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目的构建弗氏2a志贺菌301 rfbA基因缺失突变株,初步探究志贺菌脂多糖合成与糖蛋白合成通路是否相关。方法首先用PCR扩增出rfbA基因上下游同源臂,构建含有kan基因的打靶片段,采用λ-Red重组系统对rfbA基因进行缺失,用PCR进行初步验证,然后通过LPS的银染方法进行进一步验证。随后,制备野生株、rfbA缺失突变株的全菌蛋白样品,进行双向电泳后比较野生株301和rfbA缺失株的蛋白表达谱。结果成功构建了301 rfbA缺失突变株。在LPS的银染实验中,rfbA突变株在凝胶中未形成一长串的梯状条带。在双向电泳实验中,野生株和突变株没有明显的蛋白差异点。结论获得了301 rfbA基因缺失突变体,初步认为志贺菌中不存在与脂多糖O-抗原合成相关的蛋白糖基化途径。
OBJECTIVE: To construct a 301 rfbA gene deletion mutant of Shigella spp. 2a and to investigate whether Shiga lipopolysaccharide synthesis is related to glycoprotein synthesis pathway. Methods The upstream and downstream homology arms of rfbA gene were amplified by PCR, and the targeting fragment containing kan gene was constructed. The rfbA gene was deleted by λ-Red recombination system and was verified by PCR. Then, silver staining was performed by LPS verification. Subsequently, a whole-body protein sample of a wild-type strain and a rfbA-deleted mutant strain was prepared and subjected to two-dimensional electrophoresis to compare the protein expression profiles of the wild-type strain 301 and the rfbA-deleted strain. Results The 301 rfbA deletion mutant was successfully constructed. In the silver stain of LPS, the rfbA mutant did not form a long series of ladder-like bands in the gel. In the two-dimensional electrophoresis experiment, there was no obvious protein difference between the wild strain and the mutant strain. Conclusion The deletion mutant of 301 rfbA gene was obtained, suggesting that there is no glycosylation pathway associated with lipopolysaccharide O-antigen synthesis in Shigella.