[Expression and purification of membrane immunogens of Treponema pallidum in Pichia pastoris].
Genes of Treponema pallidum subsp. pallidum three membrane antigens (47kDa, 17kDa and 15kDa) were amplified by PCR with the template of the genomic DNA of Nichols strain and cloned into plasmid pPICZ B, the recombinant plasmids of pTP47, pTP17 and pTP15 were transformed into Pichia pastoris GS115. Recombinant antigens were expressed by the methanol induction and confirmed by the Western blot assay. Fusion antigens with 6 x His tag were purified using Ni-NTA agarose, and purified fusion protein yields were 4.8mg/L, 6.6mg/L and 25mg/L of cell culture for His-TP15, His-TP17 and His-TP47, respectively. Purity of all three antigens were more than 96% by SDS-PAGE assay. Particularly, His-TP17 was more about 6kDa molecular weight than that of expected probably because of glycosylation in Pichia pastoris. At last, these recombinant antigens were evaluated by ELISA assay with serum from syphilis patient and healthy blood donors, all three antigens showed strong sensitivity and specificity. So three membrane antigens of Treponema pallidum were expressed and purified fused with 6 x His tag in Pichia pastoris for the first time. The immunoreactivity results showed that all of which can be applied to diagnosis of Treponema pallidum, especially to diagnosis method based on combined two or three antigens.
Huang
Zong-Yan
ZY
National Engineering Research Center for Miniaturized Detection System, Department of Biology, Northwest University, Xi'an 710069, China. hzongyan@126.com
Wang
Ya-Ni
YN
Zhu
Juan-Li
JL
Dong
Zhao-Lin
ZL
Chen
Chao
C
D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013588:Syphilis Serodiagnosis; D014210:Treponema pallidum / Q000276:immunology*
D000942:Antigens, Bacterial; D011994:Recombinant Proteins