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Immunological Characterization of Full and Truncated Recombinant Clones of ompH(D:4) Obtained from Pasteurella multocida (D:4) in Korea

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Authors
Kim, Young Hwan; Cheong, Ki Young; Shin, Woo Seok; Hong, Sung Youl; Woo, Hee Jong; Kwon, Moo Sik
Issue Date
2006
Publisher
한국미생물·생명공학회 = The Korean Society for Microbiology and Biotechnology
Citation
J. Microbiol. Biotechnol.16: 1529-1536
Keywords
pasteurella multocida (D:4)OmpHimmune protectionvaccine candidatepig atrophic rhinitis
Abstract
We cloned a gene of ompH(D:4) from pigs infected with P. multocida D:4 in Korea [16]. The gene is composed of 1,026 nucleotides coding 342 amino acids (aa) with a signal peptide of 20 aa (GenBank accession number AY603962). In this study, we analyzed the ability of the ompH(D:4) to induce protective immunity against a wild-type challenge in mice. To determine appropriate epitope(s) of the gene, one full and three different types of truncated genes of the ompH(D:4) were constructed by PCR using pET32a or pRSET B as vectors. They were named ompH(D:4)-F (1,026 bp [1-1026] encoding 342 aa), ompH(D:4)-tl (693 bp [55-747] encoding 231 aa), ompH(D:4)-t2 (561 bp [187-747] encoding 187 aa), and ompH(D:4)-t3 (540 bp [487-1026] encoding 180 aa), respectively. The genes were successfully expressed in Escherichia coli BL21(DE3). Their gene products, polypeptides, OmpH(D:4)-F, -tl, -t2, and -t3, were purified individually using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Their Mrs were determined to be 54.6, 29, 24, and 23.2 kDa, respectively, using SDS-PAGE. Antisera against the four kinds of polypeptides were generated in mice for protective immunity analyses. Some 50 μg of the four kinds of polypeptides were individually provided intraperitoneally with mice (n=20) as immunogens. The titer of post-immunized antiserum revealed that it grew remarkably compared with pre-antiserum. The lethal dose of the wild-type pathogen was determined at 10 μl of live P. multocida D:4 through direct intraperitoneal (IP) injection, into post-immune mice (n=5, three times). Some thirty days later, the lethal dose (10 μl) of live pathogen was challenged into the immunized mouse groups [OmpH(D:4)-F, -t1, -t2, and -t3; n=20 each, two times] as well as positive and negative control groups. As compared within samples, the OmpH(D:4)-F-immunized groups showed lower immune ability than the OmpH(D:4)-t1, -t2, and -t3. The results show that the truncated-OmpH(D:4)-tl, -t2, and -t3 can be used for an effective vaccine candidate against swine atrophic rhinitis caused by pathogenic P. multocida (D:4) isolated in Korea.
ISSN
1017-7825
Language
English
URI
http://kiss.kstudy.com/search/detail_view.asp?key=2573016

https://hdl.handle.net/10371/6518
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College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Journal Papers (저널논문_수의학과)
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