Surface display of foreign epitopes on the Lactobacillus brevis S-layer
Surface display of foreign epitopes on the Lactobacillus brevis S-layer
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DOI：
10.1128/aem.68.12.5943-5951.2002
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发表时间：
2002-12-01
影响因子：
4.4
通讯作者：
Palva, A
中科院分区：
文献类型：
--
作者：
Åvall-Jääskeläinen, S;Kylä-Nikkilä, K;Palva, A
到目前为止，无法建立可行的乳酸杆菌表面层（S-层）无效突变体阻碍了乳酸杆菌S-层的生物技术应用。在这项研究中，我们证明了利用短乳杆菌S层亚基（SlpA）的表面展示外来抗原表位。利用诱导表达系统，L.在测试脊髓灰质炎病毒表位VP 1的slpA基因中的四个插入位点后，获得产生嵌合S-层的短病毒株，所述表位VP 1包含10个氨基酸。允许最佳表面表达的表位插入位点用于构建整合载体，所述整合载体携带编码来自人c-myc原癌基因的c-Myc表位的基因区，所述基因区由11个氨基酸组成。优化了L. brevis并用于用slpA-c-myc构建体替换野生型slpA基因。获得均匀的S层，在其表面上展示所有S层蛋白亚基中的所需抗原。通过PCR、Southern blotting MALDI-TOF质谱、全细胞酶联免疫吸附试验和免疫荧光显微镜证实了基因置换和均一的SlpA-c-Myc重组S层表达的成功。此外，通过电子显微镜研究了重组S层的完整性，这表明S层晶格结构不受c-Myc表位的存在的影响。据我们所知，这是第一次成功地表达外源表位在每个S-层亚基的乳杆菌S-层，同时仍然保持S-层晶格结构。
So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.