共表达ETECK99、K88菌毛蛋白重组干酪乳杆菌的构建

The Surface Display of ETEC K99,K88 Protein in Lactobacillus casei

将PCR扩增的K99和K88-LTB基因片段串联插入到干酪乳杆菌细胞表面表达载体pLA中,构建了重组表达载体pLA-K99-K88-LTB,将其电转化干酪乳杆菌CICC 6 105,获得了表达融合蛋白pLA-K99-K88-LTB的重组干酪乳杆菌表达系统。重组干酪乳杆菌在MRS培养基中表达后,经SDS-PAGE检测,约有90 KDa的融合蛋白得到表达,蛋白大小与理论值相符合。Western-blot分析表明表达的蛋白可被鼠源K99,K88抗血清所识别。间接免疫荧光技术和流式细胞术的结果表明,融合蛋白成功地利用多聚谷氨酸跨膜蛋白pgsA基因展示在干酪乳杆菌菌体表面。

Lactobacillus casei CICC 6105 was selected as antigen delivery vehicle for the expression of enterotoxigenic Escherichia coli（ETEC） protective antigen K99 and K88 protein.The gene fragments encoding the K99 fimbrial protein and the K88-LTB fusion protein were correspondingly cloned into the Lactobacillus casei surface expression vector pLA and then the recombinant plasmid（pLA-K99-K88-LTB） was electrotransformed into Lactobacillus casei CICC6 105,resulting in recombinant strain pLA-K99-K88-LTB/L.casei.The recombinant strains were induced to express interest protein,which was detected by Western blotting,immunofluorescence microscopy and flow cytometry.The results showed that a molecular weight of about 90 kDa was detected by SDS-PAGE and the result of Western-blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-K99 or K88 serum.The fluorescence microscopy and flow cytometric analysis showed the fusion protein was expressed on the cell surface of L.casei using PgsA,a membrane-anchored protein display motif.