肠道菌群中鼠李糖乳杆菌的菌落免疫印迹计数与分离

本研究通过制备鼠李糖乳杆菌(Lactobacillus rhamnosus,Lr)菌毛蛋白Fim I特异性抗体,建立了Lr菌落免疫印迹计数与分离方法。将重组Fim I蛋白(r Fim I)免疫BALB/c小鼠获得抗血清,用Western和全菌Dot-Blot鉴定抗血清的反应性和特异性,建立基于r Fim I抗血清的菌落免疫印迹方法,并对模拟样品(小鼠肠道菌群+Lr)以及灌胃小鼠粪便中的Lr进行定量分析。结果显示获得的r Fim I抗血清效价为1:51200,该血清和Lr裂解蛋白和菌体均呈强阳性反应,与小鼠肠道菌群没有交叉反应。菌落免疫印迹中r Fim I抗体最佳浓度为1:2000,所有含有Lr样品的菌落转印膜上都能呈现清晰的免疫印迹,测定的阳性菌落数和模拟样品中预设的Lr浓度一致,并能较好的反应灌服Lr的小鼠粪便中Lr的消长趋势。本研究建立的菌落免疫印迹方法为肠道样品中Lr的选择性计数和分离提供了快捷的方法。

Application of Colony Immunoblotting for Counting and Isolating Lactobacillus rhamnosus in Intestinal Flora

A specific antibody against pilus protein (FimI) of Lactobacillus rhamnosus (Lr) was prepared, to develop a colony immunoblotting (CIB) counting and isolation method for Lr. BALB/c mice were immunized with recombinant FimI (rFimI) to obtain the antiserum, and the reactivity and specificity of the antiserum were tested by western blotting and whole bacterial dot-blot analysis. The CIB counting method was developed based on the rFimI antiserum, and was used to quantitatively detect Lr in the artificial sample (mouse intestinal flora + Lr) and the fecal samples from mice fed with Lr by gavage. The titer of the obtained antiserum was 1:51,200 and this antiserum showed strong reactivity with both the lysate and whole cells of Lr, but no cross-reactivity with the intestinal flora of mouse. The optimum dilution of rFimI antiserum for CIB was 1:2000, and clear immunoblots were observed on all colony transfer membranes of samples containing Lr. The positive colony number determined using the CIB method agreed with the Lr concentration in the artificial samples, which may reflect the fluctuation tendency of Lr in fecal samples from the mouse fed with Lr by gavage. The established CIB counting method can be used to selectively count and isolate Lr in intestinal samples.