Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.
Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays. 相似文献
1. Whole body protein synthesis was measured in chick embryos cultured in vitro. On day 7 of incubation chick embryos were cultured for 60 min in synthetic serum‐free medium containing 4‐[3H]phenylalanine. Specific radioactivities in free and protein‐bound phenylaline in the whole embryo were measured, starting 2 min after commencement of the culture process.
2. The values for fractional synthesis rate (FSR) estimated in vitro at 20, 30, 45 and 60 min during the embryo culture agreed well, ranging from 35 to 40%/d, suggesting that the method would serve as a useful model for studying the effect of growth promoters in chick embryos.
3. Bovine insulin in the synthetic medium did not affect FSR of protein in chick embryos cultured in vitro. 相似文献
Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (VL) and heavy chain (VH) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location. 相似文献
L11A-Fukushima (L11A-F) derived from attenuated isolate LuA of Tomato mosaic virus (ToMV) has the highest ability to cross protect against virulent ToMV among LuA and its derivatives and is stably inherited.
Growth, yield, fruit quality and symptom attenuation of inoculated tomato plants did not differ significantly between L11A-F and L11A. The infectivity of progeny viruses in tomato infected with LuA-F was less than 4% of that with virulent ToMV. From these
results, L11A-F appears to possess the properties necessary for practical use. To manage L11A-F strictly, a PCR-based assay to detect trace contamination of virulent ToMV in L11A-F preparations was established.
Received 10 June 2002/ Accepted in revised form 30 October 2002 相似文献
DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum
production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently
infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV
originating from latently infected stock plants in chrysanthemum production fields.
Received 27 July 2001/ Accepted in revised form 27 November 2001 相似文献
Cellular fatty acids were analyzed to characterize and differentiate 34 isolates of Rhizoctonia species representing binucleate Rhizoctonia AG-D (I), AG-D (II), R. solani AG 2-2 IIIB, AG 2-2 LP, R. circinata var. circinata and var. oryzae associated with turfgrass diseases in Japan. Myristic, pentadecanoic, palmitic, palmitoleic, stearic, oleic, linoleic and
linolenic acids were consistently present in varying quantities in all isolates. Heptadecanoic and 9-heptadecenoic acids were
present in isolates of Rhizoctonia AG-D (I), AG-D (II), R. solani AG 2-2 IIIB and AG 2-2 LP but not in isolates of R. circinata var. circinata and var. oryzae. Palmitic, oleic and linoleic acids were the major fatty acids found, constituting 88.30-98.37% of the whole-cell fatty acid
content. The remaining fatty acids were present in smaller amounts. Isolates within a single group were closely clustered,
whereas isolates from different groups were clearly distinguishable based on average linkage cluster analysis of cellular
fatty acids. Principal component analysis, based on all fatty acids detected, confirmed the distinct separation of isolates
representing the six groups of Rhizoctonia species obtained from turfgrasses. These results suggested that fatty acid analysis is useful for the characterization and
differentiation of isolates of Rhizoctonia species associated with turfgrass diseases.
Received 21 May 2001/ Accepted in revised form 28 September 2001 相似文献