence GVGXP. Due to their reversible phase transition characteristics, ultra-high production, excellent biocompatibility and biodegradation, ELPs had an intensive potential in new biomedical materials. Describes principle of ELPs’ phase transition, and its applications in biomedical materials,especially gives emphasis to introduce the applications of tissue engineering, targeting tumor and constructing of drug carrier particle.
Formate dehydrogenase (FDH) is an abundant enzyme which is present widely in plants. FDH is also an NAD+-dependent enzyme which catalyzes the reversible oxidation of formate to carbon dioxide. As a component of one-carbon metabolism in plants, the enzyme played an important role in response to various environmental stresses and low oxygen or hypoxia in plants，and has great potential for application in agricultural production. Recently, considerable progresses have been made for plant FDH in gene cloning, gene regulation and physiological functions. The recent progresses on gene cloning, gene expression regulation, physiological roles of plant FDH were reviewed.
Glycerol, a common polyol metabolite, is produced during yeast cells growth, propagation and glucose metabolism. Though glycerol structure and metabolic pathway is very simple, it plays an important physiological role in yeast cells, especially which are exposed in such stress conditions as hypertonic medium, frozen temperature and anaerobic environment. Glycerol metabolism is involved in osmoregulation and redox balance regulation. Recently, physiological function of glycerol in yeast, especially for Saccharomyces cerevisiae, were focused on and investigated widely. Glycerol metabolism was introduced succinctly, and the correlations of glycerol production and osmoregulation, redox balance are emphasized on. Moreover, metabolic engineering for glycerol biosynthesis and its future research prospects are discussed.
