The 56-kDa protein of Rickettsia tsutsugamushi, which is located on the rickettsial surface, has been shown to be an immunodominant antigen. The gene that encodes the 56-kDa protein of R. tsutsugamushi Boryong (bor56) was cloned. Sequencing revealed an open reading frame of 1,602 bp encoding 534 amino acids with a molecular weight of 56,803. The 56-kDa protein ofR. tsutsugamushi Boryong (Bor56) was expressed as a fusion protein with the maltose-binding protein of Escherichia coli by deleting 252 bp from the 5' end of the open reading frame and subcloning it into the StuI site of pIH821. The recombinant fusion protein was purified by amylose column chromatography for application in an enzyme-linked immunosorbent assay to evaluate the ability of the method to detect the antibody to R. tsutsugamushi in human patient sera. By using sera from 100 patients with scrub typhus and 70 patients with other febrile diseases, a high diagnostic sensitivity (95%) and a high diagnostic specificity (100%) were demonstrated, suggesting the suitability of the recombinant antigen for use as an immunodiagnostic tool.

The genes encoding the 56-kDa polypeptides were amplified by polymerase chain reaction from the genomic DNAs of three serotypes ofRicketasia tsutsugamushi, Gifliam, Karp, and Boryong. The amplified products were cloned into expression vector pIH821, and the recombinant antigens were expressed in Escherichia conl as fusion proteins with maltose-binding protein. The recombinant 56-kDa polypeptides were purified by affinity chromatography for the sensitization of sheep erythrocytes. The recombinant 56-kDa polypeptides were * Corresponding author. epitopes are present within the protein (6, 9, 16, 17, 19, 23), it may be useful in the development of tools for serologic diagnosis.

Effort to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimeric gag-env virus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2 gag precursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1IIIB, HIV-1MN, HIV-1RF, and HIV-1SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purified gag-env VLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than the gag VLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1IIIB, HIV-1MN, HIV-1RF, and the gp120 antiserum against HIV-1SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2 gag can be used as a potential HIV vaccine against various HIVs.

To isolate a naturally occurring novel Bacillus thuringiensis strain, we investigated the distribution, toxicity, morphology, H serotype, and gene type of B. thuringiensis from residue samples of granary in Korea. A total of 163 B. thuringiensis isolates out of 411 samples producing spore and crystal were obtained. In toxicity tests, 80% of all isolates were toxic to lepidoptera, and 12% were not toxic to any of tested insects. And dipteran-active and lepidopteran/dipteran-active isolates were rare (2% and 6%, respectively). 152 B. thuringiensis isolates produced typical rhomboidal crystals, and the remainder produced parasporal inclusions with various morphologies. Serological test showed that B. thuringiensis isolates in granary represented 12 H serotypes, indicating varied distribution of B. thuringiensis. Of these, the serotype 3ab predominated, followed by the serotype 7 and 4ac. B. thuringiensis isolates of the serotype 3ab, 4ac, 5ab, 7, 8ab, 9, and 23 were toxic to lepidoptera, and the serotype 8bd, 12, 18, and 20ac were nontoxic, while 14 isolates were untypable by 33 B. thuringiensis H antisera. The frequency of toxicity against lepidoptera and diptera was primarily highly toxic. PCR analysis using cryI gene type-specific primers showed that cryIA(b) genes are frequently found and cryIE gene exists in only one isolate. Analysis of B. thuringiensis crystals and plasmid DNAs indicated a diversity of crystal and gene types.