梅毒螺旋体膜抗原基因在毕赤酵母中的表达和蛋白纯化

以梅毒螺旋体(Treponema pallidumsubsp.pallidum)Nichols菌株基因组DNA为模板,通过PCR扩增梅毒螺旋体47kDa、17kDa和15kDa 3个膜抗原基因,克隆进毕赤酵母表达载体pPICZ B,构建重组表达载体pTP47、pTP17、pTP15,转化酵母菌株GS115,甲醇诱导表达。表达菌体裂解后通过镍离子亲和层析获得3个抗原与6xHis tag的融合蛋白,重组蛋白的获得量分别为His-TP15:4.8mg/L;His-TP 17:6.6mg/L;His-TP47:25mg/L,经SDS-PAGE鉴定纯度都在96%以上,ELISA鉴定均具有很好的抗原性。从而首次在毕赤酵母中表达出梅毒螺旋体膜抗原,为梅毒血清学检测方法开辟了新的抗原制备途径。

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.