Erns is a highly glycosylated envelope protein of classical swine fever virus (CSFV) with RNase activity. Erns can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, the RNase domain of the Erns was produced in Escherichia coli. Its reactivity with CSFV-positive sera and its ability to induce antibodies and to provide protective immunity were then investigated. The serological tests showed that the prokaryotically expressed RNase domain of the Erns retained its antigenicity and induced high titers of humoral responses. However, only partial protection and a limited amount of neutralizing antibodies were demonstrated by an in vitro neutralization test and an immunization/challenge test. The results suggest that other essential factors rather than simply enhancing the immunogenicity of Erns should be taken into consideration when Erns is enrolled as one of the components of a candidate vaccine.
Human enterovirus 71 (EV71) is one of the major etiological agents of the hand-foot-and-mouth disease (HFMD) that often causes severe neurological complications. Recently,its outbreaks mainly take place in the torrid zone of the Asia-Pacific region. To study the evolution and genetic variability,we collected 532 EV71 strains with almost complete or complete VP1 sequences (891 nt) isolated worldwide from 1970 to 2004. The pairwise homologies and genetic distances were analyzed. Most strains belong to previously identified genotype B and C. However,a unique strain R13223-IND-01 appears not to fall into current three genotypes (A,B and C),and probably represents a new genotype D. Some orphan strains were observed in the genotypes B and C,and their significance in the EV71 evolution was discussed. Moreover,there is a significant co-variance of 6 discrete positions on VP1 (amino acid 43,58,164,184,240 and 249). This high co-variability is tightly related with the subgenotypes.
A multi-epitope-vaccine MEVABC consisting of two linear neutralizing determinants (BC1: aa693-716; A6: aa844-865) located on antigenic unit B/C and unit A of glycoprotein E2 was prepared to evaluate whether a combination strategy is effective in the design of peptide vaccines. After immunization, pig sera collected every one to two weeks were evaluated by enzyme linked immunosorbent assay. C-strain- induced anti-sera and hyper-immune sera cannot recognize overlapping peptides that cover the E2 N-terminus, while MEVABC is able to elicit high levels of peptide-specific antibody response. When compared with previously studied peptide vaccines PV-BC1 and PV-A6, the same dose of either component in the MEVABC increases the BC1- or A6-specific antibodies (to 1/3-1/2 of the levels of the separate vaccines). However, the synergy between the antibodies may make MEVABC much more potent. Moreover, anti-C-strain immunity pre-existing in pigs does not disturb the sequent MEVABC vaccination. Thus, MEVABC can be ad-ministrated to pigs which already possess anti-classical swine fever virus immunity. MEVABC is a promising candidate marker vaccine.
Plasma membrane calcium ATPase (PMCA) plays a critical role in transporting Ca2+ out of the cy-tosol across the plasma membrane which is essential both in keeping intracellular Ca2+ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinc-tada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further under-stand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca2+ homeostasis and the mechanism of mineralization in pearl oyster.
Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously ex-pressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridiza-tion analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium. All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.
The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily.However,the calcium-regulatory proteins in prokaryotes are still poorly understood.In this study,we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor,named CabD,which shares low sequence homology with other known helix-loop-helix EF-hand proteins.The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins.The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins,including the bending conformation of the first C-terminalα-helix,unpaired ligand-binding EF-hands and the lack of the extreme Cterminal loop region,suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes,and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.
