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Gene cloning and chracterization of a cathepsin B and cathepsin B-Like plotein from Naegleria fowleri

Lee, J; Kim, JH; Sohn, HJ; Yang, HJ; Shim, MH; Kang, HK; Song, KJ; Shin, HJ
Department of Microbiology
Naegleria fowleri, a free-living ameba, causes primary amebic meningoencephalitis (PAM) in human and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutirent uptake, excystment/encystment, host tissue invasion, larval migration, and immune evasion. Previous study, we reported important role for pathogenesis which cysteine protease such as 21.5kDa cathepsin B and 24kDa cathepsin B-like protease in N. fowleri excretory/secretory proteins. In this study, N. fowleri cathepsin B (NfCPB) and cathepsin B-Like (NfCPB-L) protein were cloned from N. fowleri cDNA library. To obtain full-length sequence, We performed 5’-RACE PCR to obtain full-length sequence of N-terminus. The open reading frame consisted 1020bp and 939bp sequence, encoded 340 and 313 amino acid and molecular weight were 37kDa and 34kDa. By BlastX analysis, they showed 56% and 49% identity with N. gruberi cathepsin B and cathepsin B-Like protein. The PCR product ligated into pEXP5-NT/TOPO expression vector and recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The 37kDa band of NfCPB and NfCPB-L protein were showed by SDS-PAGE analysis and western-blot analysis, respectively.
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