Sting these four hypotheses and discuss the significance of the results in vaccine development for A. marginale and related pathogens.Materials and Methods Amplification, cloning, expression and purification of AMAM779 was amplified from genomic DNA of the St. Maries strain of A. marginale using forward (59-GGGGACAAGTTTGTACAAAAAAGCAGGCTTATCCGAGCCTCGGGAGGAG-39) and reverse (59-GGGGACCACTTTGTACAAGAAAGCTGGGTACTAAAAATCAAAC-39) primers. Amplification consisted of 30 cycles with melting temperature at 94uC for 15 
s, annealing at 60uC for 30 s, and extension at 72uC for 1 min. The AM779 amplicon was size-separated by agarose gel electrophoresis and visualized following staining with SYBR Safe DNA GEL Stain (Invitrogen). The amplicon was cloned into the pDONR221 vector as an entry clone and then into the Gateway pDESTTM17 expression vector (Invitrogen). After overnight Tunicamycin growth in LB broth, plasmid DNA was purified from the transformed colonies (TOP10 E. coli cells) using Wizard plus Miniprep DNA purification system (Promega). Plasmid inserts were sequenced in both directions using the Big Dye kit and ABI Prism automated sequencer (Applied Biosystems). Plasmid DNA bearing correctly oriented inserts was transformed into BL21-AI E. coli. For expression, bacteria were grown in LB broth at 37uC and induced using 0.02 arabinose. Bacteria were harvested at 5 h post induction by centrifugation, pellets were re-suspended in lysis buffer, 6 M guanidine hydrochloride, 20 mM sodium phosphate, pH 7.8, and 500 mM sodium chloride and disrupted by sonication. The recombinant protein was purified from the bacterial lysates using HisPur cobalt resin (Invitrogen). The eluted recombinant protein was dialyzed against 94-09-7 site phosphate buffered saline (pH 7.3) and stored at 280uC after adding 18055761 complete Mini protease inhibitors according to the manufacturer specifications (Roche).Animals and immunizationThe immunization of calves with either native A. marginale outer membranes isolated from the St. Maries strain or cross-linked and isolated surface protein complexes of the same strain has previously been reported [7]. This prior report includes the seronegative status prior to immunization, MHC class II haplotypes of each animal (n = 5 per group), the immunization procedure, the challenge with the St. Maries strain, the infection and disease parameters, and the analysis of protection. The availability of protection data from these animals provided the opportunity to correlate AM779 immune responses with the protection induced by complex membrane immunogens containing AM779. To test whether immunization with AM779 would overcome the immunologic sub-dominance observed in context of the complex immunogens, to test recall upon in vivo challenge, and to assess induction of protective immunity, a second set of animals were screened by Msp5 c-ELISA to confirm seronegative status and the MHC class II haplotypes determined by PCR-restriction fragment length polymorphism analysis of the DRb3 locus as described in detail [25],[26]. This second set of animals was immunized with either 20 mg isolated outer membranes (n = 5) in saponin, 20 mg recombinant AM779 protein in saponin (n = 5), or the adjuvant alone. The immunization procedure including antigen and adjuvant dose and route of delivery was similar to that reported for the first set [7].Subdominant Bacterial AntigensDetermination of antibody titersAntibody titers were determined for AM779 and Msp2 by immunoblotting. Briefly, either.Sting these four hypotheses and discuss the significance of the results in vaccine development for A. marginale and related pathogens.Materials and Methods Amplification, cloning, expression and purification of AMAM779 was amplified from genomic DNA of the St. Maries strain of A. marginale using forward (59-GGGGACAAGTTTGTACAAAAAAGCAGGCTTATCCGAGCCTCGGGAGGAG-39) and reverse (59-GGGGACCACTTTGTACAAGAAAGCTGGGTACTAAAAATCAAAC-39) primers. Amplification consisted of 30 cycles with melting temperature at 94uC for 15 s, annealing at 60uC for 30 s, and extension at 72uC for 1 min. The AM779 amplicon was size-separated by agarose gel electrophoresis and visualized following staining with SYBR Safe DNA GEL Stain (Invitrogen). The amplicon was cloned into the pDONR221 vector as an entry clone and then into the Gateway pDESTTM17 expression vector (Invitrogen). After overnight growth in LB broth, plasmid DNA was purified from the transformed colonies (TOP10 E. coli cells) using Wizard plus Miniprep DNA purification system (Promega). Plasmid inserts were sequenced in both directions using the Big Dye kit and ABI Prism automated sequencer (Applied Biosystems). Plasmid DNA bearing correctly oriented inserts was transformed into BL21-AI E. coli. For expression, bacteria were grown in LB broth at 37uC and induced using 0.02 arabinose. Bacteria were harvested at 5 h post induction by centrifugation, pellets were re-suspended in lysis buffer, 6 M guanidine hydrochloride, 20 mM sodium phosphate, pH 7.8, and 500 mM sodium chloride and disrupted by sonication. The recombinant protein was purified from the bacterial lysates using HisPur cobalt resin (Invitrogen). The eluted recombinant protein was dialyzed against phosphate buffered saline (pH 7.3) and stored at 280uC after adding 18055761 complete Mini protease inhibitors according to the manufacturer specifications (Roche).Animals and immunizationThe immunization of calves with either native A. marginale outer membranes isolated from the St. Maries strain or cross-linked and isolated surface protein complexes of the same strain has previously been reported [7]. This prior report includes the seronegative status prior to immunization, MHC class II haplotypes of each animal (n = 5 per group), the immunization procedure, the challenge with the St. Maries strain, the infection and disease parameters, and the analysis of protection. The availability of protection data from these animals provided the opportunity to correlate AM779 immune responses with the protection induced by complex membrane immunogens containing AM779. To test whether immunization with AM779 would overcome the immunologic sub-dominance observed in context of the complex immunogens, to test recall upon in vivo challenge, and to assess induction of protective immunity, a second set of animals were screened by Msp5 c-ELISA to confirm seronegative 
status and the MHC class II haplotypes determined by PCR-restriction fragment length polymorphism analysis of the DRb3 locus as described in detail [25],[26]. This second set of animals was immunized with either 20 mg isolated outer membranes (n = 5) in saponin, 20 mg recombinant AM779 protein in saponin (n = 5), or the adjuvant alone. The immunization procedure including antigen and adjuvant dose and route of delivery was similar to that reported for the first set [7].Subdominant Bacterial AntigensDetermination of antibody titersAntibody titers were determined for AM779 and Msp2 by immunoblotting. Briefly, either.
