ABSTRACT
In this study, the sequences of capsid protein VPI regions of YNAs1.1 and YNAs1.2 isolates of foot-and-mouth disease virus (FMDV) were analyzed and a peptide containing amino acids (aa) 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia I was assumed to contain B and T cell epitopes, because it is hypervariable and includes a cell attachment site RGD located in the G-H loop. The DNA fragments encoding aa 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia 1 were chemically synthesized and ligated into a tandem repeat of aa 133-158-20 approximately 34-133-158. In order to enhance its immunogenicity, the tandem repeat was inserted downstream of the beta-galactosidase gene in the expression vector pWR590. This insertion yielded a recombinant expression vector pAS1 encoding the fusion protein. The latter reacted with sera from FMDV type Asia 1-infected animals in vitro and elicited high levels of neutralizing antibodies in guinea pigs. The T cell proliferation in immunized animals increased following stimulation with the fusion protein. It is reported for the first time that a recombinant fusion protein vaccine was produced using B and T cell epitopes of FMDV type Asia 1 and that this fusion protein was immunogenic. The fusion protein reported here can serve as a candidate of fusion epitopes for design of a vaccine against FMDV type Asia 1.
Subject(s)
Capsid/immunology , Foot-and-Mouth Disease Virus/immunology , Foot-and-Mouth Disease/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/biosynthesis , Base Sequence , Blotting, Western , Capsid Proteins/immunology , Disease Models, Animal , Epitopes/immunology , Foot-and-Mouth Disease/prevention & control , Guinea Pigs , Molecular Sequence Data , Plasmids/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Sequence Alignment , Viral Vaccines/geneticsABSTRACT
Two novel biosensors for urea based on immobilized corynebacterium glutamicum 617 and corynebacterium glutamicum ATCC13032 in calcium alginate gel coupled with an ammonia gas-sensing electrode, were designed and constructed. Calibration plots of measured potential difference (mV) vs. log of urea concentration were linear in the range of 5.6 x 10(-5)-1.4 x 10(-2) and 5.6 x 10(-5)-1.1 x 10(-2) mol l(-1), with slopes of 59.2 and 61.3 mV per decade respectively, in pH 8.0, 0.1 mol l(-1) phosphate buffer solution at 30 degrees C. The relationship between the initial response velocity and the substrate concentration was also discussed. The results indicate that the kinetic response process of the reaction catalyzed by bacteria is similar to that by isolated enzyme. Using an Eadie-Hofstee plot, the apparent Michaelis constant K(m) and the maximum initial response velocity V(m) for urease in the immobilized bacterial membrane were determined. The two urea biosensors were successfully applied for the actual measurement of urea in urine and were relatively stable for 20 and 40 days respectively.
