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The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification. 相似文献
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A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus 总被引:1,自引:0,他引:1
Rosana PV Brondani Emlyn R Williams Claudio Brondani Dario Grattapaglia 《BMC plant biology》2006,6(1):20-16
Background
Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus. 相似文献4.
Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse 总被引:1,自引:0,他引:1
Gonzalez P; Hernandez-Calzadilla C; Rao PV; Rodriguez IR; Zigler JS Jr; Borras T 《Molecular biology and evolution》1994,11(2):305-315
zeta-Crystallin is a novel nicotinamide adenine dinucleotide
phosphate:quinone reductase, present at enzymatic levels in various tissues
of different species, which is highly expressed in the lens of some
hystricomorph rodents and camelids. We report here the complementary DNA
(cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia
porcellus), where zeta-crystallin is highly expressed in the lens, and in
the laboratory mouse (Mus musculus), where expression in the lens occurs
only at enzymatic levels. A 5' untranslated sequence different from the one
previously reported for the guinea pig lens cDNA was found in these clones.
We also report the isolation of genomic clones including the complete
guinea pig zeta-crystallin gene and the 5' region of this gene in mouse.
These results show the presence of two promoters in the guinea pig
zeta-crystallin gene, one responsible for expression at enzymatic levels
and the other responsible for the high expression in the lens. The guinea
pig lens promoter is not present in the mouse gene. This is the first
example in which the recruitment of an enzyme as a lens crystallin can be
explained by the acquisition of an alternative lens- specific promoter.
相似文献
5.
IB Masters MM Eastburn PW Francis R Wootton PV Zimmerman RS Ware AB Chang 《Respiratory research》2005,6(1):16
Background
Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object.Methods
Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived.Results
Magnification changes as a linear function between 100 mm (×1) and 10 mm (×9.55) and then as an exponential function between 10 mm and 3 mm (×40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis.Conclusion
We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements. 相似文献6.
JB Parentes-Vieira PV Lopes-Costa CG Pires AR dos Santos JD Pereira-Filho BB da Silva 《International Seminars in Surgical Oncology : ISSO》2007,4(1):22
Background
The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.Methods
This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).Results
The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).Conclusion
ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.7.
Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents 总被引:3,自引:2,他引:1
Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase
expressed at very high levels in the lenses of two groups of mammals:
camelids and some hystricomorph rodents. It is also expressed at very low
levels in all other species tested. Comparative analysis of the mechanisms
mediating the high expression of this enzyme/crystallin in the lens of the
Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided
evidence for independent recruitment of this enzyme as a lens crystallin in
both species and allowed us to elucidate for the first time the mechanism
of lens recruitment of an enzyme- crystallin. The data presented here show
that in both species such recruitment most likely occurred through the
generation of new lens promoters from nonfunctional intron sequences by the
accumulation of point mutations and/or small deletions and insertions.
These results further support the idea that recruitment of CRYZ resulted
from an adaptive process in which the high expression of CRYZ in the lens
provides some selective advantage rather than from a purely neutral
evolutionary process.
相似文献
8.
Background
The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8+ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8+ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope.Results
Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8+ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi.Conclusions
We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response. 相似文献9.
Background
Fairy shrimps (Anostraca), tadpole shrimps (Notostraca), clam shrimps (Spinicaudata), algae (primarily filamentous blue-green algae [cyanobacteria]), and suspended organic particulates are dominant food web components of the seasonally inundated pans and playas of the western Mojave Desert in California. We examined the extent to which these branchiopods controlled algal abundance and species composition in clay pans between Rosamond and Rogers Dry Lakes. We surveyed branchiopods during the wet season to estimate abundances and then conducted a laboratory microcosm experiment, in which dried sediment containing cysts and the overlying algal crust were inundated and cultured. Microcosm trials were run with and without shrimps; each type of trial was run for two lengths of time: 30 and 60 days. We estimated the effect of shrimps on algae by measuring chlorophyll content and the relative abundance of algal species.Results
We found two species of fairy shrimps (Branchinecta mackini and B. gigas), one tadpole shrimp (Lepidurus lemmoni), and a clam shrimp (Cyzicus setosa) in our wet-season field survey. We collected Branchinecta lindahli in a pilot study, but not subsequently. The dominant taxa were C. setosa and B. mackini, but abundances and species composition varied greatly among playas. The same species found in field surveys also occurred in the microcosm experiment. There were no significant differences as a function of experimental treatments for either chlorophyll content or algal species composition (Microcoleus vaginatus dominated all treatments).Conclusions
The results suggest that there was no direct effect of shrimps on algae. Although the pans harbored an apparently high abundance of branchiopods, these animals had little role in regulating primary producers in this environment. 相似文献10.
Pavlova MV Hil'chuk PV Pokholenko IaO Nikolaiev IuS Kordium VA 《T?Sitologii?a i genetika》2008,42(2):10-15
A cDNA combinatorial antibody library of mouse variable immunoglobulin fragments has been constructed from mice immunized with rhIFN-beta1b. For this purpose, cDNAS of immunoglobulin variable heavy (V(H)) and variable light (V(L)) chains genes amplified from splenocytes were joined with linker DNA to form ScFv's (single-chain Fv-antibodies). The obtained ScFv-DNA pool was cloned into a phagemid vector and used for Esherichia coli transformation. Using the phage display technique, bacterial clones producing single-chain antibodies specific to rhIFN-beta1b were selected. The following characteristics of the combinatorial library were determined in this work: abundance, functional size, and the initial ScFv-DNA diversity in the library constructed. High specificity of interaction between phage displayed ScFv's and rhIFN-beta1b has been demonstrated. 相似文献