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KMID : 0545120010110030482
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Journal of Microbiology and Biotechnology
2001 Volume.11 No. 3 p.482 ~ p.490
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Sequencing and Baculovirus-Based Expression of the Glycoprotein B2 Gene of HSV-2(G)
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Uh, Hong Sun
Park, Jong Kuk/Kang, Hyun/Kim, Soo Young/Lee, Hyung Hoan
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Abstract
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The gene for glycoprotein B (gB2) of HSV-2 strain G was subcloned, sequenced, recombinated into the lacZ-HcNPV, expressed in insect cells, and compared with the homologous gene of other HSV-2 strains. The ORF of the gB2 gene was 2,715bp. The overall nucleotide sequence homology of the gB2 gene compared with that of the two previously reported HSV-2 strains appeared to be over 98%. A recombinant virus named Baculo-gB2 was constructed, which expressed the gB2 protein in insect cells. The recombination was confirmed by a PCR and the expression was demonstrated by radioimmunoprecipitation. Insect cells infected with the Baculo-gB2 virus synthesized and processed gB2 with approximately 120kDa in the cells, and then secreted it into the culture media, where it reacted with a monoclonal antibody to gB2. The gB2 polypeptide contained two main hydrophobic regions (a signal sequence from 1 to 23 amino acid residues, and a membrane anchor sequence from as 745 to 798), eight N-glycosylation sites evenly distributed, and was rich in alanine (11.2%). Antibodies to this recombinant protein that were raised in mice recognized the viral gB2 and neutralized the infectivity of the HSV-2 in vitro. These results show that the gB2 protein was successfully produced in insect cells and could be used to raise a protective neutralizing antibody. Accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.
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